U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
SCREENING-LEVEL HAZARD CHARACTERIZATION
Acetyl Tributyl Citrate
(CASRN 77-90-7)
The High Production Volume (HPV) Challenge Program1 was conceived as a voluntary initiative
aimed at developing and making publicly available screening-level health and environmental
effects information on chemicals manufactured in or imported into the United States in quantities
greater than one million pounds per year. In the Challenge Program, producers and importers of
HPV chemicals voluntarily sponsored chemicals; sponsorship entailed the identification and
initial assessment of the adequacy of existing toxicity data/information, conducting new testing if
adequate data did not exist, and making both new and existing data and information available to
the public. Each complete data submission contains data on 18 internationally agreed to "SIDS"
(Screening Information Data Setl1'2) endpoints that are screening-level indicators of potential
hazards (toxicity) for humans or the environment.
The Environmental Protection Agency's Office of Pollution Prevention and Toxics (OPPT) is
evaluating the data submitted in the HPV Challenge Program on approximately 1400 sponsored
chemicals by developing hazard characterizations (HCs). These HCs consist of an evaluation of
the quality and completeness of the data set provided in the Challenge Program submissions.
They are not intended to be definitive statements regarding the possibility of unreasonable risk of
injury to health or the environment.
The evaluation is performed according to established EPA guidance2'3 and is based primarily on
hazard data provided by sponsors; however, in preparing the hazard characterization, EPA
considered its own comments and public comments on the original submission as well as the
sponsor's responses to comments and revisions made to the submission. In order to determine
whether any new hazard information was developed since the time of the HPV submission, a
search of the following databases was made from one year prior to the date of the HPV
Challenge submission to the present: (ChemID to locate available data sources including
Medline/PubMed, Toxline, HSDB, IRIS, NTP, AT SDR, IARC, EXTOXNET, EPA SRS, etc.),
STN/CAS online databases (Registry file for locators, ChemAbs for toxicology data, RTECS,
Merck, etc.), Science Direct and ECHA4. OPPT's focus on these specific sources is based on
their being of high quality, highly relevant to hazard characterization, and publicly available.
OPPT does not develop HCs for those HPV chemicals which have already been assessed
internationally through the HPV program of the Organization for Economic Cooperation and
Development (OECD) and for which Screening Initial Data Set (SIDS) Initial Assessment
Reports (SIAR) and SIDS Initial Assessment Profiles (SIAP) are available. These documents are
presented in an international forum that involves review and endorsement by governmental
authorities around the world. OPPT is an active participant in these meetings and accepts these
documents as reliable screening-level hazard assessments.
1	U.S. EPA. High Production Volume (HPV) Challenge Program; http://www.epa.gov/chemrtk/index.htm.
2	U.S. EPA. HPV Challenge Program - Information Sources; http://www.epa.gov/chemrtk/pubs/general/guidocs.htm.
3	U.S. EPA. Risk Assessment Guidelines; http://cfpub.epa.gov/ncea/raf/rafguid.cfm.
4	European Chemicals Agency, http://echa.europa.eu.

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Hazard Characterization Document
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These hazard characterizations are technical documents intended to inform subsequent decisions
and actions by OPPT. Accordingly, the documents are not written with the goal of informing the
general public. However, they do provide a vehicle for public access to a concise assessment of
the raw technical data on HPV chemicals and provide information previously not readily
available to the public.
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Chemical Abstract
Service Registry Number
	(CASRN)	
Chemical Abstract Index
Name
77-90-7
1,2,3-Propanetricarboxylic acid, 2-(acetyloxy)-, 1,2,3-tributyl
ester
Structural Formula
O	^"3
SMILES: 0=C(0C(C(=0)0CCCC)(CC(=Q)0CCCC)CC(=0)0CCCC)C
Summary
Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water solubility.
It is expected to have low mobility in soil. Volatilization is expected to be high, but
adsorption to soil and sediment may attenuate the rate of volatilization. The rate of hydrolysis
is negligible. The rate of atmospheric photooxidation is moderate. Acetyl tributyl citrate is
readily biodegradable. Acetyl tributyl citrate is expected to have low persistence (PI) and low
bioaccumulation potential (Bl).
Acute oral toxicity of acetyl tributyl citrate in cats and rats is low. In a 90-day repeated-dose
oral dietary study in rats, decreased body weight and organ weight changes were observed at
1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a combined repeated-
dose/reproductive/developmental toxicity study in rats, organ weight and histopathological
changes were observed in adults at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In
a 2-generation reproductive toxicity study in rats, reduced body weight was observed in
Flmales at 300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the
same study, no other treatment related effects were observed; the NOAEL for reproductive
toxicity is 1000 mg/kg-bw/day, the highest dose tested. In the combined repeated-
dose/reproductive/developmental toxicity study in rats previously described, histopathological
changes were observed in the liver of adult males at 300 mg/kg-bw/day; the NOAEL for
systemic toxicity is 100 mg/kg-bw/day. In the same study, decreased litter size and decreased
number of implantations were observed at 1000 mg/kg-bw/day; the NOAEL for reproductive
and developmental toxicity is 300 mg/kg-bw/day. The NOAEL for maternal toxicity is 1000
mg/kg-bw/day (highest dose tested). Acetyl tributyl citrate did not induce gene mutations in
bacteria or mammalian cells in vitro and did not induce chromosomal aberrations in
mammalian cells in vitro.
For acetyl tributyl citrate, the 96-h LCso values for fish range from 38 to 60 mg/L, the 48-h
ECso value for aquatic invertebrates is 7.8 mg/L and the 72-h EC50 values for aquatic plants
are 11.5 mg/L for biomass and 74.4mg/L for growth rate, respectively. The chronic 21-d EC50
values for aquatic invertebrates are > 1.11 mg/L (survival) and >1.11 mg/L (reproduction).
No data gaps were identified under the HPV Challenge Program.
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The sponsor, Morflex, Inc., submitted a Test Plan and Robust Summaries to EPA for acetyl
tributyl citrate (CASRN 77-90-7; CA Index name: 1,2,3-propanetricarboxylic acid, 2-
(acetyloxy)-tributyl ester) on January 12, 2004. EPA posted the submission on the ChemRTK
HPV Challenge website on March 4, 2004
(http://www.epa.gov/oppt/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm). EPA comments on
the original submission were posted to the website on March 8, 2005. Public comments were
also received and posted to the website.
1. Chemical Identity
1.1	Identification and Purity
Acetyl tributyl citrate is a citric acid, trialkyl ester, acetate.
Test substance purity when noted in the Robust Summaries was > 98%.
1.2	Physical-Chemical Properties
Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water solubility.
The physical-chemical properties of acetyl tributyl citrate are summarized in Table 1.
Table 1. Physical-Chemical Properties of CASRN 77-90-71
Property
Value
CASRN
77-90-7
Molecular Weight
402.5
Physical State
Liquid
Melting Point
-59°C (measured pour point)
Boiling Point
326°C (measured)
Vapor Pressure
0.052 mm Hg at 20°C (measured)
Dissociation Constant
(pKa)
Not applicable
Henry's Law Constant
5.5><10"3 atm-m3/mole(estimated)2
Water Solubility
<100 mg/L at 25°C (measured);
5 mg/L at 25°C (measured)3
Log Kow
4.92 at 22°C (measured)
1 The Morflex Inc. 2003. Test Plan and Robust Summary for Acetyltributyl citrate. Available online at
http://www.epa.gov/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm as of April 30, 2012.
2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental
Protection Agency, Washington, DC, USA. Available online at
http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of April 30, 2012.
3SRC. 2012. The Physical Properties Database (PHYSPROP). Syracuse, NY: Syracuse Research Corporation.
Available online at http://www.svrres.com/esc/phvsprop.htm as of April 30, 2012.
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2. General Information on Exposure
2.1	Production Volume and Use Pattern
Acetyl tributyl citrate had an aggregated production and/or import volume in the United States
between 1 and 10 million pounds during calendar year 2005.
Non-confidential information in the IUR indicated that the industrial processing and uses of the
chemical include other basic organic chemical manufacturing as corrosion inhibitors and anti-
scaling agents and functional fluids; resin and synthetic rubber manufacturing, and other
chemical and allied products merchant wholesalers as functional fluids; adhesive manufacturing,
other basic inorganic chemical manufacturing, other plastic product manufacturing, paints and
coating manufacturing, pharmaceutical and medicine manufacturing, plastic packaging materials
and unlaminated film and sheet manufacturing, and paint ink manufacturing as "other." Non-
confidential commercial and consumer uses of this chemical include metal products, rubber and
plastic products and "other."
2.2	Environmental Exposure and Fate
Acetyl tributyl citrate is expected to have low mobility in soil. It achieved 82% of its theoretical
biochemical oxygen demand (BOD) using an activated sludge and the modified MITI (OECD
301C) test after 28 days and is considered readily biodegradable. It was shown to biodegrade
extensively in several other biodegradation studies and simulation tests, suggesting that it is not
persistent in the environment. Volatilization is expected to be high, but adsorption to soil and
sediment may attenuate the rate of volatilization. The rate of hydrolysis is negligible. The rate
of atmospheric photooxidation is moderate. Acetyl tributyl citrate is expected to have low
persistence (PI) and low bioaccumulation potential (Bl).
The environmental fate properties of acetyl tributyl citrate are provided in Table 2.
Conclusion: Acetyl tributyl citrate is a liquid with moderate vapor pressure and moderate water
solubility. It is expected to have low mobility in soil. Volatilization is expected to be high, but
adsorption to soil and sediment may attenuate the rate of volatilization. The rate of hydrolysis is
negligible. The rate of atmospheric photooxidation is moderate. Acetyl tributyl citrate is readily
biodegradable. CASRN 77-90-7 is expected to have low persistence (PI) and low
bioaccumulation potential (Bl).
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Table 2. Environmental Fate Properties of CASRN 77-90-71
Property
Value
CASRN
77-90-7
Photodegradation Half-life
8.9 hours (estimated)2
Hydrolysis Half-life
139 days at pH 8 (estimated)2;
3.8 years at pH 7 (estimated)2
Biodegradation
26% after 21 days;
>90% after 5 hours;
72.9%) after 42 days;
37%o after 45 days in compost soil;
82%o after 28 days (readily biodegradable)3
Bioaccumulation Factor
BAF = 12.5 (estimated)2
Log Koc
4.9 (estimated)2
Fugacity
(Level III Model)2
Air (%)
Water (%)
Soil (%)
Sediment (%)
2.8
14.8
61.6
20.9
Persistence4
PI (low)
Bi oaccumul ati on4
Bl (low)
'The Moiflcx Inc. 2003. Test Plan and Robust Summary for Acetyltributyl citrate. Available online at
http://www.epa.gov/chemrtk/pubs/summaries/acetlcit/cl5025tc.htm as of April 30, 2012.
2U.S. EPA. 2012. Estimation Programs Interface Suite™ for Microsoft® Windows, v4.10. U.S. Environmental
Protection Agency, Washington, DC, USA. Available online at
http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm as of April 30, 2012.
3National Institute of Technology and Evaluation. 2002. Biodegradation and Bioaccumulation of the Existing
Chemical Substances under the Chemical Substances Control Law. Available online at
http://www.safe.nite.go.ip/english/kizon/KIZON start hazkizon.html as of April 30, 2012.
4Federal Register. 1999. Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances.
Federal Register 64, Number 213 (November 4, 1999) pp. 60194-60204.
3. Human Health Hazard
A summary of health effects data submitted for SIDS endpoints is provided in Table 3.
Acute Oral Toxicity
(1) Five rats (strain and gender not reported) were administered acetyl tributyl citrate via gavage
(vehicle not specified) at 10 - 30 mL/kg (approximately 10,500 - 31,500 mg/kg) and observed
for 21 days. No mortalities were reported.
LDso > ~ 31,500 mg/kg
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(2) Four fasted cats (strain and gender not reported) were administered acetyl tributyl citrate by
gavage (vehicle not specified) at 30 - 50 mL/kg (approximately 31,500 - 52,500 mg/kg).
Additionally, two cats received 50 mL/kg (approximately 52,500 mg/kg) and two cats were
administered a control substance (control not identified). The animals were observed for periods
of up to 2 months. No mortalities were reported.
LDso > ~ 52,460 mg/kg
Repeated-Dose Toxicity
(1)	Sprague-Dawley rats (20/sex/group) were administered acetyl tributyl citrate (purity >98%)
via the diet at 0, 100, 300 or 1000 mg/kg-bw/day for 13 weeks. Dose levels were based on the
results of a 14-day range-finding study in which increased cytoplasmic eosinophilia was
observed in periportal hepatocytes of males and females exposed to 5000 mg/kg-bw/day. Actual
doses received in the 13-week study were 0, 101, 302 and 996 mg/kg-bw/day for males and 0,
100, 296 and 999 mg/kg-bw/day for females. Blood was collected on day 85 for comprehensive
hematology and clinical chemistry evaluations (including activities of alkaline phosphatase,
aspartate aminotransferase, alanine aminotransferase and gamma glutamyl transferase). Urine
samples were collected on days 86 - 87 for comprehensive urinalysis. All rats were killed in
week 14 and examined macroscopically. A comprehensive set of tissues (including male and
female reproductive tissues) were prepared from all rats. All tissues from control and 1000
mg/kg-bw/day rats were microscopically examined; kidney, liver, lung and gross lesions were
examined for all 100 and 300 mg/kg-bw/day rats. The following organs were weighed for all
rats: adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroid with
parathyroids. All rats survived to study termination and no clinical signs were reported. Mean
body weights were slightly reduced in males and females at 1000 mg/kg-bw/day and in females
at 300 mg/kg-bw/day (statistical significance not reported). Semiquantitative observations
revealed a decreased urinary pH in males at 1000 mg/kg-bw/day and crystals in the urine of
males at > 300 mg/kg-bw/day and in females at 1000 mg/kg-bw/day. Males exhibited increased
serum alkaline phosphatase activity at 1000 mg/kg-bw/day and females exhibited decreased
fasting glucose at > 300 mg/kg-bw/day and decreased alanine aminotransferase activity and
bilirubin concentration at 1000 mg/kg-bw/day. Males exhibited increased relative liver weights
at > 300 mg/kg-bw/day and increased relative kidney weights at 1000 mg/kg-bw/day. Females
exhibited increased relative liver weights at 1000 mg/kg-bw/day. No other treatment-related
effects were reported.
LOAEL = 1000 mg/kg-bw/day (based on decreased body weight and organ weight changes)
NOAEL = 300 mg/kg-bw/day
(2)	In a combined repeated-dose/reproductive/developmental toxicity study, Han Wistar rats (F0;
25/sex/group) were administered acetyl tributyl citrate (purity 99.9%) via the diet at 0, 100, 300
or 1000 mg/kg-bw/day for 4 weeks prior to mating, through mating (F0 males) and through
gestation and lactation (F0 females). Actual doses received in the study were 0, 103, 306, and
1013 mg/kg-bw/day for males and 0, 102, 306, and 1024 mg/kg-bw/day for females. The F1
offspring received indirect exposure in utero and from birth until the start of a 13-week toxicity
study in which selected F1 offspring (20/sex/group) received dietary doses of 0, 100, 300 or
1000 mg/kg-bw/day for 13 weeks. Additional rats (10/sex/group) were assigned to the high-dose
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group and the control group for inclusion in a 4-week recovery period following the 13-week
treatment period. Endpoints included organ and body weights, reproductive performance
variables and gross and microscopic histopathology of tissues, which were not specified in the
robust summary. Treatment at 1000 mg/kg-bw/day for 13 weeks resulted in a slight decrease in
body weight gain, increased liver weights and hepatic hypertrophy and hepatic peroxisome
proliferation in both F1 sexes. Weak peroxisome proliferation was measured in males at 300
mg/kg-bw/day. Slight variations in urine composition and plasma electrolyte composition
(indicative of an effect on renal function) were seen at the two highest doses, but the effects were
within historical control ranges, were reversible and no histopathological changes in the kidneys
were seen. No other exposure-related systemic effects (i.e., non-reproductive effects) were
mentioned in the robust summary.
LOAEL = 1000 mg/kg-bw/day (based on organ weight changes and histopathology in F1
animals)
NOAEL = 300 mg/kg-bw/day
(3)	Rats (4/concentration, mixed male and female; strain not reported) were administered acetyl
tributyl citrate via the diet at 5 or 10% (approximately 2500 and 5000 mg/kg-bw/day) for 8
weeks. A control group received basic diet only. Blood was collected for hematology after 4
and 8 weeks of exposure. After exposure, all rats were necropsied and the following tissues were
examined microscopically: heart, lungs, gastrointestinal tract, liver, pancreas, spleen and
kidneys. No clinical signs were reported. At the end of 8 weeks, no exposure-related effects
were found on hematological or histological endpoints.
NOAEL ~ 5000 mg/kg-bw/day (highest dose tested)
(4)	Rats (4/concentration, mixed male and female; strain not reported) were administered acetyl
tributyl citrate via the diet at 5 or 10% (approximately 2500 and 5000 mg/kg-bw/day) for 6
weeks. A control group received basic diet only. Body weights were measured initially and
weekly until study completion. Diarrhea was observed in animals receiving the 10% acetyl
tributyl citrate diet. These animals also exhibited reduced body weight gain (statistics not done
due to small sample size).
LOAEL -5000 mg/kg-bw/day (based on reduced body weight gain and clinical signs)
NOAEL -2500 mg/kg-bw/day
(5)	Sherman rats (20/group; gender not provided) were administered acetyl tributyl citrate
(purity 99.4%) via the diet at 0, 200, 2000 or 20,000 ppm (approximately 0, 10, 100 or 1000
mg/kg-bw) for approximately 2 years. A control group of 40 rats received the basal diet. A
transient reduction in body weight gain was observed in all three treatment groups; however, the
effect was not reproducible when two additional groups of rats were added to the study at 200
and 2000 ppm (approximately 10 and 100 mg/kg-bw/day) for 1 year. Mortalities, including 12
of 60 treated rats and 8 of 40 control rats, were not attributed to treatment. No clinical signs
were reported and lymphoid tumors of the pleural and abdominal cavities were seen in both
treated and control animals at comparable rates. There were no differences between treated and
untreated rats in comparisons of pathological findings, but reporting of experimental details and
results was sparse in the original report. The robust summary noted that the NOAEL appears to
be 20,000 ppm (approximately 1000 mg/kg-bw/day) and considered a more appropriate and
conservative NOAEL to be 2000 ppm (~ 100 mg/kg-bw/day) due to the lack of experimental
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detail in the study report and the pre-GLP timing of the study. [The original report and the
robust summary provide insufficient details to reliably designate a NOAEL or LOAEL for hazard
identification purposes. This summary is included for information purposes only.]
(6) Two cats (gender and strain not reported) were administered acetyl tributyl citrate via gavage
(vehicle not specified) at 5 mL/kg (approximately 5246 mg/kg) daily for 8 weeks. A control
group was included in the study, but information on the control substance was not provided. The
treated cats developed loose stools and lost weight during the study period. There was no change
in behavior or appearance and no effect was observed on the urine, blood chemistry (sugar and
creatinine) or blood counts. [Due to the small sample size and the use of a single dose, this study
summary is included for information purposes only.]
Reproductive Toxicity
(1)	In a 2-generation reproductive toxicity study, Sprague-Dawley rats (30/sex/group) were
administered acetyl tributyl citrate via the diet at 0, 100, 300 or 1000 mg/kg-bw/day, beginning
at 11 weeks prior to mating for F0 males and continuing through a maximum mating period of
14 days. F0 females began treatment at least 3 weeks prior to mating and continued through
mating, gestation and lactation. F1 offspring were in turn exposed to the treated (100, 300 or
1000 mg/kg-bw/day) or untreated diet from weaning, 10 weeks prior to mating and throughout
the mating period (maximum 14 days) for males and females. F1 females continued treatment
through gestation and lactation. No treatment-related clinical signs were reported for either of
the F0 or F1 parents throughout the study. Body weights of the F1 parental males in the 300 and
1000 mg/kg-bw/day groups were consistently lower than controls, while the body weights of the
F1 females and F0 parents were largely unaffected by treatment. Reduced water consumption
was noted in F0 and F1 parents in the 1000 mg/kg-bw/day group. Mating, gestation and fertility
of the F0 and F1 parents were unaffected by treatment. There were no abnormalities seen at
necropsy that were considered treatment-related. The body weights of the pups from the 300 and
1000 mg/kg-bw/day groups were slightly lower than controls and the mortality rates in these
litters were slightly higher. No other treatment-related effects were seen in the pups.
LOAEL (systemic toxicity) = 300 mg/kg-bw/day (based on reduced body weights in F1 males)
NOAEL (systemic toxicity) = 100 mg/kg-bw/day
NOAEL (reproductive toxicity) = 1000 mg/kg-bw/day (highest dose tested)
(2)	In the dietary combined repeated-dose/reproductive/developmental toxicity study in Han
Wistar rats described previously, no treatment-related clinical signs were observed in the
parental animals. No differences compared to controls were seen in estrous cycles, mating
performance, fertility, gestation length and parturition. There were no adverse effects on sperm
motility, counts or morphology. Litter size, survival and growth were similar in all groups with
the exception that litter size and the number of implantations in the 1000 mg/kg-bw/day group
were marginally lower than the control group (statistical significance not provided). At the
necropsy of parental animals, no findings were considered treatment-related. As reported in the
Repeated-Dose Toxicity section above, a 13-week exposure to 1000 mg/kg-bw/day was
associated with a slight decrease in body weight gain, increased liver weights, hepatic
hypertrophy and hepatic peroxisome proliferation in both F1 sexes. Weak peroxisome
proliferation was measured in F1 males at 300 mg/kg-bw/day.
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LOAEL (systemic toxicity) = 300 mg/kg-bw/day (based on hepatic peroxisome proliferation in
F1 males)
NOAEL (systemic toxicity) = 100 mg/kg-bw/day
LOAEL (reproductive toxicity) = 1000 mg/kg-bw/day (based on decreased litter size and
decreased number of implantations)
NOAEL (reproductive toxicity) = 300 mg/kg-bw/day
Developmental Toxicity
In the dietary combined repeated-dose/reproductive/developmental toxicity study in Han Wistar
rats described previously, no treatment-related clinical signs were observed in the parental
animals. No differences compared to controls were seen in estrous cycles, mating performance,
fertility, gestation length and parturition. There were no adverse effects on sperm motility,
counts or morphology. Litter size, survival and growth were similar in all groups with the
exception that litter size and the number of implantations in the 1000 mg/kg-bw/day group were
marginally lower than the control group (statistical significance not provided). At the necropsy
of parental animals, no findings were considered treatment-related.
NOAEL (maternal toxicity) = 1000 mg/kg-bw/day (highest dose tested)
LOAEL (developmental toxicity) = 1000 mg/kg-bw/day (based on decreased litter size and
decreased number of implantations)
NOAEL (developmental toxicity) = 300 mg/kg-bw/day
Genetic Toxicity — Gene Mutation
In vitro
(1)	Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to acetyl
tributyl citrate (purity 98.55%) in DMSO at concentrations of 50, 158, 500, 1580 or 5000
|ig/plate in the presence and absence of metabolic activation. The assay was conducted in
triplicate, with positive and vehicle controls, but the robust summary did not mention whether or
not appropriate results for controls were obtained. The test material was not cytotoxic at 5000
|ig/plate and showed no evidence of mutagenic activity with or without S-9 activation in this
assay.
Acetyl tributyl citrate was not mutagenic in this assay.
(2)	Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were exposed
to acetyl tributyl citrate in DMSO at concentrations of 333, 1000, 3333, 6667 or 10,000 |ig/plate
in the presence and absence of metabolic activation. The assay was conducted in triplicate.
Positive and negative controls were included, but the robust summary did not mention if
appropriate control results were obtained. Acetyl tributyl citrate was not cytotoxic at 10,000
|ig/plate and showed no evidence of mutagenic activity with or without S-9 activation in this
assay.
Acetyl tributyl citrate was not mutagenic in this assay.
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(3)	L5178Y TK+/- mouse lymphoma cells were exposed to acetyl tributyl citrate in DMSO at
concentrations of 10, 70, 150 or 230 |ig/mL in the absence of metabolic activation or 200, 270,
340, 410 or 480 |ig/mL in the presence of metabolic activation and assessed for forward
mutations at the TK locus. An initial toxicity assay was conducted using concentrations over a
range from 0.1 to 5140 jag/m L in the presence and absence of metabolic activation. Solvent and
positive controls tested concurrently and responded appropriately. Complete toxicity was seen in
an initial test at concentrations >514 |ag/m L for nonactivated cultures and > 1028 |ag/m L for S-9
activated cultures. There was a dose-dependent increase in cytotoxicity for treated cultures, but
there was no increase in mutant frequencies either with or without S-9 activation when compared
to solvent controls.
Acetyl tributyl citrate was not mutagenic in this assay.
(4)	Chinese hamster ovary (CHO) cells (two cultures/dose level) were exposed to acetyl tributyl
citrate (purity 99.02%) at concentrations of 0 (DMSO, solvent control), 25, 50, 100, 200 or 400
|ig/mL in the presence and absence of metabolic activation and evaluated for HGPRT mutations.
Positive and negative controls were tested concurrently and responded appropriately. An
approximate 3-fold increase in the number of mutants occurred in the second assay at the 200
|ig/mL dose, without metabolic activation (compared with controls), but the response was not
dose-related and not replicated in the first assay. Therefore, the test substance was determined to
be not mutagenic in this assay.
Acetyl tributyl citrate was not mutagenic in this assay.
Genetic Toxicity — Chromosomal Aberrations
In vitro
Rat lymphocytes were initially exposed to acetyl tributyl citrate (purity 98.55%) in DMSO at
concentrations of 4, 13.3, 40, 133 or 400 |ig/plate in the presence and absence of metabolic
activation to determine the mitotic indices. Based on those results, the concentrations selected
for determining the frequencies of chromosomal aberrations were 50, 133 and 400 |ig/mL. One
hundred cells/replicate in the treated and negative controls and 50 cells in the positive control
were scored for frequencies of chromosomal aberrations. Positive and negative controls were
tested concurrently and responded appropriately.
Acetyl tributyl citrate did not induce chromosomal aberrations in this assay.
Genetic Toxicity — Other
In vivo
In an unscheduled DNA synthesis assay, groups of five male Han Wistar rats were administered
acetyl tributyl citrate via gavage at 0 (solvent control), 800 or 2000 mg/kg in corn oil. Two
positive controls were tested concurrently. Animals were sacrificed at either 2 - 4 or 12 - 14
hours after dosing and primary hepatocyte cultures were obtained. Appropriate results were seen
in the positive and negative controls.
Acetyl tributyl citrate did not induce unscheduled DNA synthesis.
Conclusion: Acute oral toxicity of acetyl tributyl citrate in cats and rats is low. In a 90-day
repeated-dose oral dietary study in rats, decreased body weight and organ weight changes were
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observed at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a combined repeated-
dose/reproductive/developmental toxicity study in rats, organ weight and histopathological
changes were observed in adults at 1000 mg/kg-bw/day; the NOAEL is 300 mg/kg-bw/day. In a
2-generation reproductive toxicity study in rats, reduced body weight was observed in Flmales at
300 mg/kg-bw/day; the NOAEL for systemic toxicity is 100 mg/kg-bw/day. In the same study,
no other treatment related effects were observed; the NOAEL for reproductive toxicity is 1000
mg/kg-bw/day, the highest dose tested. In the combined repeated-
dose/reproductive/developmental toxicity study in rats previously described, histopathological
changes were observed in the liver of adult males at 300 mg/kg-bw/day; the NOAEL for
systemic toxicity is 100 mg/kg-bw/day. In the same study, decreased litter size and decreased
number of implantations were observed at 1000 mg/kg-bw/day; the NOAEL for reproductive and
developmental toxicity is 300 mg/kg-bw/day. The NOAEL for maternal toxicity is 1000 mg/kg-
bw/day (highest dose tested). Acetyl tributyl citrate did not induce gene mutations in bacteria or
mammalian cells in vitro and did not induce chromosomal aberrations in mammalian cells in
vitro.
Table 3. Summary of the Screening Information Data Set
as Submitted under the U.S. HPV Challenge Program -
Human Health Data
Endpoint
Acetyl Tributyl Citrate
(77-90-7)
Acute Oral Toxicity
LDso (mg/kg)
(rats)
>~ 31,500
(cats)
>-52,460
Repeated-Dose Toxicity
NOAEL/LOAEL
Oral Diet (mg/kg-bw/day)
(rat, 90-day)
NOAEL = 300
LOAEL = 1000
Reproductive Toxicity
Oral Diet (mg/kg-bw/day)
Reproductive Toxicity
(rat)
NOAEL = 300
LOAEL =1000
(rat; 2-gen)
NOAEL = 1000 (highest dose tested)
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
Table 3. Summary of the Screening Information Data Set
as Submitted under the U.S. HPV Challenge Program -
Human Health Data
Endpoint
Acetyl Tributyl Citrate
(77-90-7)
Developmental Toxicity
Oral Diet (mg/kg-bw/day)
Maternal Toxicity
(rat)
NOAEL = 1000 (highest dose tested)
Developmental Toxicity
NOAEL = 300
LOAEL =1000
Genetic Toxicity - Gene
Mutations in vitro
Negative
Genetic Toxicity - Chromosomal
Aberrations
in vitro
Negative
Genetic Toxicity - Unscheduled
DNA synthesis
Negative
4. Hazard to the Environment
A summary of aquatic toxicity data submitted for SIDS endpoints is provided in Table 4.
Acute Toxicity to Fish
(1)	Bluegill sunfish (Lepomis macrochirus) were exposed to acetyl tributyl citrate at nominal
concentrations up to 120 mg/L under flow-through conditions for 96 hours, followed by a 96-
hour observation period. Measured concentrations were not provided in the original report.
96-h LC50 = 38-60 mg/L
(2)	Mummichogs (.Fundalus heteroclitus) were exposed to acetyl tributyl citrate at nominal
concentrations up to 120 mg/L under flow-through conditions for 96 hours, followed by a 96-
hour observation period. Measured concentrations were not provided in the original report.
Flow-rate, pH, dissolved oxygen concentration and temperature were monitored.
96-h LC50 = 59 mg/L
Acute Toxicity to Aquatic Invertebrates
Water fleas (Ceriodaphnia dubia) were exposed to acetyl tributyl citrate at nominal
concentrations of 0 (control), 3.9, 7.8, 16.0, 31, 63 or 125 mg/L under static conditions for 48
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U.S. Environmental Protection Agency
Hazard Characterization Document
September, 2014
hours and assessed for immobilization. Mean measured concentrations were: less than the
detection limit (control), 3.82, 4.82, 8.70, 15.5, 17.9 or 60.2 mg/L.
48-h ECso=7.82 mg/L
Toxicity to Aquatic Plants
Green algae (Desmodesmus subspicatus) were exposed to tributyl acetyl citrate at nominal
concentrations of 10, 20, 40, 80, and 100 mg/L under static conditions for 72 hours for algae
growth inhibition test. The mean measured concentrations (arithmetic mean) of the aqueous
phase (bioavailable fraction) were for the evaluation: 4.65, 10.9, 12.9, 44.6 and 47.1 mg/L. The
measured concentrations deviated more than 20 % from the nominal values.
Therefore, the reported effect concentrations refer to mean measured concentrations (arithmetic
mean) of the measured concentrations.
72-h EC50= 74.4 mg/L (growth rate)
72-h LOEC = 10.9 mg/L (growth rate)
72-h NOEC= 4.65 mg/L (growth rate)
72-h ECso= 11.5 mg/L (yield)
72-h LOEC = 10.9 mg/L (yield)
72-h NOEC = 4.5 mg/L (yield)
http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d99c2al-cel8-2d95-e044-
00144f67d249/AGGR-2aee6091 -992c-41 fd-80c7-3e0e2740a67b PISS-9d99c2al -ce 18-2d95-
e044-00144f67d249. html# AGGR-2aee6091 -992c-41 fd-80c7-3 e0e2740a67b
Chronic Toxicity to Aquatic Invertebrates
Water fleas (Daphnia magna) were exposed to acetyl tributyl citrate at nominal concentration of
1.5 mg/L under semi-static conditions for 21 days and assessed for survival and reproduction.
The 21-day NOEC was determined to be > 1.11 mg test item/L for the reproduction rate and >
1.11 mg test item/L for the survival of the adult test animals, respectively. The EC50 for the
reproduction rate was >1.11 mg test item/L (effect value based on analytical monitoring - TWA)
21-d ECso > 1.11 mg/L (survival)
21-d EC50 > 1.11 mg/L (reproduction)
http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d99c2al-cel8-2d95-e044-
00144f67d249/AGGR-0el30278-8aab-4c90-ble8-418778935e93 DISS-9d99c2al-cel8-2d95-
e044-00144f67d249.html#AGGR-0e 13 0278-8aab-4c90-b 1 e8-41877893 5e93
Conclusion: For acetyl tributyl citrate, the 96-h LC50 values for fish range from 38 to 60 mg/L,
the 48-h EC50 value for aquatic invertebrates is 7.8 mg/L and the 72-h EC50 values for aquatic
plants are 11.5 mg/L for biomass and 74.4mg/L for growth rate, respectively. The chronic 21-d
EC50 values for aquatic invertebrates are > 1.11 mg/L (survival) and >1.11 mg/L (reproduction).
14

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Hazard Characterization Document
September, 2014
Table 4. Summary of the Screening Information Data Set
as Submitted under the U.S. HPV Challenge Program -
Aquatic Toxicity Data
Endpoint
Acetyl Tributyl Citrate
(77-90-7)
Fish
96-h LCso (mg/L)
38-60
Aquatic Invertebrates
48-h ECso (mg/L)
7.8
Aquatic Plants
72-h ECso (mg/L)
(growth rate)
(biomass)
74.4
11.5
Chronic Toxicity to Aquatic
Invertebrates
21-d ECso (mg/L)
>1.11 (survival)
>1.11 (reproduction)
(Bold) = experimentally derived data (i.e., derived from testing)
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