United States
Environmental Protection
1=1 m m Agency
EPA/690/R-10/027F
Final
9-30-2010
Provisional Peer-Reviewed Toxicity Values for
Trimethyl Phosphate
(CASRN 512-56-1)
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
Harlal Choudhury, DVM, Ph.D., DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Paul G. Reinhart, Ph.D., DABT
National Center for Environmental Assessment, Research Triangle Park, NC
Geniece M. Lehmann, 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 ii
BACKGROUND 3
HISTORY 3
DISCLAIMERS 3
QUESTIONS REGARDING PPRTVS 4
INTRODUCTION 4
REVIEW 01 PERTINENT DATA 5
HUMAN STUDIES 5
Oral Exposure 5
Inhalation Exposure 5
ANIMAL STUDIES 5
Oral Exposure 5
Subchronic Studies 5
Chronic Studies 7
Reproductive/Developmental Studies 14
Inhalation Exposure 20
OTHER STUDIES 20
Acute or Short-term Studies 20
Neurotoxicity 23
Genotoxicity 24
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RFD
VALUES I OR TRIVUTIIYI. PHOSPHATE 35
SUBCHRONIC AND CHRONIC p-RfD 35
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RFC VALUES FOR TRIVUTIIYI. PHOSPHATE 39
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR TRIMETHYL
PHOSPHATE 39
WEIGHT-OF -EVIDENCE DESCRIPTOR 39
MODE-OF-ACTION DISCI SSION 40
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK 41
Oral Exposure 41
Inhalation Exposure 43
REFERENCES 43
APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING FOR ORAL
SLOPE FACTOR 50
l
Trimethyl Phosphate
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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
IRIS
Integrated Risk Information System
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 (oral)
RfC
reference concentration (inhalation)
RfD
reference dose
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
TRIMETHYL PHOSPHATE (CASRN 512-56-1)
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
No RfD, RfC, or cancer assessment for trimethyl phosphate (see Figure 1 for the structure
of trimethyl phosphate) is included on the IRIS database (U.S. EPA, 2009) or on the Drinking
Water Standards and Health Advisories List (U.S. EPA, 2006). The HEAST reported no RfD or
RfC values (U.S. EPA, 1997). The Chemical Assessments and Related Activities (CARA) list
(U.S. EPA, 1991, 1994) included a Health and Environmental Effects Profile (HEEP) for
trimethyl phosphate (U.S. EPA, 1985) that declined to derive noncancer toxicity values due to
inadequate noncancer data and potential carcinogenicity of the chemical (see below). The
toxicity of trimethyl phosphate has not been reviewed by ATSDR (2009) or the World Health
Organization (WHO, 2009). CalEPA (2009a,b) has not derived toxicity values for exposure to
trimethyl phosphate. No occupational exposure limits for trimethyl phosphate have been derived
by the American Conference of Governmental Industrial Hygienists (ACGIH, 2009), the
National Institute of Occupational Safety and Health (NIOSH, 2009), or the Occupational Safety
and Health Administration (OSHA, 2009).
0 — P — 0
Jo CH3
h3c
Figure 1. Chemical Structure of Trimethyl Phosphate
The HEAST (U.S. EPA, 1997) reported a cancer weight-of evidence classification of
Group B2 {Probable Raman Carcinogen) and an oral slope factor (OSF) of
3.7 x 10"2 (mg/kg-day)"1 for trimethyl phosphate based on increased incidence of uterine tumors
in female mice in a 103-week gavage study (NCI, 1978). The HEAST cited the HEEP
(U.S. EPA, 1985) as the source of the OSF. Trimethyl phosphate has not been evaluated under
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the 2005 Guidelines for Carcinogen Assessment (U.S. EPA, 2005). The International Agency
for Research on Cancer (IARC, 2009) has not reviewed the carcinogenic potential of trimethyl
phosphate. Trimethyl phosphate is not included in the 11th Report on Carcinogens (NTP, 2005).
CalEPA (2009b) has not prepared a quantitative estimate of carcinogenic potential for trimethyl
phosphate.
Literature searches were conducted from the 1950s through August 2010 for studies
relevant to the derivation of provisional toxicity values for trimethyl phosphate. Databases
searched included MEDLINE, TOXLINE (with NTIS), BIOSIS, TSCATS/TSCATS2, CCRIS,
DART, GENETOX, HSDB, RTECS, Chemical Abstracts, and Current Contents (last 6 months).
The HEEP (U.S. EPA, 1985) was also reviewed for pertinent studies.
REVIEW OF PERTINENT DATA
HUMAN STUDIES
Oral Exposure
No data are available on the oral toxicity of trimethyl phosphate in humans.
Inhalation Exposure
Data on the inhalation toxicity of trimethyl phosphate in humans are limited to a single
occupational exposure study (NIOSH, 1982). The study reported no medically significant
cholinesterase depression among a group of 175 factory workers exposed via inhalation to a
mixture of chemicals including trimethyl phosphate as well as dibromochloropropane,
chloroform, Vapona, acetone, sodium hydroxide, hexane, methyl isobutyl ketone, methyl
isocyanate, methyl thioacetoldoxime, Nudrin, and Azodrin during the manufacture or
formulation of pesticide products (NIOSH, 1982). Levels of trimethyl phosphate at the plant
(based on six personal air samples) were found to be below the detection limit of analysis by gas
chromatography-mass spectrometry. These data do not support an inhalation toxicity value.
ANIMAL STUDIES
Oral Exposure
Available subchronic and chronic studies evaluating the oral toxicity of trimethyl
phosphate in animals include a 9-week feeding study in rats (Oishi et al., 1982), 7-week gavage
range-finding studies in rats and mice and follow-up lifetime gavage studies (NCI, 1978), a
lifetime drinking water study in rats (Bomhard et al., 1997), an unpublished Japanese combined
repeated dose/reproduction/developmental toxicity study (MHW, 1994), and several other
studies specifically evaluating the effects on the male reproductive system and processes
(Takizawa et al., 1998; Cho and Park, 1994; Hanna and Kerr, 1981; Harbison et al., 1976).
Subchronic Studies—Male JCL-Wistar rats (18 controls and 6 treated) were fed diets
containing trimethyl phosphate (purity not reported) at concentrations of either 0 or 0.5%
continuously for up to 9 weeks (Oishi et al., 1982). Based on a default body weight for male
Wistar rats of 217 g and a default food consumption rate of 0.02 kg/day (U.S. EPA, 1988), the
approximate equivalent doses are 0 and 461 mg/kg-day. Rats were weighed at study termination,
and blood was collected for hematology (prothrombin time, kaolin-activated partial
thromboplastin time [kaolin-PTT], leukocyte counts [white blood cells, or WBCs], erythrocyte
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counts [red blood cells, or RBCs], hemoglobin concentration [Hgb], hematocrit [Hct], and mean
corpuscular volume [MCV]) and clinical chemistry (total protein, urea nitrogen, cholesterol,
glutamic oxaloacetic transminase [GOT or aspartate aminotransferase, AST] activity, glutamic
pyruvic transaminase [GPT or alanine aminotransferase, ALT] activity, alkaline phosphatase
[ALP] activity, total bile acids, sodium, and potassium). At sacrifice, liver, kidneys, spleen, and
testes were removed and weighed; all but the testes were examined histologically.
As shown in Table 1, treated rats gained statistically significantly (p < 0.05) less weight
than controls during the exposure period (12%). Significant differences (p < 0.05) in hematology
and serum chemistry findings between treated and control rats included shorter prothrombin time
and longer kaolin-PTT, lower RBC and Hgb, and decreased AST and ALT activities. In treated
animals, both mean absolute and relative kidney weights were statistically significantly
(p < 0.05) elevated over controls, and mean absolute testes weight was statistically significantly
(p < 0.05) lower than controls. However, no treatment-related histological changes were
reported. The only dose tested of 461 mg/kg-day is identified as a LOAEL for rats in this study
based on a reduction in body weight and statistically significant (p < 0.05) hematological and
biochemical changes as described above.
Table 1. Significant Changes in Male JCL-Wistar Rats Treated with
Trimethyl Phosphate via Oral Administration for 9 Weeks
Parameter
Control
461 mg/kg-day
Number of animals examined
18
6
Terminal body weight
446.2 ± 10.7a
392.5 ± 3.9b
Hematology
RBC (xl06/mm3)
6.94 ± 0.072
6.63 ± 0.076b
Hgb (g/100 mL)
13.3 ±0.12
12.7 ± 0.13b
Prothrombin time (second)
20.1 ±0.54
17.6 ± 0.4b
Kaolin-PTT (second)
37.4 ± 1.2(17)
43.2 ± 1.0b
Clinical chemistry
AST (Karmen units)
79 ±4.9 (16)
59 ± 2.9b
ALT (Karmen units)
32 ± 1.8 (15)
25 ± 1.4b
Absolute organ weights
Kidneys (g)
3.36 ± 0.11
3.73 ± 0.051b
Testes (g)
3.69 ±0.051
3.01 ±0.19b
Relative organ weights
Kidneys (g/100 gbw)
0.75 ±0.017
0.95 ± 0.017b
aMean ± standard error (n. if different from group size).
bSignificantly different from control atp< 0.05.
Source: Oishi et al. (1982).
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The National Cancer Institute (NCI, 1978) conducted 7-week dose range-finding studies
in rats and mice to estimate the low and high doses for use in chronic studies (described below).
Trimethyl phosphate (purity >99% in one batch and >95% in a second batch) was administered
by gavage in distilled water to groups of five rats or mice per sex at 0, 100 (rats only), 147, 215,
316, 464, 681, 1000, 1470, or 2150 (mice only) mg/kg-day, 3 days/week, for 7 weeks. Animals
were monitored for survival and changes in body-weight gain. All rats dosed with trimethyl
phosphate at >681 mg/kg-day died, and one male rat exposed to 464 mg/kg-day died. Distended
bladders and gastrointestinal hemorrhage were observed in these rats. At 464 mg/kg-day, males
gained approximately 44%, and females gained approximately 32% less weight than controls
(data not shown). Male and female rats dosed with <316 mg/kg-day gained approximately
20% less weight than controls (data not shown). Five male mice and one female mouse died at
2150 mg/kg-day, and two female mice died at 1470 mg/kg-day. A slight depression in mean
body weights was observed among male mice at >681 mg/kg-day (data not shown). According
to the study authors, body weights among female mice were not greatly affected. Surviving
animals were killed and necropsied 1 week after the end of the exposure period, but the results
were not reported. Frank effect levels (FELs) of 464 and 1470 mg/kg-day are identified for rats
and mice, respectively, based on mortality.
Chronic Studies—Lifetime exposure studies were conducted in rats and mice to assess
the carcinogenicity of trimethyl phosphate (NCI, 1978). In the rat study, trimethyl phosphate
(99% pure) was administered by gavage in distilled water to groups of F344 rats (50/sex) at
doses of 50 or 100 mg/kg-day, 3 days/week, for 104 weeks. Rats were observed for an
additional week following the exposure period. Vehicle control groups consisted of 20 male and
20 female rats given distilled water by gavage, 3 days/week, for 105 weeks.
Rats were observed twice daily for clinical signs, and body weights were measured at
regular intervals, although the study does not report the frequency of this observation. At each
weighing, rats were palpated for masses. Rats found in a moribund condition during the study
and surviving rats at study termination were killed and necropsied. Pathological evaluation
included gross and microscopic examination of all major tissues, organs, and gross lesions. No
rats died prior to 52 weeks. Survival at the end of the study was 40% for control males, 56% for
low-dose males, 35% for high-dose males, 60% for control females, 12% for low-dose females,
and 55%) for high-dose females. Statistical tests for a dose-related increase in mortality did not
achieve significance in either sex (p > 0.05, Tarone's test).
NCI (1978) reported that the mean body weights among both sexes for both dose groups
were slightly lower than controls. Body-weight data were presented as growth curves. Based on
visual inspection of the growth curve, it appears that terminal body weights of high-dose rats of
both sexes were more than 10% lower than controls. No clinical signs of toxicity were reported.
Histopathology revealed a variety of degenerative and inflammatory conditions related to aging,
but no treatment-related nonneoplastic lesions were observed. Doses of 50 and 100 mg/kg-day
(duration-adjusted doses of 21 and 43 mg/kg-day by multiplying 3/7) are identified as NOAEL
and LOAEL values, respectively, based on reduced body weights.
Table 2 shows a summary of tumor incidence as reported in NCI (1978) for F344 rats.
There was a statistically significant dose-related increase (p < 0.01.by Cochran-Armitage test)
for the incidence of subcutaneous fibromas in males across all dose groups, and the incidence of
fibromas in high-dose males was statistically significantly (p < 0.05 by Fisher's exact test)
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increased compared with controls. These benign tumors were characterized by layers of
well-differentiated fibroblastic cells separated by dense bands of mature collagen. Apparent
dose-related increases in the incidences of several other tumors were observed in male rats (as
shown in Table 2), but none of these were statistically significant in trend or pairwise tests.
Tumor incidence was not significantly different from controls among female rats, although the
authors did note several 'unusual' tumors in female rats including glioblastoma multiforme in
1/48 high-dose females, myxosarcoma in 2/49 high-dose females, and malignant reticulosis in
1/50 low-dose females. NCI (1978) concluded that trimethyl phosphate treatment was associated
with the induction of benign fibromas of the subcutaneous tissue in male rats and did not appear
to be carcinogenic in female rats.
Table 2. Significant Changes in Male F344 Rats Treated with Trimethyl Phosphate via
Oral Administration for up to 105 Weeks
Parameter
Control
50 mg/kg-day
100 mg/kg-day
Neoplastic lesions
Subcutaneous tissue, fibroma
0/20 (0)a'b
2/50 (4)
9/49 (18)°
Alveolar/bronchiolar, adenoma or carcinoma
0/19 (0)
2/49 (4)
5/46(11)
Hematopoietic system, leukemia or lymphoma
8/20 (40)
20/50 (40)
25/49 (51)
Adrenal, pheochromocytoma
1/20 (5)
4/48 (8)
7/47 (15)
aNumber of tumor-bearing animals/number of animals examined at site (percent).
Significant dose-related increase by Cochran-Armitage test at p < 0.01.
Significant pairwise difference from control by Fisher's exact test atp< 0.05.
Source: NCI (1978).
In the corresponding mouse study, groups of B6C3F1 mice (50/sex/dose) were treated by
gavage with trimethyl phosphate (99% pure) in distilled water at doses of 250 or 500 mg/kg-day,
3 days/week, for 103 weeks (NCI, 1978). No observation period followed treatment. Similar to
the rat study, vehicle controls consisted of 20 male and 20 female mice treated by gavage with
distilled water. Mice were evaluated for the same endpoints as outlined above in the rat study.
Survival at the end of the study was 70% for control mice, 88% for low-dose males, 80% for
high-dose males, 90% for control females, 62% for low-dose females, and 59% for high-dose
females. Statistical tests for a dose-related increase in mortality did not achieve significance in
either sex (p > 0.05, Tarone's test).
A dose-related decrease in mean body weights was observed among female mice, while
mean body weights of male mice were comparable to controls throughout the study (data
presented as growth curves). Based on an evaluation of the growth curve for this assessment, it
appears that terminal body weight among high-dose female mice was at least 10% lower than
controls. Most nonneoplastic lesions observed in treated mice were considered to be either
spontaneous or common in mice in long-term studies. The 500 mg/kg-day dose
(duration-adjusted dose of 214 mg/kg-day) is identified as a NOAEL for male mice and as a
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LOAEL for female mice based on reduced body weights. The corresponding NOAEL for female
mice is 250 mg/kg-day (duration-adjusted dose of 107 mg/kg-day by multiplying 3/7).
No significant increase in tumor incidence was observed in male mice (NCI, 1978). In
female mice, a statistically significant (p = 0.01, Cochran-Armitage test) dose-related trend in the
incidence of endometrial adenocarcinomas of the uterus was observed. The incidence of these
tumors in the high-dose group was statistically significantly higher (p = 0.01, Fisher's exact test)
than that in the controls (see Table 3). Microscopic examination of these tumors revealed
vascular involvement and pulmonary metastases in four high-dose females and one low-dose
female. NCI (1978) reported that this tumor has never been observed among historical controls
(100 female B6C3F1 mice) at this laboratory. Extensive thrombosis of the pulmonary arteries
was observed in three of these mice. In addition, endometrial squamous-cell carcinoma of the
uterus occurred in one high-dose female, and leiomyosarcoma of the uterus was observed in one
low-dose female. Hydronephrosis was observed in five of the female mice with uterine tumors.
NCI (1978) concluded that trimethyl phosphate was carcinogenic to female mice in this study.
Table 3. Uterine Tumor Incidences in Female B6C3F1 Mice Treated with
Trimethyl Phosphate via Oral Administration for 103 Weeks
Parameter
Control
250 mg/kg-day
500 mg/kg-day
Neoplastic lesions
Uterus, adenocarcinoma
0/16 (0)
7/40 (18)b
13/37 (35)°
aNumber of tumor-bearing animals/number of animals examined at site (percent).
Significant dose-related increase by Cochran-Armitage test at p < 0.01.
Significant pairwise difference from control by Cochran-Armitage test at p < 0.01.
Source: NCI (1978).
In a more recent chronic toxicity/carcinogenicity study, weanling Wistar rats
(60/sex/dose) were exposed by gavage to trimethyl phosphate (99% pure) in drinking water at
doses of 0, 1, 10, or 100 mg/kg-day for up to 30 months (Bomhard et al., 1997). Due to high
mortality at 100 mg/kg-day, this dose was reduced to 50 mg/kg-day at Week 54. At 12 months,
10 rats/sex/dose were sacrificed and necropsied. At 24 months, surviving rats in the high-dose
group were terminated and necropsied. All other surviving rats were terminated and necropsied
at 30 months.
During the exposure period, rats were monitored for changes in appearance and behavior
daily. Body weights were recorded weekly during the first 3 months and once every other week
for the remainder of the study. Food and water consumption were monitored weekly.
Ophthalmological examinations were performed in Weeks 98/99 and 128 on 10 rats/sex/dose.
Clinical laboratory investigations were also conducted on 10 randomly selected animals per
group. These included hematology (RBC, reticulocytes, leukocytes, differential leukocyte, and
platelet counts, Hgb, Hct, MCV, mean corpuscular hemoglobin [MCH], mean corpuscular
hemoglobin concentration [MCHC], and thromboplastin time) and urinalyses (volume, total
protein, specific gravity, pH [Month 14 only], sediment [microscopically examined], and
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semiquantitative measurements for blood, glucose, bilirubin, protein, ketone bodies, and pH
[except Month 14]) on blood and urine samples collected at 6, 12, 14, 18, 23/24, and 30 months,
and clinical chemistry (ALP, lactate dehydrogenase, AST, and ALT activity, total bilirubin,
cholesterol, creatinine, albumin, total protein, urea nitrogen, triglycerides, inorganic phosphate,
calcium, potassium, sodium, and chloride) on serum samples collected at 6, 12, and 14 months.
Necropsies were performed on all rats found dead or terminated in extremis, and on
10 rats/sex/group 12 months after study initiation, all high-dose rats at 24 months, and on all
other surviving rats at 30 months. Organ weights (adrenals, brain, heart, kidneys, liver, lungs,
ovaries, spleen, and testes) were recorded at scheduled necropsies. All rats were subjected to
complete gross and histopathological evaluations.
Clinical signs characterized by the study authors as hind limb weakness, sunken flanks
(especially in males), distended abdomen (especially in females), and poor general condition
were observed in high-dose animals beginning in Week 46 (Bomhard et al., 1997). Reduction in
the dose level for this group at Week 54 did not appear to alter these effects. Hind limb
weakness was also observed in the other dose groups and controls starting around Week 120; this
observation was attributed to old age. As mentioned above, high mortality rates were observed
following treatment with 100-mg/kg-day trimethyl phosphate in both males and females. In both
sexes, the increase in mortality became evident during the latter half of the first year of treatment
(data presented as a survival curve), and continued, despite the dose reduction to 50 mg/kg-day
at 54 weeks, until only approximately 30% of animals in this group survived to Week 100
(versus 85% or more of animals in other groups surviving to that point). Slightly more animals
dosed at 10 mg/kg-day died toward the end of the study compared with controls, but a
substance-related effect was questionable since Bomhard et al. (1997) reported that the mortality
rate was within the range of historical controls, and no indications of substance-specific causes
of death were reported. There was no effect on survival at 1 mg/kg-day. The authors reported
reductions in body weights among high-dose males (-20%) and females (-15%) throughout the
study and among mid-dose males towards the end of the study (about 10%) when compared with
controls (body-weight data presented as growth curves). Food consumption was slightly
decreased among high-dose males but was slightly higher than controls when adjusted to body
weight.
No treatment-related effects were observed in any dose group upon ophthalmological
examination. Hematology, clinical chemistry, and urinalysis data were not shown, but the
authors reported that significant findings in the high-dose group (males and females unless
otherwise noted) included reductions in Hgb, Hct, and RBC counts; increased proportion of
reticulocytes (males up to Month 18) and higher thrombocyte counts (up to Month 18); and an
increase in segmented neutrophils and a corresponding decrease of lymphocytes. At 12 months,
a relative increase in the al-globulin fraction above the historical range was observed,
accompanied by significant decreases in albumin and y-globulin fractions. A similar, but not
statistically significant, trend was reported at 18 and 24 months. Increased urinary protein
excretion (especially in males at Months 18 and 23) and decreased urine pH (females at
6 months) were also reported. No significant effects were noted in the other dose groups, with
the exception of decreases in urine pH among all treated female groups at 12 months.
As shown in Table 4, absolute organ weights at interim sacrifice (12 months) among
treated rats were comparable to controls except for a statistically significant (p < 0.05) decrease
in liver weight among high-dose males and statistically significantly (p < 0.01) decreased lung
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weight among mid-dose females (Bomhard et al., 1997). Relative heart, lungs, liver, and kidney
weights were statistically significantly (p < 0.05) elevated over controls among high-dose males.
Similarly, relative heart, liver, and kidney weights were statistically significantly (p < 0.01)
elevated over controls among high-dose females at 12 months. The organ weight changes in
high-dose rats occurred in parallel with decreased body weights in these rats. Statistically
significant (p < 0.05) increases in relative liver and kidney weights were also observed among
low-dose females. Since statistically significant differences between mid-dose females and
controls in liver and kidney weights were not observed, the findings in low-dose females could
possibly be related to sample size and may not be treatment-related. Gross examination of rats
sacrificed at 12 months showed changes only in high-dose rats: muscle wasting and changes in
lungs (mottled, reddish, pale), heart (thick, hard, abnormal color), liver (thick, scarring), and
kidneys (scarring) in males and females; small seminal vesicles and testes in males; and skin
edema in females. Microscopic examination at interim sacrifice revealed higher incidence of
peripheral nerve and spinal cord degeneration in high-dose males and females only (see Table 4).
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Table 4. Significant Changes in Wistar Rats Treated with Trimethyl Phosphate
via Oral Administration for 12 Months
Parameter
Control
1 mg/kg-day
10 mg/kg-day
100 mg/kg-day
Males
Number of animals examined
10
10
10
10
Absolute organ weights
Liver (mg)
16,089 ± 1738.7a
16,728 ± 1750.1
14,850 ± 1499.1
14,160 ± 1528. lb
Relative organ weights
Heart (mg/100 g bw)
253 ± 11.7
255 ±21.3
251 ± 17.8
325 ± 43.4°
Lungs (mg/100 gbw)
336 ± 15.8
329 ±28.7
352 ±32.5
438 ± 44.8°
Liver (mg/100 g bw)
3801 ± 152.1
3850 ±426.9
3609 ±331.1
4241 ± 360.9b
Kidneys (mg/100 gbw)
659 ±30.9
635 ±47.9
656 ±73.8
807 ± 88.7°
Nonneoplastic lesions
Degeneration of peripheral
nerve fiber
0/10d
0/10
0/10
8/10e
Degeneration of spinal cord
nerve fiber
0/10
0/10
0/10
4/10
Females
Number of animals examined
10
10
10
10
Absolute organ weights
Lungs (mg)
1091 ± 102.0a
1005 ±45.6
963 ± 112.6°
1057 ±75.3
Relative organ weights
Heart (mg/100 g bw)
301 ± 17.2
321 ±29.6
316 ±24.2
352 ± 21.0°
Liver (mg/100 g bw)
3668 ± 164.8
4034 ± 269. lb
3746 ±231.0
4300 ± 449.0°
Kidneys (mg/100 gbw)
695 ± 52.4
760 ± 65.lb
745 ±63.6
851 ±43.6°
Nonneoplastic lesions
Degeneration of peripheral
nerve fiber
0/10d
0/10
1/10
9/10°
Degeneration of spinal cord
nerve fiber
0/10
0/10
0/10
4/10
aStudy did not indicate whether data reflect mean ± standard deviation or mean ± standard error.
bSignificantly different from control atp< 0.05.
><0.01.
dNumber affected/number examined.
"Significantly different from control atp< 0.05 by Fisher's exact test performed for this review.
Source: Bomhard et al. (1997).
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Necropsy of animals from the main study groups revealed a slight increase in the
frequency of small hindlimbs, scarring of the kidneys, and small, soft testes in high-dose
animals. No treatment-related gross changes were observed at necropsy in the low- and
mid-dose animals. Microscopy revealed a statistically significantly (p < 0.05 by Fisher's exact
test) increased incidence of degeneration and loss of spinal cord nerve fibers of high-dose male
and female rats (see Table 5). Fiber damage in the peripheral nerves of these animals was
associated with reactive cell proliferation, resulting in hypercellularity, which was statistically
significantly increased in high-dose male and female rats (p < 0.05 by Fisher's exact test, see
Table 5). Although incidence data were not shown, the authors described additional findings that
they considered to be suggestive of dysfunction of the cardiovascular and respiratory system and
possibly related to perturbation of the nervous system; these findings included chronic
congestion of the lungs and kidneys, formation of thrombi in the heart atria in some males and
females, necrosis and lymphocytic infiltration of the liver in some males, and edema of
subcutaneous tissue and increased hematopoietic activity in the adrenal glands and bone marrow
in some females at the high dose.
Table 5. Significant Changes in Wistar Rats Treated with Trimethyl Phosphate
via Oral Administration for up to 24-30 Months
Parameter
Control
1 mg/kg-day
10 mg/kg-day
76 mg/kg-daya
Males
Nonneoplastic lesions
Peripheral nerve hypercellularity
0/5 0b
0/49
1/48
11/47°
Degeneration of spinal cord nerve
0/50
2/49
1/48
6/47°
fiber
Loss of spinal cord nerve fiber
0/50
0/49
0/48
15/47°
Females
Nonneoplastic lesions
Peripheral nerve hypercellularity
0/49
2/49
1/50
6/50°
Loss of spinal cord nerve fiber
0/49
0/49
0/50
10/50°
aTime-weighted average (100 mg/kg-day for 54 weeks and 50 mg/kg-day for 50 weeks).
bNumber affected/number examined.
Significantly different from control atp< 0.05 by Fisher's exact test performed for this review.
Source: Bomhard et al. (1997).
No significant treatment-related differences in the incidence, time of occurrence,
spectrum of types, or localizations of tumors were observed among treated rats when compared
with concurrent controls (Bomhard et al., 1997). Although the early termination of the high-dose
group limits the interpretation of those results, tumor incidence in this group was within the
range of historical controls at this laboratory, and a survival-adjusted statistical analysis did not
reveal any significant increase in any tumors in this group. Tumor incidences among the 10 and
1 mg/kg-day groups were also comparable to historical controls. Bomhard et al. (1997)
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identified a NOAEL of 1 mg/kg-day for this study, based on suppression of body-weight gain in
males at 10 mg/kg-day.
Reproductive/Developmental Studies—In an unpublished Japanese study, trimethyl
phosphate (99.9% pure) was administered to groups of Sprague-Dawley rats (13/sex/dose) via
gavage at 0, 40, 100, or 250 mg/kg-day, dosing commenced 14 days prior to mating and
continued through the 2-week mating period and for an additional 2 weeks postmating. For
females, dosing commenced 14 days before mating and was continued through mating, gestation,
and until Lactation Day 3 (MHW, 1994). This study is reported in Japanese, but an English
abstract and English data tables are available. This study was also submitted as a combined
repeated dose/reproductive/developmental toxicity study under the Organisation for Economic
Co-Operation and Development (OECD) high production volume (HPV) Chemicals Programme
and was included in the SIDS Initial Assessment Report (IPCS, 1996). Based on the data tables
in the Japanese report and the information included in the SIDS report, toxicological endpoints
evaluated in this study appeared to include mortality, behavior, body weight, food consumption,
organ weights (liver, kidneys, thymus, testes, epididymides) and histopathology (kidneys, liver,
thymus, reproductive organs, and nerve fibers). Blood and serum samples were also collected
from males for hematology (RBC, WBC, and platelet counts; Hgb; Hct; MCV; MCH; MCHC;
and differentiation of leukocytes) and clinical chemistry (total protein, albumin, A/G ratio, ALP,
GPT [ALT], GOT [AST], gamma-glutamyl transpeptidase [GGT], total bilirubin, cholinesterase,
cholesterol, glucose, blood urea nitrogen [BUN], creatinine, inorganic phosphorous, sodium,
potassium, chloride, and calcium). Reproductive endpoints evaluated in this study appeared to
include copulation rate, fertility index, number of implantations, embryonic mortality, pup
viability, pup weights, and morphological abnormalities among pups.
High-dose male rats gained significantly less weight (p < 0.01) and consumed
significantly less food (p < 0.01) than controls starting on the first week of treatment (MHW,
1994). Clinical signs, including progressive paralytic gait and decreased motor activity, were
observed in this group starting on the second week of exposure. Mortality in this group was high
(12/13), with the first deaths occurring during the fourth week of treatment. No mortality or
significant signs of toxicity were reported among low- or mid-dose males.
However, changes in hematology, clinical chemistry, organ weights, and histopathology
were observed among these rats, as summarized in Table 6. As shown, the hematological
changes observed in males at >100 mg/kg-day included decreased RBC counts, Hgb, and Hct,
and an increase in platelet count and percent of segmented neutrophils. Statistically significant
changes in clinical chemistry included decreases in A/G ratio and increases in cholinesterase,
total cholesterol and calcium levels at >40 mg/kg-day and decreased glucose levels, increased
total protein, and elevated sodium levels at 100 mg/kg-day. Males also exhibited significant
increases in absolute and relative kidney weights at >40 mg/kg-day and significant increases in
absolute and relative thymus and epididymis weights and relative liver weights at
100 mg/kg-day. In males, histopathology revealed nephropathy (slight to moderate), atrophy of
the thymus (severe), liver (moderate), spleen (severe), and testis (moderate to severe);
hypertrophy of the adrenal gland (slight); decreased sperm count (severe); and degeneration of
peripheral nerve fibers (slight). Most of these lesions occurred only at the high dose of
250 mg/kg-day, but degeneration of sciatic nerve was increased also at 100 mg/kg-day, and renal
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lesions were increased at all doses. Nephropathy in treated rats was characterized by
slight-to-moderate tubular alterations, including increased eosinophilic droplets in the tubular
epithelium, regenerated tubules, and increased eosinophilic bodies.
Table 6. Significant Changes in Male Sprague-Dawley Rats Treated with
Trimethyl Phosphate via Gavage
Parameter
Control
40 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Group size
13
13
13
13
Mortality
0
0
0
12
Terminal body weight (g)
479.7 ±48.7a
480.7 ± 27.2
468.8 ±28.8
244.8
Hematology
Number examined
13
12
13
1
RBC count (x 104/mm3)
781 ±37
768 ± 34
739 ± 24b
702
Hgb (g/dl)
14.5 ±0.5
14.2 ±0.5
13.7 ± 0.4b
13.2
Hct (%)
42.7 ± 1.5
41.4 ± 1.7
40 ± l.lb
37.6
Segmented neutrophils (%)
8 ± 4
9 ± 4
16 ± 8°
52
Platelet count (x 104/mm3)
100.3 ±6.7
105.9 ±7.2
114.5 ± 10.8b
106.4
Biochemistry
Number examined
13
12
13
1
Total protein (g/dl)
5.6 ±0.3
5.8 ±0.2
6.1 ± 0.3b
6.3
A/G ratio
1.17 ± 0.13
1.07 ± 0.07°
1.02 ± 0.10b
0.80
Cholinesterase (U/l)
288 ± 47
340 ± 42°
422 ± 93b
485
Total cholesterol (mg/dl)
56 ±9
70 ± 13°
75 ± 16b
145
Glucose (mg/dl)
203 ± 19
190 ± 15
183 ± llb
152
Na (mEq/1)
140.2 ±0.5
140.9 ±0.7
141.8 ± 1.3b
142.8
Ca (mg/dl)
8.9 ±0.4
9.3 ± 0.2°
9.5 ± 0.3b
9.2
Absolute organ weights
Number examined
13
13
13
1
Kidney (g)
2.97 ±0.32
3.49 ± 0.44b
3.46 ± 0.34b
2.87
Thymus (mg)
337.7 ±94.2
409.2 ±91.1
458.7 ± 116.9b
222.6
Testes (g)
3.03 ±0.19
3.27 ± 0.19°
3.07 ±0.35
1.30
Epididymides (g)
1.15 ± 0.11
1.08 ±0.08
0.89 ± 0.07b
0.50
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Table 6. Significant Changes in Male Sprague-Dawley Rats Treated with
Trimethyl Phosphate via Gavage
Parameter
Control
40 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Relative organ weights
Number examined
13
13
13
1
Liver (g/100 gbw)
3.82 ±0.28
4.07 ±0.36
4.26 ± 0.29°
4.05
Kidney (g/100 gbw)
0.62 ± 0.04
0.73 ± 0.08b
0.74 ± 0.05b
1.17
Thymus (mg/100 gbw)
69.8 ± 15.9
85.6 ±20.6
98.4 ± 27.2b
90.9
Epididymides (g/100 gbw)
0.24 ±0.03
0.22 ± 0.02
0.19 ± 0.02b
0.20
Histopathology
Thymus: atrophy
0/13 d
0/13
0/13
12/13b
Liver: hepatocellular atrophy
0/13
0/13
0/13
12/13b
Kidney: eosinophilic droplet in tubular
epithelium
1/13
13/13b
13/13b
2/13
Kidney: regenerated tubule
6/13
13/13b
13/13b
12/13b
Kidney: eosinophilic body
5/13
13/13b
13/13b
1/13
Adrenal: hypertrophy of cortical cell
0/13
0/13
0/13
8/13b
Spleen: atrophy of follicle
0/13
0/13
0/13
13/13b
Testes: atrophy
0/13
1/13
1/13
13/13b
Epididymis: decreased number of sperm
0/13
0/13
1/13
13/13b
Skeletal muscle: atrophy of myofiber
0/13
0/13
0/13
ll/12b
Skeletal muscle: degeneration of nerve
fiber
1/13
0/13
4/13
10/12b
Sciatic nerve: degeneration of nerve fiber
0/13
0/13
9/13b
12/12b
aMean ± standard deviation.
V<0.01.
Significantly different from controls atp< 0.05.
dNumber affected/number examined.
Source: MHW (1994).
As in the males, high-dose females typically showed decreased motor activity starting on
the second week of the study, but progressive paralytic gait was only infrequently reported, and
only one animal died during the study (MHW, 1994). Fertility was significantly (p < 0.01)
affected by trimethyl phosphate. As shown in Table 7, only two high-dose pairs copulated, and
neither female became pregnant. Mid-dose pairs copulated, but only 2/13 females from this
group became pregnant. Body weight of pregnant females was reduced compared to controls in
both the low- (-12%) and mid-dose (-21%) groups on Gestation Day (GD) 20. Neither of the
two pregnant mid-dose females achieved parturiency. In the low-dose group, the fraction of
pregnant females delivering litters with live pups was lower than controls (10/12 versus 13/13),
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and the average number of live pups per litter was markedly reduced (-43%, p < 0.01). The
English abstract specifically describes a significant increase in intrauterine mortality in this
group. There was no additional effect on pup viability between Days 0 and 4 of lactation, and
pup weights were statistically significantly (p < 0.01) higher than controls from birth to terminal
necropsy at Lactation Day 4. Terminal examination of the F0 females showed statistically
significant (p < 0.01) increases in absolute and relative thymus weights in the low-dose group
(not measured in mid- or high-dose groups) but without any corresponding histopathology. The
only noteworthy histopathology finding in females was significant (p < 0.01) degeneration of
nerve fibers in the high-dose group.
Table 7. Significant Changes in Female Sprague-Dawley Rats Treated with
Trimethyl Phosphate via Gavage
Parameter
Control
40 mg/kg-day
100 mg/kg-day
250 mg/kg-day
Number mated
13
13
13
13
Number copulated
13
13
13
2b
Number pregnant
13
12
2b
0
Body weight on GD 20 (g)
404.2 ± 24.4a
357.2 ±26.9b
319.5
NA
Day 0 of lactation (birth)
Number of litters with live pups
13
10
0
0
Average number of live pups per
litter
13.4 ±3.7
7.6 ±3.8b
NA
NA
Pup weight—male (g)
6.3 ± 1.1
7.8 ± 0.6b
NA
NA
Pup weight—female (g)
5.9 ±0.9
7.5 ±0.5b
NA
NA
Day 4 of lactation
Number of litters with live pups
13
9
0
0
Average number of live pups per
litter
12.8 ±4.4
8.4 ±2.9b
NA
NA
Pup weight—male (g)
9.8 ±2.0
12.8 ± 1.8b
NA
NA
Pup weight—female (g)
8.9 ±2.4
12.5 ±1.8b
NA
NA
Terminal examination of F0
Thymus wt (mg)
138.1 ±70.2
261.9 ±51.2b
NA
NA
Thymus wt—relative
(mg/100 gbw)
44.7 ±22.3
82.5 ± 15.4b
NA
NA
Skeletal muscle: degeneration of
nerve fiber
0/13
0/13
0/13
9/13b
Sciatic nerve: degeneration of
nerve fiber
0/13
0/13
0/13
ll/13b
aMean ± standard deviation.
bSignificantly different from control at/? < 0.01.
Source: MHW (1994).
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Based on the effects described above, MHW (1994) identified the NOAEL as
<40 mg/kg-day for both repeated dose toxicity and reproductive/developmental toxicity.
OECD/Screening Information Data Sets (SIDS) (ICPS, 1996) also identified <40 mg/kg-day as
the NOAEL and 40 mg/kg-day as the LOAEL for both repeated dose toxicity and
reproductive/devel opmental toxi city.
Hanna and Kerr (1981) administered trimethyl phosphate (purity not reported) to male
albino Sprague-Dawley rats (number not specified) at 0 or 250 mg/kg-day via gavage
5 days/week, for 30 days, or 6 days/week, for 60 days. After treatment, males were mated with
untreated females to assess fertility. Upon cessation of treatment, semen was collected from two
rats of the 30-day exposure group for evaluation of sperm presence and morphology. Testes
from three animals from each treatment group and from controls were examined histologically.
Following termination of treatment, virgin females mated with treated males lacked vaginal
plugs, indicating impaired mating ability of the treated males. Spermatozoa from treated rats
appeared abnormal, exhibiting detached heads and abnormalities of the head, middle piece, and
principal piece. Histological examination of testes from rats exposed for 30 days revealed
indications of impaired spermatogenesis due to abnormal spermiogenesis and depletion of the
numbers of mature spermatids. Round spermatids showed vacuoles within their nuclei and
extensive extracellular spaces between the germ cells and Sertoli cells. Evaluation of the testes
from rats exposed for 60 days revealed absent germ cells in the seminiferous tubules, resulting in
collapse and shrinkage of the tubules and the presence of only Sertoli cells. The only dose
tested, 250 mg/kg-day (duration-adjusted dose of 179 mg/kg-day, based on exposure
5 days/week), is identified as a LOAEL for male reproductive effects.
Cho and Park (1994) administered trimethyl phosphate (99% pure) to male albino
Sprague-Dawley rats (20/group) at 0, 400, 500, 750, 1000, or 1500 mg/kg-day via gavage
5 days/week for up to 5 weeks. Four surviving rats per group were sacrificed weekly. Testes
were collected for microscopic examination and evaluation of spermatogenic stages, and
seminiferous tubules were examined for maturation staging. Immediately following dosing,
effects on spermatogenesis characterized by aggregation of multinucleated giant cells and
maturation arrest at the spermatid level were observed. Peak frequency in the emergence of
multinucleated giant cells occurred 1 week after treatment ended, and maturation arrest was
prominent at 3 weeks following treatment. Dead rats found during the exposure period were
subjected to gross and microscopic examination. Mortality rates were 0, 10, 90, 100, 100, and
100% at 0, 400, 500, 750, 1000, and 1500 mg/kg-day, respectively. Almost all dead rats were
anuric and anorexic prior to death. Gross and microscopic examination of the kidneys, liver,
lungs, and heart from these rats was unremarkable. However, gross examination revealed
severely distended bladders. Microscopic evaluation of the bladders of these animals revealed
multifocal ulceration, loss of urothelial epithelium with marked thinning, and atrophy of the
muscle proper. The 400 mg/kg-day dose (lowest dose tested) is identified as a FEL for this study
based on mortality.
Takizawa et al. (1998) administered trimethyl phosphate (purity not reported) to male
Sprague-Dawley rats (10/dose) at 0 or 100 mg/kg-day via gavage once daily for 28 days. Rats
were monitored for changes in body weight and food consumption during the exposure period.
Twenty-four hours after the final dosing, the rats were sacrificed, and the testes, epididymides,
seminal vesicles, and prostrate were removed and weighed. Sperm samples were collected from
the cauda epididymis and analyzed for evaluation of sperm viability and counts (using flow
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cytometry) as well as motility and morphology (using light microscopy). Testes and
epididymides were also examined microscopically. No significant changes in body weights,
food consumption, or organ weights were observed among treated rats. No significant effects
were observed on sperm viability or counts, but sperm motility was reduced. Degenerative
spermatogenic cells and degenerative sperm were observed in the ducts of the epididymides in
1/10 and 3/10 rats, respectively. No notable histological changes were observed in the testes,
seminal vesicles, or prostate. The 100 mg/kg-day dose (the only dose tested) is identified as a
LOAEL for reduction in sperm motility for this study.
Harbison et al. (1976) conducted numerous experiments in rats, mice, and rabbits,
including short-term studies that are described in further detail below under the section Acute or
Short-term Studies, and longer-term studies in which trimethyl phosphate (purity 97%) was
injected intraperitoneally (i.p). or administered by gavage. The report was unclear as to which
method of administration was used in any given experiment. For all species, treated males were
mated with untreated females. The females were later sacrificed for determination of pregnancy
as a measure of male fecundity (calculated as the number of pregnancies per total breeding
population). Control males were also mated with untreated females. Animals were monitored
for differences between treated and control groups in general behavior, skeletal muscle activity,
mating behavior, and frequency of vaginal plugs and mountings. Testicular biopsies and caudal
spermatozoa were obtained at various periods during and following treatment. Treatment and
findings reported by Harbison et al. (1976) for rats, mice, and rabbits are described briefly
below.
Male albino Sprague-Dawley rats (number not specified) were treated with trimethyl
phosphate at 0 and 100 mg/kg-day, 5 days/week, for 1 month or 0 and 750 mg/kg-day,
1 day/week, for 12 weeks (Harbison et al., 1976). In rats treated with 750 mg/kg-day, fecundity
was reduced by 50% during the first week of treatment and, by 94-100%), on Weeks 3 through
12. Rats treated with 100 mg/kg-day for 1 month demonstrated a reduction in fecundity by about
71%) during the first week following termination of treatment. Fertility returned to normal
during the next week. There were no differences between treated and control groups in general
behavior, skeletal muscle activity, mating behavior, or frequency of vaginal plugs. In addition,
there were no significant histological changes in the testes of treated animals, and
spermatogenesis appeared normal. The 100 mg/kg-day dose (the lowest dose tested,
duration-adjusted dose of 71 mg/kg-day) is identified as a LOAEL for rats based on the effects
on fecundity.
Male albino Swiss mice (number not specified) were treated with trimethyl phosphate at
1500 mg/kg-day, for 5 days/week, for 1 month (Harbison et al., 1976). In these mice, sterility
persisted for 2 weeks following termination of treatment and gradually returned to normal
4 weeks later. There were no differences between treated and control groups in general
behavior, skeletal muscle activity, mating behavior, or frequency of vaginal plugs. In addition,
there were no significant histological changes in the testes of treated animals, and
spermatogenesis appeared normal. The 1500 mg/kg-day dose (the only dose tested,
duration-adjusted dose of 1071 mg/kg-day) is identified as a LOAEL for mice based on observed
persistent sterility.
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Male New Zealand white rabbits (number not specified) were treated with trimethyl
phosphate at 200 mg/kg-day every 5 days, for 9 weeks, or at 325 mg/kg-day, once a week, for
13 weeks, and 750 mg/kg-day for dogs (Harbison et al., 1976). In rabbits treated with
200 mg/kg-day, fecundity was reduced by 50% by the 3rd week of treatment and by 75% by the
9th week. Among the rabbits treated with 325 mg/kg-day, fecundity was reduced by about 63%
by the 2nd week of treatment and by 87% in the 750-mg/kg-day group in the first week
following treatment. Sterility persisted from Week 5 after the start of treatment through
Week 13 in these rabbits. Within 1 week following the end of the treatment (Week 13), fertility
returned to normal in these animals. There were no differences between treated and control
groups in general behavior, skeletal muscle activity, mating behavior, or frequency of vaginal
plugs. In addition, there were no significant histological changes in the testes of treated animals,
spermatogenesis appeared normal, and mating behavior was unaffected in any species tested.
The 200 mg/kg-day dose (the lowest dose tested, duration-adjusted dose of 143 mg/kg-day) is
identified as a LOAEL for rabbits based on the effects on fecundity.
Based on the results across all of the experiments conducted by Harbison et al. (1976)
(including the shorter-term studies described below), trimethyl phosphate was found to induce a
dose-dependent reversible sterility, which was also dependent on duration of exposure, in male
rats, mice, and rabbits. However, the lack of information regarding route of exposure limits the
interpretation of these data for risk-assessment purposes.
Inhalation Exposure
No data are available on the inhalation toxicity of trimethyl phosphate in animals.
OTHER STUDIES
Acute or Short-term Studies
Acute oral studies (exposure duration of <5 days) have been conducted on male animals
that support the findings from subchronic-duration and reproduction studies that trimethyl
phosphate affects reproductive parameters and, in particular, have shown effects on the male
reproductive tissues and processes (see Table 8). These studies have shown that acute oral
exposure of male animals to trimethyl phosphate results in reversible functional infertility at
doses of >100 mg/kg-day in rats and at 1000 mg/kg-day in mice. Trimethyl phosphate has also
been shown to cause a significant reduction in sperm count and sperm motility; reduced prostate,
testes, and epididymide weights; increased cauda weights; reduced testosterone levels; and
increased numbers of immature Leydig cells in male rats. As shown in Table 8, the lowest dose
associated with acute reproductive effects is 200 mg/kg-day in male rats.
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Table 8. Acute Oral Studies of Male Reproductive Effects
Animal
Dose/Route/Duration
Parameters
Examined
Results
Comments
Reference
Rats
Male rats
(5/group, strain not
specified)
100 or 250 mg/kg-day via
gavage for 5 d
Number of offspring from
matings with untreated
females (determined
weekly)
Dose-related reduction in the
number of offspring; offspring
were absent or markedly
reduced in number at Wks 2-5,
but fertility was restored at
Wks 6-12 in both dose groups.
No controls were reported;
few data were presented.
Jackson and
Jones, 1968
Male rats
(number and strain not
specified)
500 or 2500 mg/kg via an
unspecified oral route for
an unspecified duration
Effects on spermatogenesis
Sterility for 3 wks posttreatment
at 500 mg/kg; complete
disorganization of
spermatogenesis without
damage to the tubular
architecture at 2500 mg/kg
accompanied by infertility for
20-25 wks posttreatment.
Lacking study details; no
further data were presented.
Jones and
Jackson, 1969
Male Wistar rats
(10-17/group)
0 or 100 mg/kg-day via
gavage for 5 d
Organ weights and
histology of testes, prostate,
seminiferous tubules,
pituitary, adrenal glands,
liver, and kidney,
testosterone levels in
plasma and testes tissue,
and histochemistry of the
testes
Decreased prostate weight,
decreased testosterone
concentration in plasma and
testes, positive histochemical
reaction for 3/;-hydro\ystcroid
dehydrogenase by the sperm
tails, increased number of
immature Leydig cells, and
increased interstitial fluid in the
testicular interstitial tissue.
Carstensen,
1971
Male Long-Evans hooded
rats
(20/group)
0, 100, 250, or
600 mg/kg-day, via gavage
for 5 d
Body weight, organ weights
(testis, cauda epididymis,
epididymis), sperm counts,
and sperm motion
Dose-related reduction in
weight gain, increased cauda
weight, decreased sperm count,
and altered sperm motion
Tothetal., 1992
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Table 8. Acute Oral Studies of Male Reproductive Effects
Animal
Dose/Route/Duration
Parameters
Examined
Results
Comments
Reference
Male Wistar rats
(number not given)
250 or 500 mg/kg-day via
an unspecified oral route
for 5 d
Sperm motility and count
Decreased sperm motility and
count at 500 mg/kg-day.
This publication is an
abstract.
Suzuki et al.,
1996
Male Sprague-Dawley rats
(number not given)
0 or 600 mg/kg single dose
via an unspecified oral
route
Testes weight and sperm
counts, motility, swimming
speed, and pattern
No effect on testes weight or
sperm count. Significant
decrease in sperm swimming
speed, path velocity,
straight-line velocity,
curvilinear velocity, and lateral
amplitude. Significant increase
in beat frequency.
Rats were observed for
3 wks following treatment.
This publication is an
abstract.
Fukunishi et al.,
2000
Male Sprague-Dawley rats
(number not given)
0, 60, 200, or
600 mg/kg-day, via gavage
for 5 d
Testes and epididymides
weights, and sperm motion
Decreased testes and
epididymides weights at
600 mg/kg-day, decreased
sperm count at 600 mg/kg-day,
and altered sperm motion at
>200 mg/kg-day.
Data for trimethyl
phosphate originally
presented in an abstract
presented at the 26th annual
meeting of the Japanese
Society of Toxicology.
Fukunishi et al.,
1999 (as cited
inKato et al.,
2001)
Mice
Male mice, strain not
specified
(8/dose)
1000 mg/kg-day via gavage
for 5 d
Number of offspring from
matings with untreated
females (determined
weekly)
Reduced number of offspring.
Study is lacking
information on controls and
study methods; few data
were presented.
Jones and
Jackson, 1969;
Jackson and
Jones, 1968
Male Swiss mice
(number not given)
0, 500, or 1000 mg/kg-day
via gavage for 5 d
Number of pregnancies
from matings with
untreated females
(determined weekly)
One male at 1000 mg/kg-day
died; no other signs of systemic
effects in males; reduced
pregnancy rate at
1000 mg/kg-day.
Epstein et al.,
1972, 1970
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Additional acute studies by Harbison et al. (1976) in male rats, mice, and rabbits
demonstrated that treatment with trimethyl phosphate daily for 5 days produced temporary
infertility in rats at 600 mg/kg-day, and in mice at 750 and 1500 mg/kg-day. Rabbits treated
with a single dose of trimethyl phosphate at 750 mg/kg also exhibited temporary infertility.
However, these studies are limited because the authors do not indicate which animals or in which
experiments gavage dosing was used instead of dosing by i.p. injection.
Neurotoxicity
In an older study specifically designed to assess the neurotoxicity of trimethyl phosphate in dogs,
Schaeppi et al. (1984) fed gelatin capsules containing 1-mL trimethyl phosphate to five adult
beagle dogs (two males and three females) daily for 1-4 months. Based on the reported
individual body weights of the dogs during the course of the study, the approximate daily doses
for each dog were 88 and 121 mg/kg-day for males exposed for 29 and 50 days, and 105, 89, and
106 mg/kg-day for females exposed for 71, 101, or 121 days, respectively. An additional female
dog received a capsule containing 2-mL trimethyl phosphate orally 5 days/week, for 150 days.
Based on an average body weight of 9.45 kg, the duration-adjusted equivalent dose for
continuous daily exposure was approximately 181 mg/kg-day. No control group was tested
simultaneously with these dogs. Neurological tests, including examination of tonic neck
reflexes, righting response, standing on a straight line, pain reflex, cornea reflex, and pupil light
response, were conducted weekly. Electrodiagnostic tests (maximum nerve conduction velocity
[MNCV]) were conducted biweekly. Electrophysiological control values were available from
pretest examination of the treated dogs and from previous studies on untreated control dogs.
Treated dogs were also subjected to neuropathology examination.
All dogs treated daily with 1-mL trimethyl phosphate (88-121 mg/kg-day) developed
signs of neurotoxicity including impaired gait, hopping, tactile placing and landing, persistence
in abnormal posture, and decreased muscle tone; these signs became progressively more severe
with duration of treatment (Schaeppi et al., 1984). The dogs that received >50 treatments had
prolonged distal latency for neuromuscular impulse transmission compared with pretest values.
Sensory MNCV was decreased for the dog that received 120 treatments. No changes in
peripheral motor MNCV occurred in any dogs receiving 1 mL/day when compared with
pretreatment control values or untreated dogs from previous studies. No neuropathologic
changes were observed in dogs treated for <71 days. Neuropathology examination revealed
degenerative changes in nerve fibers and demyelination of axons among the female dogs treated
the longest (101 and 121 days).
The dog treated with 181 mg/kg-day exhibited notable weight loss after 85 days of
treatment and inactivity after Day 88 (Schaeppi et al., 1984). Treatment was discontinued on
Days 93-112, resumed during Days 113-149, and terminated following Day 149 due to severe
morbidity. This dog was sacrificed on Day 151 in poor general condition. Signs of
neurotoxicity in this animal increased in severity with increasing duration of exposure. These
signs included enhanced patellar reflex (Day 18), attenuated extensor postural thrust (Day 25),
atactic gait (Day 39), decreased muscle force and persistence in abnormal posture (Day 46), and
decreased muscle tonus and impaired hopping and landing (Day 53). Neurophysiologic testing
revealed attenuated sensory MNCV and a progressive decrease of central motor MNCV to as
low as 50% of the pretreatment value by Day 150. Neuropathology examination revealed
advanced distal degeneration of the long spinal tracts and the peripheral nerve fibers, and
demyelination of nerve fibers.
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The findings reported by Schaeppi et al. (1984) are uncertain due to the small number of
animals, lack of concurrent control group, and variable dosing regimen; however, this study does
provide suggestive evidence for neurotoxicity associated with oral trimethyl phosphate. The
evidence for neurotoxicity is bolstered by the observations of Bomhard et al. (1997), who
reported peripheral nerve damage manifested as muscle wasting of the hind limbs in Wistar rats
treated with 100-mg/kg-day trimethyl phosphate in the drinking water for 54 weeks. This was
the highest dose tested; however, this dose was reduced during the course of the study due to
intolerance. Neurological effects were not observed at the lower test does of 1 and
10 mg/kg-day.
Genotoxicity
Trimethyl phosphate has been studied extensively in mutagenicity and genotoxicity
assays in vivo and in vitro. As shown in Tables 9 (in vitro studies) and 10 (in vivo studies),
trimethyl phosphate generally gave mixed or equivocal results in bacterial reverse-mutation
assays, mixed results in bacterial tests of DNA repair, and consistently positive results for
genotoxicity in mammalian cell systems and in mammals and D. melanogaster tested in vivo.
Additionally, there are numerous in vivo studies where trimethyl phosphate has been used as a
positive control substance (Sinha et al., 1983; Moutschen and Degraeve, 1981; Valencia, 1981;
Hanna and Dyer, 1975; Legator et al., 1973).
As shown in Table 9, trimethyl phosphate was positive for reverse mutation in
Salmonella typhimurium strain TA100 in the presence and absence of metabolic activation
(Zeiger et al., 1992, 1982; De Flora et al., 1990, 1984; De Flora, 1981; Bruce and Heddle, 1979;
Anderson and Styles, 1978; Farrow et al., 1976). For the most part, results in other
S. typhimurium strains (TA98, TA1538, TA1537, TA1535, TA1530, TA1531, TA1532, TA1534,
TA1536, TA1537, TA1538, GS46) were either equivocal or negative (De Flora et al., 1990,
1984; Zeiger et al., 1982; De Flora, 1981; Bruce and Heddle, 1979; Anderson and Styles, 1978;
Farrow et al., 1976; Hanna and Dyer, 1975; MacPhee, 1973). However, some studies reported
positive results in S. typhimurium strains TA102, TA2638 (Watanabe et al., 1996), TA98
(Farrow et al., 1976), hisC117 (Hanna and Dyer, 1975), LT2hisG46 (MacPhee, 1973), and
TA1535 (Anderson and Styles, 1978). Positive results for reverse mutation were also obtained in
Escherichia coli strains WP2, WP2uvrA, CM611, CM891, and WP12 (Watanabe et al., 1996;
Li et al., 1993; Hanna and Dyer, 1975; Dean, 1972) and in the bacterium Serratia marcesans
strains Hy/al3 and Hy/a21 (Dean, 1972). Trimethyl phosphate was positive with and without
S9-activation in a DNA-repair assay using E. coli strains WP2 and WP67 or CM871
(DeFlora et al., 1990, 1984) and positive with S9-activation in strains uvrB+/recA/lac- and
lvrB-/recA-/lac+ (Hellmer and Bolcsfoldi, 1992). Results were negative without activation in a
similar assay using E. coli strains P3110 and P3478 (Fluck et al., 1976). Only a few assays using
mammalian cell systems are available. A dose-related increase in the incidence of DNA
single-strand breaks was observed in rat hepatocytes without metabolic activation (Sina et al.,
1983). However, a similar assay was negative for DNA double-strand breaks (Storer et al.,
1996). Trimethyl phosphate was positive for micronuclei in Chinese hamster lung cells
(Ni et al., 1993) and chromosome aberrations in human lymphocytes (Soderman, 1972).
24
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Table 9. Genotoxicity Studies of In Vitro Trimethyl Phosphate Exposure
Test System
Endpoint
Concentration
Activating
System
Results"
Comments
Reference
Without
Activation
With
Activation
S. typhimurium
TA1535, TA1538, TA98,
TA100
Reverse
mutation
2500 (ig/plate
+S9
NA
+/-
Positive for TA1535 and TA100,
negative for TA1538 and TA98.
Anderson and
Styles, 1978
S. typhimurium
TA1535, TA1537, TA98,
TA100
Reverse
mutation
0.05,0.5,5, 50, or
500 (ig/plate
±S9
-/=
+/=
Positive for TA100 in presence of
S9, equivocal for other strains; S9
from Aroclor-induced rats.
Bruce and
Heddle, 1979
S. typhimurium
TA1535, TA1537, TA1538,
TA98, TA100
Reverse
mutation
0.6-1.1 x 106
nmol/plate
±S9
+/-
+/-
Positive for TA100
(0.0003 revertants/nmol) and S9
slightly enhanced the response;
negative for other strains; S9
from Aroclor-induced rats.
DeFlora et al.,
1990, 1984;
DeFlora, 1981
S. typhimurium
LT2hisG46, LT2hisG46
(R-Utrecht), TA1530
Reverse
mutation
Not reported
None
+/-
NA
Positive for LT2hisG46 and
LT2hisG46 (R-Utrecht), negative
for TA1530.
MacPhee, 1973
S. typhimurium
GS46
Reverse
mutation
1250, 1500, or
1700 mg/kg
Host-mediated
NA
Trimethyl phosphate did not
induce revertants in the bacteria
from the peritoneum, mouse host
treated intramuscularly.
Farrow, 1975
S. typhimurium
hisC117, hisG46, TA1530,
TA1535, TA1531, TA1532,
TA1534, TA1536, TA1537,
TA1538
Reverse
mutation
Not reported
None
+/-
NA
Positive for hisCl 17 strain only.
Hanna and Dyer,
1975
S. typhimurium
GS46, TA92, TA100, TA98
Reverse
mutation
125 mg/plate
Liver
microsomes
(mouse, rat, or
monkey)
NA
+/-
Positive for TA100 (greatest
number of revertants regardless
of species) and TA92.
Farrow et al.,
1976
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Table 9. Genotoxicity Studies of In Vitro Trimethyl Phosphate Exposure
Test System
Endpoint
Concentration
Activating
System
Results"
Comments
Reference
Without
Activation
With
Activation
S. typhimurium
GS46, TA92, TA100, TA98
Reverse
mutation
1750 mg/kg
administered to
mice (urine added
to S. typhimurium
cultures)
NA
NA
+/-
Weakly positive for TA98 and
TA100.
Farrow et al.,
1976
S. typhimurium
TA1535, TA100
Reverse
mutation
0, 1, 10, 50, 100,
or 250 mmol/plate
±S9
=/+
=/+
Equivocal for TA1535, positive
for TA100; metabolic activation
slightly enhanced the response in
TA100; S9 from Aroclor-induced
rats.
Zeiger et al.,
1982
S. typhimurium
TA98, TA100
Reverse
mutation
0 (solvent control),
333, 1000, 3333,
6666, 10,000, or
15,000 (ig/plate
±S9
NR
+/-
Positive for TA100, negative for
TA98.
Zeiger et al.,
1992
S. typhimurium
TA97, TA98, TA100, TA102
Reverse
mutation
Not reported
±S9
-
-
Negative in all strains; Chinese
study.
Li et al., 1993
S. typhimurium
TA102, TA2638
Reverse
mutation
Not reported
±S9
+
NR
Results given for S9 mix.
Watanabe et al.,
1996
E. coli
WP2
Reverse
mutation
Not reported
None
+
NA
Weakly mutagenic; paper disc
method.
Dean, 1972
E. coli
WP2, WP2uvrA, CM561,
CM571, CM611, WP67,
WP12
Reverse
mutation
Not reported
None
+/-
NA
Positive for strains WP2,
WP2uvrA, CM611, WP67, and
WP12.
Hanna and Dyer,
1975
E. coli
WP2, WP2uvrA, CM891,
ND160, MR2-102
Reverse
mutation
Not reported
±S9
+/-
+/-
Positive for strains WP2,
WP2uvrA, and CM891; response
potentiated by S9 activation;
Chinese study.
Li et al., 1993
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Table 9. Genotoxicity Studies of In Vitro Trimethyl Phosphate Exposure
Test System
Endpoint
Concentration
Activating
System
Results"
Comments
Reference
Without
Activation
With
Activation
E. coli
WP2/pKM101,
WP2uvrA/pKM 101
Reverse
mutation
Not reported
±S9
+
NR
Results given for S9 mix.
Watanabe et al.,
1996
E. coli
WP2 (repair proficient)
WP67 or CM871 (repair
deficient)
DNA repair
Not reported
±S9
+
+
Positive for all strains; S9 from
Aroclor-induced rats.
DeFlora et al.,
1990, 1984
E. coli
P3110 (polA+)
P3478 (polA-)
DNA repair
25 |iL
None
NA
Negative in both strains.
Flucketal., 1976
E. coli
uvrB+/recA/lac- (repair
proficient) or
243/59 luvrB-/recA-/lac+
(repair deficient)
DNA repair
±S9
+
S9 from Aroclor-induced rats.
Hellmer and
Bolcsfoldi, 1992
Rat hepatocytes
DNA damage
0.03, 0.3, or 3 mM
None
+
NA
Evaluated for DNA single strand
breaks; dose-related response;
negative at lowest dose and
positive at other doses; <30%
cytotoxicity.
Sina et al., 1983
Rat hepatocytes
DNA damage
0.03,0.1,0.3, 1, 3,
7, or 10 mM
None
-
NA
Negative for DNA double-strand
breaks.
Storer et al.,
1996
Serratia marcesans
HY/al3, HY/a21
Reverse
mutation
25, 50, or
100 mg/mL
None
+
NA
Positive dose-related results in
both strains, significant effect at
all doses inHY/al3, significant
effect at 50 and 100 mg/mL in
HY/a21; paper disc method.
Dean, 1972
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Table 9. Genotoxicity Studies of In Vitro Trimethyl Phosphate Exposure
Test System
Endpoint
Concentration
Activating
System
Results"
Comments
Reference
Without
Activation
With
Activation
Chinese hamster lung cells
Micronucleus
test
Not reported
None
+
NA
English data table; positive in
vitro; no other details available in
English.
Nietal., 1993
(published in
Chinese)
Human lymphocytes
Chromosome
aberrations
0,0.01,0.1, 1,2.5,
5, 10, 25, 50, 75,
or 100 mM
None
+
NA
Dose-related increase in the
percentage of anaphases with
aberrations and in the number of
metaphase breaks.
Soderman, 1972
a+ = positive; - = negative; = = equivocal; NA = not available; NR = not reported.
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As shown in Table 10, gavage treatment and i.p. injection with trimethyl phosphate
induced chromosomal aberrations in bone marrow cells of rats and mice (Sinha et al., 1983;
Moutschen and Degraeve, 1981; Anderson and Richardson, 1981; Sheu, 1979; Weber et al.,
1975; Farrow, 1975; Legator et al., 1973; Adler et al., 1971) and in spermatocytes of Chinese
hamsters and mice (Katoh and Matsuda, 1985; Degraeve et al., 1984; Moutschen and Degraeve,
1981; Machemer and Lorke, 1975). Katoh and Matsuda (1985) suggested that trimethyl
phosphate may act on the first cleavage metaphase of postmeiotic male germ cells to produce a
high rate of heritable translocations in the F1 progeny males. Trimethyl phosphate induced
micronuclei in vivo in mouse bone marrow cells (Bruce and Heddle, 1979; Farrow et al., 1976;
Weber et al., 1975, 1974). However, in a study published in Chinese, Ni et al. (1993) reported
no increased incidence of micronuclei in bone marrow cells of mice; however, few details were
available in the English abstract.
During a recessive lethal test with Drosophila melanogaster, trimethyl phosphate fed to
developing larvae resulted in reversible sterility in males (Dyer and Hanna, 1972). Trimethyl
phosphate was used as a positive control in two other recessive lethal studies in D. melanogaster
(Valencia, 1981; Hanna and Dyer, 1975). Other positive results in D. melanogaster included
dose-dependent induction of somatic mutations in a wing-spot test (Graf et al., 1989) and in an
eye mosaic assay (Vogel and Nivard, 1993).
Trimethyl phosphate was positive in dominant lethal assays in mice, resulting in
increases in early fetal deaths and preimplantation losses within the first 3 weeks after mating
(Moutschen and Degraeve, 1981; Degraeve et al., 1979; Newell et al., 1976; Lorke and
Machemer, 1975; Farrow, 1975; Epstein et al., 1972, 1970; Dean, 1972; Dean and Thorpe,
1972). An increased incidence in translocation heterozygotes was observed in male ICR/SIM
mice (Rushbrook et al., 1985), and a dose-related increase in the frequency of translocation
carrier mice was observed in male C3H mice (Tezuka et al., 1985; Sasaki et al., 1984) following
treatment with trimethyl phosphate during the postmeiotic stage of late spermatids.
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Table 10. Genotoxicity Studies of In Vivo Trimethyl Phosphate Exposure
Test System
Endpoint/
Indicator
Application
Concentration or Dose
Response"
Comment
Reference
Male CD rats
Chromosome
aberrations in
bone marrow
cells
i.p. injection
2000 mg/kg (single dose) or
at single doses of 0, 500, 750,
1000, 1250, 1500, or
1750 mg/kg, or at
500 mg/kg-day for 4 d
+
Trimethyl phosphate induced
chromatid aberrations including open
breaks and reunion figures following
single dose (>750 mg/kg) and repeated
exposure (500 mg/kg), a dose-response
was observed with maximal response at
48 hrs.
Adleretal. 1971
Male Osborne-
Mendel rats
Chromosome
aberrations in
bone marrow
cells
i.p. injection or
gavage
0 or 2000 mg/kg (single dose)
or at 0 or 100 mg/kg-day for
5 d
+
Trimethyl phosphate used as a positive
control by multiple laboratories;
induced aberrations at both
1000 mg/kg-day for 5 days and 2000
mg/kg (single dose) by both routes.
Legator et al., 1973
Male Osborne-
Mendel rats
Chromosome
aberrations in
bone marrow
cells
i.p. injection or
gavage
0 (solvent control) or single
unspecified dose level as a
single dose or as five daily
doses
+
Trimethyl phosphate used as a positive
control by multiple laboratories;
induced chromosomal aberrations by
both routes and with both single dose
and repeated exposure.
Sheu, 1979
Male Wistar rats
Chromosome
aberrations in
bone marrow
cells
i.p. injection
0 or 3000 mg/kg (single dose)
or 0 or 1500 mg/kg 5 times in
Id
+
Trimethyl phosphate induced
chromosome aberrations including
gaps, breaks, and fragments, and
induced significantly greater numbers
of abnormal cells following single and
multiple doses.
Anderson and
Richardson, 1981
Male and female
Sprague-Dawley
rats
Chromosome
aberrations in
bone marrow
cells
Gavage
0 or 2000 mg/kg (single dose
24 hrs prior to sacrifice)
+
Trimethyl phosphate used as a positive
control; induced chromatid gaps (males
only), breaks, and exchanges,
chromosome breaks in males, and
severely damaged cells in both sexes;
no effect on mitotic index.
Sinha et al., 1983
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Table 10. Genotoxicity Studies of In Vivo Trimethyl Phosphate Exposure
Test System
Endpoint/
Indicator
Application
Concentration or Dose
Response"
Comment
Reference
Male mice (strain
not specified)
Chromosome
aberrations in
bone marrow
cells
Not reported
1250, 1500, or 1750 mg/kg
+
Abstract; no mention of control;
maximum number of chromosome
aberrations observed at 48 hrs and
maximum changes (breaks, gaps, and
fragments) seen at highest dose; data
not shown.
Farrow, 1975
B6D2Fi/J mice (sex
not specified)
Chromosome
aberrations in
bone marrow
cells
i.p. injection
0, 500, 750, 1000, or
2000 mg/kg-day for 5 d
+
Dose-related increase in chromatid
breaks (>500 mg/kg-day); similar
results at 750 and 1000 mg/kg-day;
2000 mg/kg-day was lethal.
Weber etal., 1975
Male mice
(Q strain)
Chromosome
aberrations in
bone marrow
cells
i.p. injection
Not reported
+
Trimethyl phosphate used as a positive
control; induced chromosomal
aberrations including breaks,
exchanges, and gaps.
Moutschen and
Degraeve., 1981
Male Chinese
hamsters
Chromosome
aberrations in
spermatocytes
Gavage
0 or 500 mg/kg-day for 2 d, or
0 or 1000 mg/kg-day for 5 d
+
Significant increase in the number of
aberrant metaphases when gaps were
included, not significant (but still
higher) when gaps excluded; three
translocations were observed
(500 mg/kg-day); marked mitotic
inhibition (1000 mg/kg-day).
Machemer and
Lorke, 1975
Male mice
(Q strain)
Chromosome
aberrations in
spermatocytes
i.p. injection
Not reported
+
Trimethyl phosphate used as a positive
control; induced chromosomal
aberrations including breaks,
exchanges, and gaps.
Moutschen and
Degraeve, 1981
Male mice
(Q strain)
Chromosome
aberrations in
spermatocytes
i.p. injection
0 or 1000 mg/kg (single dose)
+
Primarily positive for breaks (24 total
across Recovery Days 10-15, 1
exchange on Recovery Days 12-13,
and 1 gap on Recovery Days 10-11
and 14-15).
Degraeve et al., 1984
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Table 10. Genotoxicity Studies of In Vivo Trimethyl Phosphate Exposure
Test System
Endpoint/
Indicator
Application
Concentration or Dose
Response"
Comment
Reference
Male mice (strain
not reported)
Chromosome
aberrations in
spermatocytes
i.p injection
3000 mg/kg
+
Abstract; no mention of control;
chromosome aberrations induced in the
postmeiotic stages, late spermatid stage
was the most sensitive; data not shown.
Katoh and Matsuda,
1985
B6D2F,/J mice (sex
not specified)
Micronuclei in
bone marrow
cells
i.p. injection
0, 500, 750, 1000, or
2000 mg/kg-day for 5 d
+
Dose-related increase in the frequency
of micronuclei (>500 mg/kg-day);
2000 mg/kg-day was lethal.
Weber etal., 1974,
1975
Mice (sex and
strain not reported)
Micronuclei in
bone marrow
cells
Not reported
0, 1250, 1500, or 1750 mg/kg
+
Abstract; time- and dose-related
increase in micronuclei; data not
shown.
Farrow et al., 1976
Female hybrid
(C57BL/6 x
C3H/He) mice
Micronuclei in
bone marrow
cells
i.p. injection
0-10,000 mg/kg
+
Doses were not explicitly reported, but
the range on the figure plotting
micronuclei was from 0 to
10,000 mg/kg; increases in micronuclei
observed at and above 6000 mg/kg.
Bruce and Heddle,
1979
Mouse (sex and
strain not specified)
Micronuclei in
bone marrow
cells
i.p. injection
Not reported
English data table; negative in vivo; no
other details available in English.
Ni et al., 1993
(published in
Chinese)
Male C3H mice
Heritable
translocation
assay
i.p. injection
0, 1000, or 1500 mg/kg
(single dose)
+
Significant dose-related increases in the
frequency of translocation carrier mice
at >1000 mg/kg.
Tezuka et al., 1985;
Sasaki et al., 1984
Male Swiss mice
Dominant lethal
i.p. injection or
gavage
0, 200, 700, or 1000 mg/kg
(single dose i.p.); 0, 500, 850,
1250, 1500, or 2000 mg/kg
(single dose i.p.); 0, 500, or
1000 mg/kg-day for 5 d
(gavage)
+
Early fetal deaths observed during first
2 wks of mating at >1000 mg/kg
following i.p. injection; early fetal
deaths at 700 mg/kg during Mating
Wk 8 following i.p. injection; early
fetal deaths during first 2 wks of
mating at >500 mg/kg following oral
exposure; reduction in total implants
during first 3 wks of mating at
>200 mg/kg following i.p. injection.
Epstein etal., 1972,
1970
32
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Table 10. Genotoxicity Studies of In Vivo Trimethyl Phosphate Exposure
Test System
Endpoint/
Indicator
Application
Concentration or Dose
Response"
Comment
Reference
Male Swiss CF1
mice
Dominant lethal
i.p. injection
0 or 1000 mg/kg
+
Increased number of early fetal deaths
at 2nd wk of mating.
Dean and Thorpe,
1972
Male NMRI mice
Dominant lethal
Oral
(unspecified)
0 or 1000 mg/kg (single dose)
+
Used as a reference material; no effect
onpreimplantationloss; marked
increase in postimplantation loss in the
2nd wk of mating.
Lorke and
Machemer, 1975
Male mice (strain
not specified)
Dominant lethal
i.p. injection or
gavage
1250 mg/kg (i.p.) or
500 mg/kg-day for 5 d
(gavage)
+
Abstract; no mention of controls;
significant lethality occurred
maximally in the 2nd wk of mating
(i.p.); dominant lethality in the 1st and
2nd wks of mating (gavage); data not
shown.
Farrow, 1975
Mouse (strain not
specified)
Dominant lethal
Oral
(unspecified)
Not reported, 5-d oral dosing
+
Abstract; no mention of controls;
dominant lethal effects for 2 wks after
5-d treatment.
Newell etal., 1976
Mouse (strain not
specified)
Dominant lethal
Not reported
1000 mg/kg
+
Abstract; no mention of control; high
mutagenicity particularly at postmeiotic
stages; data not shown.
Degraeve et al., 1979
Male mice
(Q strain)
Dominant lethal
i.p. injection
0 or 1000 mg/kg
+
Trimethyl phosphate used as a positive
control; significant increase in the
frequency of preimplantation and
postimplantation losses 2 wks after
injection.
Moutschen-Dahmen
etal., 1981
Male C3H mice
Dominant lethal
i.p. injection
0, 1000, 1250 or 1500 mg/kg
(single dose)
+
Significant decreases in the number of
implants and living embryos and
increases in early fetal deaths at
>1000 mg/kg; no significant effect on
the number of corpora lutea.
Tezuka et al., 1985;
Sasaki et al., 1984
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Table 10. Genotoxicity Studies of In Vivo Trimethyl Phosphate Exposure
Test System
Endpoint/
Indicator
Application
Concentration or Dose
Response"
Comment
Reference
Male
D. melanogaster
Recessive lethal
(second
chromosome)
Feeding
0,0.01,0.015 or 0.2 M
+
Dose-related increase in the percentage
of males with lethal mutations;
significant increase in lethal mutations
at >0.01 M.
Dyer and Hanna,
1972
Male
D. melanogaster
Recessive lethal
(second
chromosome)
Feeding
Not reported
+
Used as a positive control; induced a
high level of accumulated mutations
(83%) compared with negative control
(9%).
Hanna and Dyer,
1975
Male
D. melanogaster
Recessive lethal
Feeding
0, 100, 300, or 1000 mg/kg
+
Used as a positive control;
dose-response; negative at 100 and
300 ppm, positive at 1000 mg/kg.
Valencia, 1981
Male and female D.
melanogaster
Mwh-flr3 cross
(larvae)
Somatic mutation
(Wing-spot test)
Feeding
0, 5, 10, or 20 mM for 48 hrs
+
Dose-dependent induction of all types
of spots (small, large, and twin); results
inconclusive at 5 mM and positive at
>10 mM.
Grafetal., 1989
Male and female D.
melanogaster
Eye mosaic assay
Feeding
2 or 10 mM for 3 d or 10, 50,
100, or 200 mM on the
surface of food given for
48 hrs
+
Positive at both doses following the 3-d
treatment; positive at >50 mM
following surface treatment.
Vogel and Nivard,
1993
a+ = positive; - = negative
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DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RFD
VALUES FOR TRIMETHYL PHOSPHATE
Oral studies of trimethyl phosphate include a sub chronic-duration feeding study in rats
(Oishi et al., 1982), sub chronic-duration range-finding gavage studies in rats and mice (NCI,
1978), chronic-duration gavage studies in rats and mice (NCI, 1978), a chronic-duration drinking
water study in rats (Bomhard et al., 1997), a neurotoxicity study in dogs (Schaeppi et al., 1984),
a combined repeated dose/reproductive/developmental study in rats (MHW, 1994), and
numerous acute, short-term, and subchronic-duration studies evaluating effects on male
reproduction (Fukunishi et al., 2000, 1999, as cited in Kato et al., 2001; Takizawa et al., 1998;
Suzuki et al., 1996; Cho and Park, 1994; Toth et al., 1992; Hanna and Kerr, 1981;
Harbison et al., 1976; Carstensen, 1971; Jones and Jackson, 1969; Jackson and Jones, 1968).
These studies reported effects on body weight in rats and mice, neurotoxic effects in dogs, and
reproductive effects in rats, mice, and rabbits following oral treatment with trimethyl phosphate.
Male animals appeared particularly susceptible to the reproductive effects of trimethyl
phosphate. Oral studies considered adequate for derivation of a subchronic or chronic p-RfD are
summarized in Table 11.
SUBCHRONIC AND CHRONIC p-RfD
As shown in Table 11, the lowest LOAEL among the subchronic-duration and
reproductive toxicity studies is the LOAEL of 40 mg/kg-day in rats (MHW, 1994). This value is
from an unpublished, combined systemic/single-generation reproduction study in rats that
evaluated a sufficient number of animals and endpoints (MHW, 1994). The most prominent
effects at this dose level were decreased gestational body weights in females at GD 20, decreased
fraction of pregnant dams delivering litters with live pups, and markedly decreased number of
live pups per litter. Also, at this dose level, treated males evaluated at study termination showed
increases in absolute and relative kidney weights accompanied by histological changes
(regenerated tubules and eosinophilic bodies). This study is limited by the fact that it has not
been published and is available only in Japanese with an English abstract and data tables.
The lowest dose in the chronic and reproductive toxicity studies is 10 mg/kg-day for
depressed body-weight gain in male rats given drinking water with trimethyl phosphate for
30 months (Bomhard et al., 1997). Body-weight was also depressed in female rats at a higher
dose in this study; this body-weight change (10%) at this dose is considered a biologically
relevant critical endpoint; thus, it is considered the POD for derivation of oral p-RfDs for both
subchronic- and chronic-durations. BMD modeling cannot be conducted because the
body-weight data were not provided in the principal study (the data were shown graphically).
The NOAEL of 10 mg/kg-day is protective of the reproductive effects observed at
>40 mg/kg-day and was chosen as the POD for deriving the chronic p-RfD.
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Table 11. Summary of Noncancer Oral Dose-response Information Considered
Adequate for Toxicity Value Derivation
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Duration-
adjusted"
NOAEL
(mg/kg-day)
Duration-
adjusted"
LOAEL
(mg/kg-day)
Responses at the
LOAEL
Comments
Reference
Subchronic oral animal studies
Rats
(6 males/group)
Diet
0, 461 mg/kg-day,
7 d/wk for 9 wks
NA
461
NA
461
Significant reduction
in body-weight gain,
significant changes in
hematology and
serum chemistry,
elevated absolute and
relative kidney
weights and absolute
testes weight
No treatment-related
histological changes were
observed.
Oishi et al., 1982
Chronic oral animal studies
Rats
(50/sex/group)
Gavage
0, 50, or
100 mg/kg-day,
3 d/wk for 104 wks
50
100
21
43
Significant reduction
in body weights
NCI, 1978
Rats
(60/sex/group)
Drinking water
0, 1, 10,
100 mg/kg-day for
30 mos
(100 mg/kg-day
dose reduced to
50 mg/kg-day in
Wk 54)
10
100
10
100
Significant reduction
in body weight of
males
Bomhard et al.,
1997
Mice
(50/sex/group)
Gavage
0, 250, or
500 mg/kg-day,
3 d/wk for 103 wks
250
500
107
214
Significant reduction
in body weight of
females
NCI, 1978
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Table 11. Summary of Noncancer Oral Dose-response Information Considered
Adequate for Toxicity Value Derivation
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Duration-
adjusted"
NOAEL
(mg/kg-day)
Duration-
adjusted"
LOAEL
(mg/kg-day)
Responses at the
LOAEL
Comments
Reference
Oral studies of male reproductive effects
Rats
(13/sex)
Gavage
0, 40, 100, or
250 mg/kg-day,
2 wks prior to
mating through
mating for a total
of 42 d (males);
2 wks prior to
mating through
gestation to
Postpartum Day 3
(females)
NA
(parental)
NA
(repro)
40
(parental)
40 (repro)
NA
(parental)
NA (repro)
40
(parental)
40 (repro)
Parental: evidence of
renal toxicity in males
characterized by
significant changes in
clinical chemistry and
kidney weights
accompanied by
histological changes
Reproduction:
decreased gestational
body weights in
females at GD 20,
decreased fraction of
pregnant dams
delivering litters with
live pups; markedly
decreased number of
live pups per litter
Unpublished Japanese
combined repeated
dose/reproductive/
developmental toxicity.
MHW, 1994
Rats
Male
(number not given)
Gavage
0 or
250 mg/kg-day,
5 d/wk for 30 d or
6 d/wk for 60 d
NA
250
NA
179
Impaired mating
ability, abnormal
sperm, and impaired
spermatogenesis
Hanna and Kerr,
1981
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Table 11. Summary of Noncancer Oral Dose-response Information Considered
Adequate for Toxicity Value Derivation
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Duration-
adjusted"
NOAEL
(mg/kg-day)
Duration-
adjusted"
LOAEL
(mg/kg-day)
Responses at the
LOAEL
Comments
Reference
Rats
Male
(number not given)
Gavage
0 or
100 mg/kg-day,
5 days/week, for
4 wks
NA
100
NA
71
Reduced sperm
motility
Takizawa et al.,
1998
NA = not applicable.
aAdjusted for continuous exposure as follows: NOAELadj = NOAEL x exposure days/7 days.
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A chronic p-RfD was derived for trimethyl phosphate by dividing the NOAEL of
10 mg/kg-day by a UF of 1000, as shown below. The value derived for the chronic p-RfD is also
adopted for the subchronic p-RfD.
Chronic and subchronic p-RfD = NOAEL UF
= 10 mg/kg-day ^ 1000
= 0.01 or 1 x 10"2 mg/kg-day
The composite UF of 1000 is composed of the following UFs:
• UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating a susceptible human response are
insufficient.
• UFa: A factor of 10 is applied for animal-to-human extrapolation because data for
evaluating relative interspecies sensitivity are insufficient.
• UFd: A factor of 10 is applied for database inadequacies because no studies
evaluating developmental or multi-generational reproductive toxicity are available.
The available reproduction study is not multigenerational.
• UFl: A factor of 1 is applied for extrapolation from a LOAEL to a NOAEL because a
NOAEL was used for the POD.
Confidence in the principal study (Bomhard et al., 1997) is medium. The chronic study
evaluated multiple dose levels using an adequate number of rats (60/sex/dose) and evaluated
several endpoints for assessing chronic toxicity. However, only limited data was reported.
Confidence in the database is low. In addition to the principal chronic rat study, NCI (1978)
evaluated the chronic effects of trimethyl phosphate in rats and mice and also observed effects on
body-weight gain. The chronic database also consists of a single-generation reproduction study
in rats and two studies specifically designed for evaluating effects on the male reproductive
system. There are no developmental or multi-generational reproductive toxicity studies
available. Available data indicate the occurrence of neurotoxicity; but, no comprehensive
neurotoxicity study is available, and the neurotoxicity study in dogs is limited by the lack of a
concurrent control group. Overall confidence in the chronic and subchronic p-RfD is low.
FEASIBILITY OF DERIVING PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RFC VALUES FOR TRIMETHYL PHOSPHATE
No data are available on the effects of trimethyl phosphate in humans or animals exposed
via inhalation. Derivation of provisional RfC values for trimethyl phosphate is precluded by the
absence of data.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR TRIMETHYL PHOSPHATE
WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), trimethyl
phosphate is "Likely to be Carcinogenic to Humans." This descriptor is appropriate when an
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agent "has tested positive in animal experiments in more than one species, sex, strain, site, or
exposure route, with or without evidence of carcinogenicity in humans" (U.S. EPA, 2005). No
information was located on the carcinogenicity of trimethyl phosphate in humans. NCI (1978)
observed a significant positive dose-related increase in the incidence of subcutaneous fibromas in
male rats (see Table 2) given trimethyl phosphate by gavage at 50- and 100-mg/kg-day
(duration-adjusted doses of 21 and 43 mg/kg-day) for 104 weeks. No historical control data
were provided for this tumor in the study, but a study published a year later indicated that the
background rate of this tumor during that time period was only 2.6% (Goodman et al., 1979).
This compares with incidences of 0, 4, and 18% at the control, low-, and high-doses in the NCI
(1978) study. In a more recent drinking water study, no evidence of carcinogenicity was
observed among rats dosed with trimethyl phosphate up to 100 mg/kg-day (Bomhard et al.,
1997). Significant mortality occurred at the highest dose tested in this study (100 mg/kg-day),
but survival-adjusted statistical analysis performed by the study authors did not indicate evidence
of carcinogenicity in this group. NCI (1978) also observed a significant increase in the incidence
of uterine endometrial adenocarcinomas among female mice administered trimethyl phosphate
by gavage at 500 mg/kg-day (duration-adjusted dose of 214 mg/kg-day) for 103 weeks (see
Table 3). NCI (1978) reported that this tumor had never been observed in female mice based on
historical controls. Genotoxicity data on trimethyl phosphate generally resulted in mixed or
equivocal findings in bacterial mutagenicity assays and consistently positive findings in
genotoxicity tests among animals exposed in vivo. In in vitro tests, trimethyl phosphate was
mutagenic in bacterial mutation assays with S. typhimurium and E. coli (Watanabe et al., 1996;
Li et al., 1993; Zeiger et al., 1992, 1982; De Flora et al., 1990, 1984; De Flora, 1981; Bruce and
Heddle, 1979; Anderson and Styles, 1978; Farrow et al., 1976; Hanna and Dyer, 1975; Dean,
1972), impaired DNA repair in E. coli (DeFlora et al., 1990, 1984; Hellmer and Bolcsfoldi,
1992), and induced DNA damage in rat hepatocytes (Sina et al., 1983). A dose-related increase
in the frequency of anaphases with aberrations and metaphase breaks was observed in human
lymphocytes following trimethyl phosphate treatment at concentrations ranging from 0.01 to
100 mM. In vivo, trimethyl phosphate induced chromosome aberrations and micronuclei in rat
and mouse bone marrow cells and aberrations in mouse sperm cells. Trimethyl phosphate was
found to produce dominant lethal effects in several strains of mice (Moutschen and Degraeve,
1981; Degraeve et al., 1979; Newell et al., 1976; Lorke and Machemer, 1975; Farrow, 1975;
Epstein et al., 1972, 1970).
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.
Apart from genotoxicity data, there is no other information on the potential mode(s) of
action by which trimethyl phosphate induces subcutaneous fibromas or uterine adenocarcinomas.
There is strong evidence that trimethyl phosphate induces micronuclei and chromosomal
aberrations in laboratory animals tested in vivo; it is often used as a positive control substance in
such assays. In vitro tests for mutagenicity have given mixed results.
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QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
Oral Exposure
Data for trimethyl phosphate are sufficient to perform dose-response modeling. The
incidences of subcutaneous fibromas in male F344 rats and endometrial adenocarcinomas in
female B6C3F1 mice administered trimethyl phosphate by gavage for up to 104 weeks (NCI,
1978) were modeled. In order to determine a POD for OSF derivation, animal doses in the NCI
(1978) study were first adjusted (see Table 12) for continuous exposure as follows:
DoseADj = dose (mg/kg-day) x (days per week 7)
= dose (mg/kg-day) x (3 -h 7)
The duration-adjusted values were then converted to human equivalent doses (HEDs) by
adjusting for differences in body weight between humans and rats. In accordance with EPA
(2005) Guidelines for Carcinogen Risk Assessment, a factor of bw3 4 was used for cross-species
scaling. Using this scaling factor, the dose (mg) in humans is obtained by multiplying the animal
dose (mg) by the ratio of human:animal body weight raised to the 3/4 power. For doses
expressed per unit of body weight (mg/kg or mg/kg-day), the relationship is reciprocal, and the
human dose (mg/kg) is obtained by multiplying the animal dose (mg/kg) by the ratio of
animal :human body weight raised to the 1/4 power. Because NCI (1978) did not report any
information on the body weights of the animals used in the principal study, default body-weight
values (0.380 kg for male F344 rats exposed chronically and 0.0353 kg for female B6C3F1 mice
exposed chronically, as reported by U.S. EPA, 1988) were used to calculate the animal:human
body-weight ratios. The equation used to calculate the HED values (see Table 12) is shown
below, and the Table 12 presents HED.
Dose[HED] = Dose^Dj] x (animal bw:human bw)14
where:
Dose[ADj] = average daily animal dose adjusted for continuous exposure
(mg/kg-day)
animal bw = average rat or mouse body weight (kg), based on default values
(U.S. EPA, 1988)
human bw = reference human body weight, 70 kg (U.S. EPA, 1988)
Appendix A describes the modeling approach and results. Table 13 shows the
BMD io[hed] and BMDLio[hed] values predicted by the multistage cancer model for these tumor
data. The results of modeling the two tumor types are very similar. A BMDL of 5.74 mg/kg-day
associated with adenocarcinoma in female mice is considered the POD for the cancer
assessment. The endometrial adenocarcinoma in mice is most likely associated with the
carcinogenic potential of trimethyl phosphate exposure. As the mode of action of trimethyl
phosphate-induced tumorigenicity is not clearly defined, a linear extrapolation from the POD to
the origin was applied.
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Table 12. Dose-response Data for the Incidence of Subcutaneous Fibromas in
Male F344 Rats and Endometrial Adenocarcinomas in Female B6C3F1 Mice Administered
Trimethyl Phosphate by Gavage for up to 104 Weeks
Male F344 rats
Female B6C3F1 mice
Dose
(mg/kg-day)
Adjusted
Dose3
(mg/kg-day)
HEDb
(mg/kg-day)
Incidence
Dose
(mg/kg-day)
Adjusted
Dose3
(mg/kg-day)
HEDb
(mg/kg-day)
Incidence
0
0
0
0/20
0
0
0
0/16
50
21
6
2/50
250
107
16
7/40
100
43
12
9/49
500
214
32
13/37
aAdjusted for continuous (7 d/wk) exposure.
bHED = animal dose x (animal bw/human bw)°25, where animal body weights = 0.380 kg (male rats) and 0.0353 kg
(female mice) (default values from U.S. EPA, 1988), and human body weight = 70 kg.
Source: NCI (1978).
Table 13. Summary of Human Equivalent BMDs and BMDLs Based on
Incidence of Subcutaneous Fibromas in Male F344 Rats and Uterine
Adenocarcinomas in Female B6C3F1 Mice Administered Trimethyl Phosphate by Gavage
for up to 104 Weeks
BMDio[hed]
(mg/kg-day)
BMDLio[hed]
(mg/kg-day)
Male rat subcutaneous fibromas
8.84
5.81
Female mouse uterine adenocarcinoma
8.13
5.74
The p-OSF for trimethyl phosphate was calculated as the ratio of the benchmark
response (BMR) to the POD (BMDLi0[hed]) as shown below:
p-OSF = BMR BMDLio[hed]
= 0.1 ^ 5.74 mg/kg-day
= 0.017 or 2 x 10"2 (mg/kg-day)"1
The OSF for trimethyl phosphate should not be used with exposures exceeding the POD
(BMDLio[hed] = 5.74 mg/kg-day) because, at exposures above this level, the fitted dose-response
model better characterizes what is known about the carcinogenicity of trimethyl phosphate.
Table 14 shows the doses associated with specific levels of cancer risk based on the p-OSF
estimated herein.
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Table 14. Doses of Trimethyl Phosphate Associated with
Specific Levels of Cancer Risk
Risk Level
Dose (mg/kg-day)
lO"4
0.006
lO"5
0.0006
lO"6
0.00006
Inhalation Exposure
Lack of suitable data precluded derivation of quantitative estimates of cancer risk
following inhalation exposure to trimethyl phosphate.
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APPENDIX A. DETAILS OF BENCHMARK DOSE MODELING
FOR ORAL SLOPE FACTOR
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
multistage cancer model in the EPA Benchmark Dose Software (BMDS) is fit to the incidence
data using the extra risk option. The multistage cancer model is run for all polynomial degrees
up to n - 1 (where n is the number of dose groups including control). Adequate model fit is
judged by three criteria: goodness-of-fit p-walue (p > 0.1), visual inspection of the dose-response
curve, and scaled residual at the data point (except the control) closest to the predefined
benchmark response. Among all of the models providing adequate fit to the data, the lowest
BMDL is selected as the point of departure when the difference between the benchmark dose
lower bound 95% confidence intervals (BMDLs) estimated from these models are more than
3-fold; otherwise, the BMDL from the model with the lowest Akaike's information criterion
(AIC) is chosen. In accordance with EPA (1999 guidance, BMDs and BMDLs associated with
an extra risk of 10% are calculated.
MODEL-FITTING RESULTS FOR SUBCUTANEOUS FIBROMAS IN MALE F344
RATS (NCI, 1978)
Table 12 shows the incidence data for subcutaneous fibromas in male F344 rats
administered trimethyl phosphate via gavage 3 days/week, for 104 weeks (NCI, 1978).
Modeling was performed according to the procedure outlined above using BMDS version 2.1
with default parameter restrictions based on the duration-adjusted human equivalent dose (HED)
shown in Table 12. Table A-l shows model predictions. The multistage cancer model provided
adequate fit (goodness-of-fit p-w alue > 0.1), and the 2-degree polynomial model had the lowest
AIC, yielding a BMDio[hed] value of 8.57 mg/kg-day with an associated 95% lower confidence
limit (BMDLio[hed]) of 5.63 mg/kg-day. The fit of the 2-degree multistage cancer model to the
incidence data for male rats is shown in Figure A-l.
Table A-l. Model Predictions for Subcutaneous Fibromas in Male F344 Rats
Model
Degrees
of
Freedom
x2
2
X
Goodness-of-Fit
/>-Valuc
AIC
BMDiohed
(mg/kg-day)
BMDL10hed
(mg/kg-day)
Multistage Cancer Model
(2-degree polynomial)13
2
0.08
0.961
65.61
8.57
5.63
Multistage Cancer Model
(1-degree polynomial)13
2
1.46
0.482
67.15
7.67
4.85
aValues <0.10 fail to meet conventional goodness-of-fit criteria.
bBetas restricted to >0.
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Multistage Cancer Model with 0.95 Confidence Level
Dose
01:55 09/15 2009
BMDs and BMDLs indicated are associated with an extra risk of 10% and are in
units of mg/kg-day as HED.
Figure A-l. Fit of 2-Degree Multistage Cancer Model to Data on Incidence
of Subcutaneous Fibromas in Male F344 Rats
Source: NCI (1978).
MODEL-FITTING RESULTS FOR ENDOMETRIAL ADENOCARCINOMAS IN
FEMALE B6C3F1 MICE (NCI, 1978)
Table 12 shows the incidence data for endometrial adenocarcinomas in female B6C3F1
mice administered trimethyl phosphate via gavage 3 days/week, for 103 weeks (NCI, 1978).
Modeling was performed according to the procedure outlined above using BMDS version 2.1
with default parameter restrictions based on the duration-adjusted HED shown in Table 12.
Table A-2 shows model predictions. The multistage cancer model provided adequate fit
(goodness-of-fit /;-value > 0.1), and the 1-degree polynomial model had the lowest AIC, yielding
a BMD 10[hed] value of 8.13 mg/kg-day with an associated 95% lower confidence limit
(BMDLio[hed]) of 5.74 mg/kg-day. The fit of the 1-degree multistage cancer model to the
incidence data for female mice is shown in Figure A-2.
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Table A-2. Model Predictions for Uterine Adenocarcinomas
in Female B6C3F1 Mice
Model
Degrees
of
Freedom
2
X
x2
Goodness-of-Fit
/>-Valuc
AIC
BMDiohed
(mg/kg-day)
BMDLiohed
(mg/kg-day)
Multistage Cancer Model
(2-degree polynomial)13
1
0
1
89.07
9.25
5.76
Multistage Cancer Model
(1-degree polynomial)13
2
0.06
0.969
87.13
8.13
5.74
aValues < 0.10 fail to meet conventional goodness-of-fit criteria.
bBetas restricted to > 0.
Multistage Cancer Model with 0.95 Confidence Level
Dose
12:52 11/10 2009
BMDs and BMDLs indicated are associated with an extra risk of 10% and are in
units of mg/kg-day as HED.
Figure A-2. Fit of 1-Degree Multistage Cancer Model to Data on Incidence
of Uterine Adenocarcinomas in Female B6C3F1 Mice.
Source: NCI (1978).
52 Trimethyl Phosphate
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