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EPA/63 5/R-10/005C
www.epa.gov/iris
&EPA
TOXICOLOGICAL REVIEW
OF
UREA
(CAS No. 57-13-6)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
June 2010
NOTICE
This document is an Interagency Science Consultation draft. This information is distributed
solely for the purpose of pre-dissemination peer review under applicable information quality
guidelines. It has not been formally disseminated by EPA. It does not represent and should not
be construed to represent any Agency determination or policy. It is being circulated for review
of its technical accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and
should not be construed to represent any Agency determination or policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
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CONTENTS—TOXICOLOGICAL REVIEW OF UREA (CAS No. 57-13-6)
LIST OF TABLES vi
LIST OF FIGURES vi
LIST 01 ABBREVIATIONS AND ACRONYMS vn
FOREWORD x
AUTHORS, CONTRIBUTORS, AND REVIEWERS xi
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 6
3.1. ABSORPTION 7
3.2. DISTRIBUTION 10
3.3. METABOLISM 12
3.4. ELIMINATION 15
3.5. PHYSIOLOGICALLY BASED TOXICOklNLTIC MODELS 18
4. HAZARD IDENTIFICATION 23
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS 23
4.1.1. Oral Exposure 23
4.1.2. Inhalation Exposure 24
4.1.2.1. Cohort Studies 25
4.1.2.2. Experimental Studies 26
4.1.3. Dermal Exposure 27
4.1.4. Additional Human Studies 30
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN
ANIMALS—ORAL AND INJECTION 33
4.2.1. Oral Exposure 33
4.2.1.1. Subchronic Studies 33
4.2.1.2. Chronic Studies 33
4.2.2. Inhalation 34
4.2.3. Other routes of exposure 34
4.2.3.1. Subchronic Studies 34
4.2.3.2. Chronic Studies 35
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES 35
4.3.1. Oral Exposure 35
4.3.2. Intrauterine, Intraperitoneal, or Intravenous Exposure 38
4.3.3. Other Studies 39
4.4. OTIILR ENDPOINT - SPECIFIC STUDIES 41
4.4.1. Acute Studies 41
4.4.2. Short-Term Studies 44
4.4.3. Cardiotoxicity 47
4.4.4. Pituitary Effects 50
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4.4.5. Dermal Toxicity 50
4.4.6. Intracranial and Intraocular Effects 51
4.4.7. Urea Toxicity in Ruminants and Non-Laboratory Animals 51
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION 52
4.5.1. Mechanistic Data from In Vivo and In Vitro Studies 52
4.5.1.1. Neurological Effects 52
4.5.1.2. Effects on the Renal System 55
4.5.1.3. Hematological Effects 58
4.5.2. Role of Urea Transporters 58
4.5.2.1. In Vivo Studies in Rats 59
4.5.2.2. In Vivo Studies in Mice 62
4.5.3. Gene Expression Studies 62
4.5.4. Genotoxicity 66
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 70
4.6.1. Oral Exposure 70
4.6.2. Inhalation Exposure 71
4.6.3. Dermal Exposure 72
4.6.4. Additional Studies 73
4.6.5. Mode of Action 73
4.7 EVALUATION 01 CARCINOGENICITY 75
4.7.1. Summary of Overall Weight of Evidence 75
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 76
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 77
5. DOSE-RESPONSE ASSESSMENTS 78
5.1. ORAL REFERENCE DOSE (RID) 78
5.1.1. Choice of Principal Study and Critical Effect with Rationale and Justification.... 78
5.1.2. Previous RfD Assessment 79
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 79
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 80
5.2.2. Previous RfC Assessment 80
5.3. CANCER ASSESSMENT 81
5.3.1. Choice of Study/Data - with Rationale and Justification 81
5.3.2. Previous Cancer Assessment 81
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF 82
HAZARD AND DOSE RESPONSE 82
6.1. HUMAN HAZARD POTENTIAL 82
6.2. DOSE RESPONSE 83
6.2.1. Noncancer / Oral 83
6.2.2. Noncancer / Inhalation 83
6.2.3. Cancer / Oral 83
6.2.4. Cancer / Inhalation 83
7. REFERENCES 84
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APPENDIX A: SU LY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND Disposrnoi
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LIST OF TABLES
Table 2-1. Chemical and physical properties of urea 3
Table 3-1. The percentage of phenol red and urea recovered in the saliva and chewed gum 8
Table 3-2. Pharmacokinetic parameters for plasma distribution of urea in fasted and nonfasted
rats 9
Table 3-3. Excretion of radiolabeled urea in urine, feces, and air in fasted and nonfasted
rats 17
Table 4-1. Liver and kidney function tests from workers exposed to urea and urea-containing
mixtures 25
Table 4-2. Composition of 3% and 10% urea creams used for assessment of urea skin-irritating
effects 27
Table 4-3. Relationship between mean blood pressure and plasma urea concentrations 31
Table 4-4. Early and late effects of urea injection on plasma and brain metabolite
concentrations 44
Table 4-5. Effect of exogenous urea on brain and plasma urea concentration 53
Table 4-6. Genotoxicity and mutagenicity data from in vitro and in vivo assays of urea 66
Table 5-1. Incidence of testicular tumors in male F344 rats and hematopoietic tumors in female
C57B1/6 mice exposed to urea in drinking water for 2 years 83
LIST OF FIGURES
Figure 3-1. Urea cycle 6
Figure 3-2. Two compartmental model of human urea kinetics 19
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LIST OF ABBREVIATIONS AND ACRONYMS
AFP
alpha-fetoprotein
ALT
alanine aminotransferase
AST
aspartate aminotransferase
ATF3
activating transcription factor 3
AUC
area under the curve
BAE
bovine aortic endothelial cells
CA
chromosomal aberration
CASRN
Chemical Abstract Services Registry Number
CEA
carcinoembryonic antigen
CI
confidence interval
cLDL
carbamylated low-density lipoprotein
CNS
central nervous system
CRF
chronic renal failure
CSF
cerebrospinal fluid
EPA
Environmental Protection Agency
EGF
epidermal growth factor
FEVi
forced expiratory volume in 1 second
FSH
follicle stimulating hormone
FVC
forced vital capacity
G3PDH
glyceraldehyde-3-phosphate dehydrogenase
GC
gas chromatography
GD
gestational day
GnRH
gonadotropin-releasing hormone
IEG
immediate early gene
IGF
insulin-like growth factor
ILR
irreversible loss rate
iNOS
inducible nitric oxide synthase
l.p.
intraperitoneal(ly)
IP3
inositol 1,4,5-triphosphate
IRIS
Integrated Risk Information System
IUGR
intrauterine growth retardation
i.v.
intravenous(ly)
MAPK
mitogen-activated protein kinase
MDCK
Madin-Darby canine kidney
mIMCD3
murine inner medullary collecting duct
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mOsm
mOsm/kg
MS
MSDS
NHE
nLDL
NLM
OECD SIDS
OR
P-gP
P wave
PEF
PEFR/min
PGF2a
PI3K
PKC
PLC
PND
p.o.
PSA
PUN
QRS wave
RBC
RfC
RfD
RME
ROS
s.c.
SD
SEM
SMR
SNP
ST segment
SUN
milliosmol
milliosmol per kilogram
mass spectrometry
material safety data sheet
Na+/H+ exchange
native low-density lipoprotein
National Library of Medicine
Organisation for Economic Cooperation and Development Screening
Information Data Set
odds ratio
P-glycoprotein
represents atrial depolarization of the heart in an electrocardiogram
tracing
peak expiratory flow
peak expiratory flow rate per minute
prostaglandin F2a
phosphatidylinositol-3 kinase
protein kinase C
phospholipase C
postnatal day
per os, oral(ly)
prostate-specific antigen
plasma urea nitrogen
represents ventricular depolarization of the heart in an electrocardiogram
tracing
red blood cell
reference concentration
reference dose
rat mesangial cells
reactive oxygen species
subcutaneous(ly)
standard deviation
standard error of the mean
standardized mortality ratio
single nucleotide polymorphism
isoelectric period following the QRS wave is the time at which the entire
ventricle of the heart is depolarized
serum urea nitrogen
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TPA
12-O-tetradecanoylphorbol-13 -acetate
UER
urea entry rate
UF
uncertainty factor
UT
urea transporter
VC
vital capacity
w/v
weight/volume
w/w
weight/weight
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to urea. It is
not intended to be a comprehensive treatise on the chemical or toxicological nature of urea.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of data and related uncertainties. The discussion is intended to convey the limitations
of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk
assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Amanda S. Persad, Ph.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
AUTHORS
John Cowden, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Andrew K. Hotchkiss, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Channa Keshava, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Janice S. Lee, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Allan Marcus, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Andrew Rooney, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
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Reeder Sams, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
CONTRACTOR SUPPORT
Neepa Choksi, Ph.D.
Integrated Laboratory Systems, Inc.
Research Triangle Park, NC
Claudine Gregorio, M.A.
Integrated Laboratory Systems, Inc.
Research Triangle Park, NC
Marc Jackson, B.A
Integrated Laboratory Systems, Inc.
Research Triangle Park, NC
Gloria Jahnke, D.V.M.
Integrated Laboratory Systems, Inc.
Research Triangle Park, NC
INTERNAL EPA REVIEWERS
Marion Hoyer, Ph.D.
Office of Air and Radiation
Office of Transportation and Air Quality
Karen Hammerstrom, J.D.
National Center for Environmental Assessment
Office of Research and Development
Samantha Jones, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
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1. INTRODUCTION
This document presents background information and justification for the Integrated Risk
Information System (IRIS) Summary of the hazard and dose-response assessment of urea. IRIS
Summaries may include oral reference dose (RfD) and inhalation reference concentration (RfC)
values for chronic and other exposure durations, and a carcinogenicity assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both the respiratory system (portal-of-entry) and for
effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
acute (<24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
lifetime) exposure durations, all of which are derived based on an assumption of continuous
exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
derived for chronic exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on
the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a
plausible upper bound on the estimate of risk per (ig/m3 air breathed.
Development of these hazard identification and dose-response assessments for urea has
followed the general guidelines for risk assessment as set forth by the National Research Council
(1983). EPA Guidelines and Risk Assessment Forum Technical Panel Reports that may have
been used in the development of this assessment include the following: Guidelines for the Health
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Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk
Assessment (U.S. EPA, 1986b), Recommendations for and Documentation of Biological Values
for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for Developmental Toxicity Risk
Assessment (U. S. EPA, 1991), Interim Policy for Particle Size and Limit Concentration Issues in
Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for
Reproductive 'toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998), Science Policy Council Handbook: Risk Characterization (U.S.
EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b),
Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (U.S.
EPA, 2000c), A Review of the Reference Dose and Reference Concentration Processes (U.S.
EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), Supplemental
Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA,
2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework
for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006b).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through May 2010.
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2. CHEMICAL AND PHYSICAL INFORMATION
Urea (CASRN 57-13-6) is also known as carbamide. Other names include Aquacare,
Aquadrate, Basodexan, Carbonyldiamide, Hyanit, Keratinamin, Nutraplus, Onychomal,
Pastaron, Ureaphil, and Urepearl. Table 2-1 lists some chemical and physical properties of urea.
Table 2-1. Chemical and physical properties of urea
Chemical formula
ch4n2o
Molecular weight
60.06
Color
colorless to white
State
tetragonal prisms
Odor
develops slight ammonia odor
Taste
cooling, saline taste
Melting point
132.7°C; upon further heating decomposes to ammonia, biuret,
and cyanuric acid
Boiling point
not applicable
Density
1.3230 g/mL @ 20°C
Vapor pressure
1.2 x 10~5 mm mercury (Hg) @ 25°C
Flash point
72.7 ± 22.6°C
Log Pow
-1.59 @ 20-25°C (experimental)
Water solubility
5.45 x 105 mg/L @ 25°C
Solubility
1 gram in 10 mL 95% alcohol, 1 mL boiling 95% alcohol, 20 mL
absolute alcohol, 6 mL methanol, 2 mL glycerol; also soluble in
concentrated hydrochloric acid
pH
7.2 (10%) aqueous solution)
Dissociation constant (pKa)
0.10 @ 21°C
Henry's law constant
1.74 x 10~12 atm-m3/mol @ 25°C (estimated)
Atmospheric OH rate constant
4.00 x 10~n cm3/molecule-sec (2) 25°C (estimated)
Bioconcentration factor
1.0 (2> pH 1-10 and 25 °C
Impurities
biuret 0.3-2 weight%o; cyanates
Sources: NLM (2008a,b,c); OECD SIDS (2008); Registry (2008); O'Neil et al. (2006).
Urea is an endogenous product of protein and amino acid catabolism. It is formed in the
liver from ammonia, which is a deamination product of amino acids. Approximately 20-
35 grams of urea are excreted in human urine per day.
Urea was the first organic compound to be synthesized from inorganic reagents. It is
prepared in methanol from a combination of ammonia, carbon monoxide, and sulfur. Urea can
be produced as granules, flakes, pellets, crystals, and in solutions. It is pelletized or prilled to
avoid caking. Production in the United States was reported at 15.66 and 15.2 billion pounds in
1993 and 1996 respectively (NLM, 2008a, b; O'Neil et al., 2006).
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Urea is nonvolatile in solid form and highly water soluble. It is not expected to volatilize
from moist or dry soil surfaces or to evaporate from water based on its Henry's law constant
—12 3
(1.74 x 10 atm-m /mol @ 25°C), which is based upon vapor pressure and water solubility.
Urea leaches from the soil into surface and groundwater due to its weak adsorption to the soil,
high water solubility, and low soil-water partition coefficient. In both soil and water, urea is
hydrolyzed quickly to ammonia and carbon dioxide by urease, an extracellular enzyme that
originates from microorganisms and plant roots. It biodegrades rapidly and is not expected to
bioaccumulate. In semi-continuous activated sludge, urea degraded, on average, 93-98% in a
24-hour cycle (NLM, 2008a; OECD SIDS, 2008).
If urea is released into the air, it is expected to be found in both the vapor and particulate
phases of the ambient atmosphere. Vapor phase urea is degraded by photochemical reaction
with a half-life estimated by the American Chemistry Council's MSDS at 9.6 hours. Particulate
phase urea may be removed from the atmosphere by wet and dry deposition (NLM, 2008a;
OECD SIDS, 2008).
Urea is used in a variety of products and applications, including as a:
• component of fertilizer and animal feed, plastics, flame-proofing agents, and
adhesives,
• chemical intermediate (e.g., preparation of biuret),
• redundant in selective catalytic reduction (SCR) systems to lower emissions of
nitrogen oxides from stationary and mobile sources
• stabilizer in explosives
• stabilizer in medicine, pharmaceuticals, cosmetics, and dentifrices
• viscosity modifier for starch or casein-based paper coatings
• roadways and airport runway deicing product
• flavoring agent
• humectant and dehydrating agent
• component in consumer goods such as skin care products, liquid soaps, detergents,
and household cleaning products
• food additive in formulation and fermentation of yeast-raised baked goods,
alcoholic beverages, and gelatin products
• insect repellent
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• medical product for reducing cerebral edema and brain mass before and after
neurosurgery (NLM, 2008a; OECD SIDS, 2008; O'Neil et al., 2006).
Inhalation and dermal exposure to urea are the most probable routes of occupational urea
exposure. The National Institute for Occupational Safety and Health National Occupational
Exposure Survey (conducted from 1981-1983) estimated that 783,504 nonfarm workers
(326,824 females) were exposed to urea in the United States (NLM, 2008a). Exposure occurs in
workplaces where urea is produced or used, particularly in places where urea-based fertilizers are
applied. The general population may be exposed to urea in food and drinking water and/or by
dermal contact with urea-containing products (NLM, 2008a). Urea exposure of livestock,
ruminants, pets, and wild animals may occur from spreading of fertilizers to fields, accidental
spills on land into ponds, and other water sources.
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3. TOXICOKINETICS
Urea is an endogenous product of protein and amino acid catabolism. In animals, it is
formed during normal physiological processes that occur primarily in the liver for removal of
nitrogen from the body. Nitrogen, present in the form of ammonia, is a deamination product of
amino acids. The removal of nitrogen is a metabolic process that is part of the Krebs-Henseleit
cycle, also known as the urea or ornithine cycle (illustrated in Figure 3-1). Five key products
that perpetuate the cycle are: arginine, urea, ornithine, carbamoyl-P(hosphate), and aspartate.
The reactions occur intracellularly and are distributed between the mitochondrial matrix and the
cytosol.
+ HCO3
2 , ATP
2 ADP + P
Carbamoyl-P
Orninthine
4rginase ;
sej
Citrulline
Aspartate
Arginine
AMP j 4- (PP)
Arginosuccinate
Fumarate
Figure 3-1. Urea cycle
Source: Adapted from http://www.lhsc.on.ca/programs/rmgc/met/arginase.htm.
Arginine is hydrolyzed by arginase in the cytosol to form urea and ornithine.
Subsequently, a new urea molecule is produced starting with the ornithine product after it enters
the mitochondria. Carbamoyl phosphate, the product of ammonia and HCO3, reacts with
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ornithine in the mitochondria to produce citrulline, which is released into the cytosol where it
reacts with aspartate to form arginosuccinate. Fumarate is then cleaved by argininosuccinate
lyase to form arginine and the cycle begins again. Urea is eliminated from the body primarily
through the urinary system and accounts for approximately half of the total urinary solids.
Approximately 20-35 grams of urea are excreted in human urine per day. Blood concentrations
range from 200-400 mg/L (3.3-6.7 mmol/L) (OECD SIDS, 2008).
Because urea is a naturally occurring product in mammals and other biological
organisms, the majority of the literature identified during the search process pertained to urea
production in vivo and factors affecting its production. Relatively few studies that assessed the
absorption, distribution, excretion, and/or metabolism of exogenous urea were found. This
section will only present results from studies of exogenously administered urea.
3.1. ABSORPTION
The primary route of exposure to urea is through oral exposure and, in simple stomach
animals, such as humans, nonhuman primates, rodents, and pigs, ingested urea is primarily
absorbed into the blood in the upper gastrointestinal tract. No studies that investigated
absorption via inhalation were found. Dawes (2006) reported data for the absorption of urea
through the oral mucosa in 10 adults (five males and five females; age range 24-68 years; mean
age 36 years) who chewed gum that contained urea as an additive. Study participants signed a
consent that had been approved by the Health Research Ethics Board of the University of
Manitoba and avoided eating, drinking, chewing gum, or any type of oral hygiene activities for at
least one hour prior to the study. One group simultaneously chewed two pieces of sugar-free
gum, one contained 27.30 ± 0.64 mg urea and the other 0.496 mg phenol red (i.e., 0.3444 ±
0.0110 mg/g gum x 1.443 g mean gum weight), which is not absorbed by the oral mucosa. The
second group simultaneously chewed two pieces of gum containing only phenol red to establish
the endogenous urea concentration in saliva for use as a sham control. The mean urea content (±
standard deviation [SD]) was determined in saliva and residual chewed gum from each of ten
participants in the two different groups. Saliva samples were collected from each group during a
10-minute chewing time. Participants were instructed to spit into a collection vessel without
swallowing any saliva. Saliva was also collected from each participant for a 5-minute period
prior to initiating gum chewing. The saliva-urea content from chewing gum with only phenol
was 123 ± 38 mg/L (2.05 ± 0.63 mmol/L). Based on a paired /-test, this concentration was
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statistically lower (p = 0.0015) than that in samples collected prior to gum chewing, 198 ± 100
mg/L (3.30 ± 1.68 mmol/L) saliva. The investigators suggested the difference was due to the
higher rate of saliva production in gum chewers and longer collection time resulting in sample
dilution.
The total content of urea and/or phenol red in the residual chewed gum from each
participant was assayed. The concentrations were not reported, only the percentage (mean ± SD)
of urea recovered was given (calculated as a function of total urea recovered in the saliva plus
residual gum, after adjusting for sham control values, and urea content of the unchewed gum).
Urea absorption was determined as the percentage of urea recovered relative to the percentage of
unabsorbable phenol red recovered (theoretically 100%) to adjust for sample loss due to
swallowing during saliva collection. The results summarized in Table 3-1 show the percentage
of phenol red and urea recovered from the saliva samples and the chewed gum residues plus
saliva obtained from sham control and urea exposed participants (Dawes, 2006).
Table 3-1. The percentage of phenol red and urea recovered in the saliva
and chewed gum
Group3
Gum
Saliva
Chewed gum + saliva
Concentration (mg)b
Volumeb
(mL)
% Recoveryb
% Recoveryb
Phenol red
Urea
Phenol red
Urea
Phenol red
Urea
Control
0.497
ND
26.31 ±6.55
69.74 ±8.23
91.04 ± 6.51
96.43 ±6.43
ND
Treated
0.497
27.30 ±0.64
24.63 ±6.42
73.36 ±8.34
92.72 ±3.59
96.92 ±6.45
85.66 ±5.64
"Control = saliva from simultaneous chewing of two pieces of gum containing only phenol red;
Treated = saliva from simultaneous chewing of one piece of gum containing phenol red and one containing urea.
bValues = mean ± SD; ND = not done.
Source: Dawes (2006).
Of the total urea and phenol red recovered, 91.04 ± 6.51% and 69.74 ± 8.23%,
respectively, were attributed to release from the gum into the saliva. The mean total recovery of
phenol red was 96.92 ± 6.45% (three of the ten values were below the lower 95% confidence
limit of the assay assuming a theoretical recovery of 100%), suggesting some participants
swallowed a small amount of saliva during sample collection. By comparison, the mean total
recovery of urea was 85.66 ± 5.64% (values from nine of the ten participants were below the
lower 95% confidence limits of the assay). Based on the observation that the percentage of urea
recovery was less than the non-absorbed marker, phenol red, Dawes (2006) postulated that when
the salivary urea concentration is higher than that in the plasma, urea may be absorbed through
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the oral mucosa. Dawes (2006) noted that calculation of an absorption coefficient was not
possible since (1) saliva urea concentrations were not maintained at a constant level and (2) the
mucosa surface area was not measured.
Nomura et al. (2006) investigated the absorption, distribution, metabolism, and excretion
(ADME) of radiolabeled urea administered by intravenous (i.v.) injection or orally (p.o.) to
fasted and nonfasted male Sprague-Dawley rats (206-359 grams; n = 60). Fasted rats were
included in this study design as the authors were interested in the effects of diet on the
disposition of urea. Uptake of urea was measured in plasma samples after i.v. injection of
[14C]-urea (specific activity 32.2-35.2 MBq/mg) into either the saphenous vein or the tail vein
(2 mg/1.85-3.7 MBq-kg) or p.o. administration via a stomach tube (2-1000 mg/1.85-
3.7 MBq-kg). Nonfasted rats received food ad libitum but food was removed 15 hours prior to
dosing and withheld for eight hours after treatment of the fasted rats. Urine, feces, blood, and/or
tissue samples were collected at 30 minutes, 1, 4, 8, 24, 48, 72, and/or 96 hours. In both fasted
and nonfasted rats given 2 mg urea/kg body weight, plasma concentrations decreased
biphasically as a function of time in both the oral and i.v. treatment groups. The
pharmacokinetic parameters for the concentration and half-life of urea within the plasma are
shown in Table 3-2.
Table 3-2. Pharmacokinetic parameters for plasma disposition of urea in
fasted and nonfasted rats
Route
Diet
Dose
(mg/kg)
V/
(mL/kg)
C b
max
(fig eq/mL)
Tmax
(hr)
ti/2 (hr)c
AUC„J
(fig eq X hr/mL)
a
P
Intravenous
Fasted
2
749
2.0
(0.083-10)
3.5
(10-24)
8.06
Nonfasted
2
741
1.7
(0.083-10)
6.2
(10-24)
7.00
Oral®
Fasted
2
1.96 ± 0.17
0.5
2.1
(0.5-10)
3.4
(10-24)
7.43
Nonfasted
2
1.10 ± 0.16
1
2.5
(1-10)
7.5
(10-24)
5.18
62.5
32.1 ±9.7
1
2.0
(2-10)
10.7
(10-24)
123.00
250
100 ±42
1
2.1
(2-10)
9.0
(10-24)
515.00
1,000
470 ±53
2
1.9
(2-10)
8.4
(10-24)
2,374.00
aVd - apparent volume of distribution after i.v. injection.
bCmax - maximal concentration (mean ± SD, n = 3).
°t 1/2 - calculated half-life in each phase of the biphasic curve. In parentheses: time period (in hours) for each phase.
Area under the curve (expressed as (ig equivalents x hours/mL).
Calculated from mean plasma concentrations of three rats.
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Source: Nomura et al. (2006).
The half-life of urea elimination in the initial phase (a) was approximately two hours
regardless of route of exposure or fasting condition, while the half-life in the second phase ((3)
was shorter in fasted animals for both routes of exposure (3.5 and 3.4 hours for i.v. and p.o.,
respectively) compared with that of the nonfasted animals (6.2 and 7.5 hours for i.v. and p.o.,
respectively). The a phase included the time period from approximately 0.5-10 hours and the (3
phase was from 10-24 hours. The decrease in plasma concentrations in nonfasted rats given
62.5, 250, or 1000 mg urea/kg p.o. was also biphasic and similar to that of the 2 mg/kg dose.
The half-life calculated for each phase of the curve was also similar at all four doses (see Table
3-2). The maximum concentration (Cmax) and area under the curve (AUG,..) increased
proportionally with increasing dose indicating that urea fits a linear pharmacokinetic model
across the wide range of doses tested for both routes of exposure. The authors claim that the
disposition of exogenous urea is similar to that of endogenous urea and suggests that rats have a
sufficiently large capacity for disposition.
3.2. DISTRIBUTION
The disposition of urea administered exogenously is not well characterized. Two in vivo
studies in rats were conducted to examine the uptake and distribution kinetics of exogenous
[14C]-urea administered p.o. or intraperitoneally (i.p.) (Nomura et al., 2006; Johanson and
Woodbury, 1978). The results from these studies, along with data from a more recent study by
Sahin and Rowland (2007) of the hepatic kinetics of [14C]-urea in situ and the effect of
erythrocytes on uptake and elimination, are presented here.
In the study by Nomura et al. (2006) described in Section 3.1, the tissue distribution of
urea given p.o. was assessed in fasted (food removed 15 hours prior to treatment and withheld
for 8 hours afterwards) and nonfasted (food received ad libitum) male Sprague-Dawley rats.
Urine, feces, blood, and/or tissue samples (i.e., urinary bladder, kidney, gastrointestinal tissues,
pancreas, liver, heart, aorta, lung, trachea, thyroid, tongue, eye ball, brain, thymus, adrenal,
testes, prostate, skin, bone, and bone marrow) were collected at 30 minutes up to 96 hours after
[14C]-urea (2 mg/1.85-3.7 MBq-kg) dissolved in sterilized distilled water was administered via
stomach tube. In general, fasting had little effect on the tissue distribution but did produce a
slight increase in the overall concentrations. With the exception of the brain and eyeball, the
maximum tissue concentrations were recorded 30 minutes to an hour after urea administration
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(plasma concentration reached Cmax at 30 minutes in both the nonfasted and fasted animals; 1231
±319 and 1675 ± 938 ng eq/mL, respectively). Excluding the gastrointestinal tract (site of
administration), the tissues with the highest radiolabel concentration were the kidney and urinary
bladder (-2.5- and 3.2-fold higher than plasma concentrations). Fat and brain had the lowest
urea concentrations (225 ±138 and 263 ± 182 ng eq/mL, respectively) at this time point. Urea
concentrations in the remaining tissues were similar to or below that in the plasma. After 24
hours, all tested tissues, with the exception of the large intestine and the Harderian gland, had
below detectable levels of radiolabeled urea after 24 hours. At 72 hours, none of the tested
tissues had detectable levels of radiolabeled urea.
The distribution of [14C]-urea from plasma into the lateral ventricular choroid plexus, the
tissue responsible for production of cerebrospinal fluid (CSF), was investigated in adult rats to
determine the permeability characteristics of the choroid plexus epithelial membrane (Johanson
and Woodbury, 1978). Forty-four nephrectomized male Sprague-Dawley rats (300-425 grams)
were anaesthetized and injected, via i.p., with [14C]-urea (1.7 MBq [45 |iCi]; dose not reported).
Bilateral nephrectomy of each animal was accomplished by ligation of both renal pedicles to
allow urea to reach a steady-state concentration of 370 mg/L in the plasma eight hours after
ligation. Plasma urea concentrations for control Sprague-Dawley rats range between 240-260
mg/L (Hardy et al., 1983; Kamm et al., 1987). Rats were sacrificed 0.5, 1, 2, 3, 5, 8, 11.5, and
16 hours after injection of the isotope and samples of blood, CSF, cerebral cortex, and the lateral
ventricular choroid plexus were collected. [3H]-H20 was administered, as a tracer label in the
sample fluid spaces to assist in interpreting the distribution of radiolabeled urea to the brain, to
12 rats that were sacrificed 3, 6, 9, and 15 minutes after injection. The radiolabeled water and
urea distribution was calculated as the ratio of radiolabel in tissue or CSF to that in plasma.
Results showed that the uptake and distribution of [14C]-urea in the choroid plexus was much
slower than in the skeletal muscle. A steady-state distribution was observed approximately
eight hours after injection in the choroid plexus, as well as in both the cerebral cortex and the
CSF, compared with only one hour required to reach steady state concentrations in muscle
tissue. Evaluation of the relative concentrations (at steady-state) of radiolabeled urea in plasma,
choroid epithelial cells, and CSF showed that the concentrations in the choroid epithelial cells
and CSF reached a maximum concentration that was -70% of that observed in the plasma. The
authors stated that these observations, along with the lack of a concentration gradient for
[14C]-urea from choroid cell to CSF, imply that the basolateral membrane of the choroid
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epithelial cells substantially hinders urea molecules in the plasma from entering into the
epithelial cell compartment.
Sahin and Rowland (2007) evaluated the hepatic distribution kinetics of urea compared to
thiourea. The effect of the presence of erythrocytes on the distribution of urea was assessed in
situ using isolated perfused liver from male Sprague-Dawley rats (200-400 grams). Livers were
perfused in a single-pass mode (15 mL/minute) via the portal vein with Krebs-bicarbonate buffer
(pH 7.4) containing 3 g/L glucose, 6 mg/L sodium taurocholate, saturated with humidified
95% 02-5% CO2. The experiments were conducted in the absence or presence of red blood cells
(RBCs). In experiments with RBCs, [14C]-labeled urea (0.001 MBq [0.03 |lCi]/50 |lL) and the
RBC suspension were incubated 30 minutes prior to injection into the liver (n = 4). The model
consisted of two parallel components each representing the free and RBC-associated portions of
the compound. Effluent curves were calculated based on the volume of free and RBC-associated
compound. Analysis of the data showed that the presence of RBCs had no effect on urea
distribution (the effluent curves from both experiments were unimodal and superimposable).
These results indicate that a barrier effect by the RBCs on rapidly penetrating substances like
water or urea is not expected and therefore RBCs could be viewed as an extension of the plasma
compartment (Sahin and Rowland, 2007).
3.3. METABOLISM
In published reports, there is little evidence that endogenous urea is metabolized. Walser
and Bodenlos (1959) observed that there was no evidence that urea undergoes any metabolic
transformation in humans other than hydrolysis in the gut. Results from the ADME study by
Nomura et al. (2006) (previously described in Sections 3.1 and 3.2) also showed no evidence of
urea metabolism. Methanol extracts of plasma and urine samples collected at time Tmax from
fasted (Tmax = 30 minutes) and nonfasted (Tmax = 1 hour) rats given [14C]-urea p.o. were analyzed
by radiochromatography to identify urea metabolites. Only a single peak of unchanged urea was
observed on the chromatographs regardless of time after administration or fasting condition.
In vivo colonic metabolism of urea in humans was studied by placing a tracer dose of a
double labeled [15N15N]-urea into the colon of study subjects then collecting urine and stool
samples for up to 72 hours to measure radiolabel (Moran and Jackson, 1990a, b). The authors
noted that the Southampton Hospitals' Ethical Committee approved these studies.
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Moran and Jackson (1990a) administered 1.5 mg/kg [15N15N]-urea through the biopsy
channel of a colonoscope to 12 male patients considered to have normal colons. Urine and stool
samples were obtained prior to administering the radiolabeled urea to establish baseline urea
concentrations. Urea was administered into the right colon (cecum) of one group of six patients
and into the left colon (distal to the splenic fixture) of the remaining six patients—because it had
been proposed that different segments of the colon may function differently. Urine and stool
were collected for the next 72 hours and urea nitrogen in the urine was measured by mass
spectrometry (MS). The concentrations of urea, ammonia nitrogen, and free nitrogen content
were evaluated in samples from both groups. Unmetabolized urea excreted in the urine was
detected as [15N15N]-urea. Detection of [15N14N]-urea in the urine indicated that urea had been
metabolized producing [15N]-ammonia that was reincorporated into newly synthesized urea.
Following digestion of the stool samples, the nitrogen content was measured using an automatic
analyzer (Kjeltec Auto 1030, Tecator Sweden).
The results showed that with the exception of one subject, less than 4% of the study dose
(regardless of where it was administered) was recovered in the stool, indicating that over 90%
was absorbed. The amount of [15N14N]-urea in the urine samples ranged from 3.0-34.0% of the
total urea dose administered to either study group. There was no indication of functional
differences between the two colon segments to which urea had been administered.
In a second, similar study, Moran and Jackson (1990b) administered [15N15N]-urea
(1.5 mg/kg) directly into the defunctional colons of 11 subjects (10 males and 1 female, 55-
74 years old) previously diagnosed with rectal carcinomas or diverticulitis. Six of these subjects,
patients that had previously received a transverse colon loop colostomy (also called a
defunctioning colostomy), were in two studies. In the first study, urea was administered into the
left defunctioning colon (distal to the splenic fixture) while in the second study, the test
substance was infused into the right functioning colon (ascending colon). The remaining five
subjects received urea via the left functioning colon (descending colon). Urine and stool samples
were collected over a 72-hour period and prepared as previously described above for analysis of
[15N15N]- and [15N14N]-urea by MS. Urine and stool samples were also obtained prior to
administering the radiolabeled urea to determine the baseline urea concentrations.
Nonhydrolyzed urea ([15N15N]-urea) was recovered in greater proportion in the urine of
patients after placement in the defunctioned colon (median recovery 29%; range 22-74%) than it
was in urine from the same patients after placement of urea in the functioning colon (median
recovery 9%; range 5-14%). This was attributed to a decrease in bacterial activity in the
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defunctioned colon and, consequently, a decrease in urea hydrolysis. However, the recovery of
hydrolyzed [15N14N]-urea in urine (expressed as a percentage of the initial dose) was similar
irrespective of whether urea was administered in the patients' left defunctioned or right
functioning colon (median =10 and 11% with ranges of 6-14% and 4-20%, respectively),
indicating metabolism of the exogenously added urea. On the other hand, recovery of
hydrolyzed [15N14N]-urea in urine from the five patients who were administered urea in the left
functioning colon (median 21%; range 14-66%) was significantly greater (p < 0.05) than the
urea recovery from the six patients in given urea in the left defunctioned colon. However,
recovery after placement in the left functioning colon was not significantly different from that
observed after placement of urea in the right functioning colon (median recovery 11%; range 4-
20%) The authors concluded that urea, when present in a functioning colon, is rapidly
hydrolyzed by the bacteria present.
Forsythe and Parker (1985) studied urea synthesis and degradation in the digestive tract
using New Zealand White and cross-bred Black and Brindle rabbits (2.4-3.5 kg). A cecal probe
and two catheters (one in the carotid artery and one in the jugular vein) were implanted in each
animal and food and water were provided ad libitum. [14C]- or [15N]-urea (0.2 MBq [5 |iCi]/hour
and 976 jj.g urea nitrogen/hour, respectively) were infused separately into the jugular vein over a
period of 7-10 hours. Samples of arterial blood from the carotid catheter and of cecal dialysate
were collected over the infusion period. Urine was also collected over a 48-hour period. All
values were reported as mean ± SEM. The concentrations of plasma urea and cecal dialysate
ammonia remained constant (20 mg/L [0.35 mM]) throughout the infusion period implying that
urea metabolism was at steady-state. The mean irreversible loss rate (ILR) of plasma
urea-carbon (26.3 ± 2.0 mg carbon urea/hour; n = 7) was calculated from the [14C]-urea infusion
rate and the plateau specific radioactivity in the plasma, a value that had to be predicted because
plasma 14C did not plateau during the infusion period. The cecal dialysate 15N-enrichment time
curve also had to be calculated because 15N-enrichment did not plateau. The relationship
between plasma 14C activity and cecal dialysate 15N-enrichment time was defined by a single
exponential function suggesting that little of the urea-carbon was recycled. Therefore, the
authors concluded that the ILR represented urea synthesis. Most of the [14C]-urea dose was
excreted in the urine (mean fraction = 0.62 ± 0.03; n = 6), indicating that a majority of the
infused urea was not metabolized but rather was absorbed directly by the gastrointestinal tract.
Based on these values, the degradation rate in the gastrointestinal tract was determined to be 63
mg urea/hour. Analysis of the cecal dialysate revealed that radiolabeled urea was not present
14 DRAFT—DO NOT CITE OR QUOTE
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during the infusion time period. To assess urea entry into the cecum, three animals were given
[14C]-urea (0.02 MBq [0.6 |iCi]/hour) intracecally along with a noncompetitive urease inhibitor.
The mean cecal urea ILR was 16.8 ± 2.4 mg urea/hour (n = 3), which would represent the plasma
urea entry rate (UER) if all urea in the cecum were derived from the plasma only. However, the
actual calculated rate was 12.8 ± 2.3 mg urea/hour (n = 7) which represents the product of cecal
ammonia nitrogen multiplied by the proportion of cecal ammonia nitrogen derived from plasma
urea nitrogen. The mean plasma urea nitrogen ILR from infusion of [15N]-urea was 40.2 ±
4.6 mg nitrogen urea/hour (n = 9). Comparison of the radiolabeled enriched nitrogen in the cecal
ammonia pool with the plasma urea enriched nitrogen levels indicated that the mean proportion
of cecal ammonia obtained from plasma urea nitrogen was 0.25 ± 0.03 (n = 8). The rate of urea
nitrogen reutilization after degradation in the gastrointestinal tract, calculated from the nitrogen
urea synthesis rate and plasma nitrogen urea ILR, was 18.6 ± 3.5 mg nitrogen/hour (n = 7).
These data show that, in the rabbit, plasma urea can enter the cecum through the blood stream
where a major portion is degraded and the nitrogen is eliminated or reutilized. However, with
the methodology applied here for measuring the movement and degradation of urea, only 14% of
the total degraded urea could be accounted for by ileal flow into the large intestine.
3.4. ELIMINATION
A dilution technique was used to determine the urea kinetic parameters, distribution
volume, production rate, and clearance using a healthy 57-year-old male (182 cm, 86 kg)
compared with a 38-year-old male (177 cm, 71.5 kg) with renal failure who was receiving
hemodialysis three times a week (Kloppenburg et al., 1997). The study was conducted with
approval from the Medical Ethics Committee of the University of Groningen and informed
consent from both participants. A single i.v. injection of [13C]-urea was administered to both
subjects; 24 mg (0.4 mmol) to the healthy subject and 48 mg (0.8 mmol) to the patient with renal
failure. Blood samples were collected at 0, 2, 5, 10, 15, and 30 minutes and every 30 minutes
thereafter up to four hours post-injection. Plasma was separated from the RBCs and [13C]-urea
content analyzed by headspace chromatography-isotope ratio MS. The reproducibility of the
method used was assessed by conducting the study in the healthy volunteer four times over a
period of four months. Clearance was determined by plotting logarithmic radiolabeled urea
concentration versus time and doing a least squares linear regression analysis. Results show that
the endogenous urea concentration in the patient with renal failure was elevated compared with
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the healthy subject. In addition, elimination was about six-fold greater in the healthy volunteer
when compared with the renal failure volunteer (0.0674/hour vs. 0.0120/hour). Calculated urea
clearance rate in the healthy subject (61.7-72.6 mL/minute; n = 4) was also higher than in the
renal failure patient (7.4 mL/minute; n = 1), and the half-life (8.4-10.4 hours; n = 4) was shorter
in the healthy subject compared with the renal failure patient (58.6 hours; n = 1).
In the Moran and Jackson (1990a, b) studies, described in Section 3.3, the excretion of
[15N15N]-urea in urine and stool samples was reported. Briefly, one group of six patients
received [15N15N]-urea into different segments of the colon in two separate studies.
Concentrations of urea, ammonia nitrogen, and nitrogen in urine and stools collected over the
next 72 hours were measured. Of the total administered dose, unmetabolized ([15N15N]) and
metabolized ([15N14N]) urea recovered in the urine ranged from 1.0 to 9.6% and 3.0 to 34.0%,
respectively. The overall excretion of radiolabel in the stool was relatively low (0.4 to 12%)
compared with the urinary concentration. The mean whole-body retention of [15N15N]-urea
72 hours after administration in the right and left colon was 74 and 82%, respectively (Moran
and Jackson, 1990a).
A second study by Moran and Jackson (1990b) assessed the elimination of labeled urea in
the urine and stool by MS using samples collected for 72 hours after treatment of 11 subjects
(10 males and 1 female, 55-74 years old) previously diagnosed with rectal carcinomas or
diverticulitis. The results showed that the majority of the radiolabeled urea was excreted through
the urine-and that its elimination appeared to depend upon the functional status of the colon. No
radiolabel was recovered in the stool when [15N15N]-urea was placed in the left defunctioned
colon. However, when [15N15N]-urea was placed into the right functioning colon of the same
individuals, 1-24% of the administered radiolabel was excreted in the stool. Urinary excretion of
radiolabel in these individuals ranged from 9-29% and 35-80% of the administered dose for the
left defunctioned and right functioning colon, respectively. Urinary excretion of urea from
individuals with a left functioning colon was similar to that observed when urea was excreted
from the right functioning colon. These results show that colonic metabolism of urea and
excretion in the urine is noticeably reduced in the absence of intestinal microflora, as is the case
in dysfunctional segments of the colon.
Nomura et al. (2006) studied of the elimination of [14C]-urea (specific activity 32.2-
35.2 MBq/mg) administered p.o. or i.v. to fasted and nonfasted male Sprague-Dawley rats (206-
359 grams; n = 60). Rats were given 2 mg/kg (1.85-3.7 MBq/kg) urea dissolved in sterilized
distilled water through a stomach tube or by i.v. injection into either the saphenous vein or the
16 DRAFT—DO NOT CITE OR QUOTE
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tail vein (see Section 3.1 for study details). Nonfasted rats were provided food ad libitum; the
food for the fasted rats was removed 15 hours prior to dosing and withheld for eight hours after
treatment. [14C] derived from radiolabeled urea was analyzed in samples of urine, feces, and
expired air collected up to 96 hours after dosing. The results from this analysis are presented in
Table 3-3. The total percentage of radiolabel recovered from fasted rats in urine, feces, and
expired air 24 hours after dosing was comparable for each route of exposure.
Table 3-3. Excretion of radiolabeled urea in urine, feces, and air in fasted
and nonfasted rats
Route
Time
(hours)
Recovery of radioactivity (% of dose)a
Urine
Feces
Expired air
Fasted
Nonfasted
Fasted
Nonfasted
Fasted
Nonfasted
Intravenous
0-4
2.4 ± 1.2
10.5 ± 1.0
4-8
0.6 ±0.1
5.4 ±1.5
8-24
1.6 ± 1.4
4.7 ±2.6
0-24
90.8 ±3.6
72.6 ±5.3
0.3 ±0.2
1.6 ± 0.4
4.6 ±2.6
20.5 ±2.7
24-48
0.2 ± 0.1
0.3 ±0.1
-
-
0.1 ±0.1
0.3 ±0.2
48-72
0.1 ±0.1
0.2 ±0.1
-
-
-
-
72-96
0.0 ±0.0
0.1 ±0.1
-
-
-
-
Washing15
-
0.0 ±0.0
0-96
91.1 ±3.6
73.1 ±5.5°
0.3 ±0.2
1.6 ± 0.4
4.7 ±2.7
20.9 ±2.8
Oral
0-4
1.8 ± 0.4
33.7 ± 11.2
4-8
0.6 ±0.1
5.3 ±0.7
8-24
1.1 ± 1.4
3.8 ± 4.0
0-24
94.9 ±6.0
53.8 ± 9.5
1.2 ±1.0
1.0 ±0.1
3.5 ± 0.8
42.7 ±8.1
24-48
0.2 ±0.0
0.1 ±0.0
-
-
-
0.2 ±0.1
48-72
0.0 ±0.0
-
-
-
-
-
72-96
0.0 ±0.0
-
-
-
-
-
Washing
-
-
0-96
95.1 ±6.0
54.0 ±9.6
1.2 ±1.0
1.0 ±0.1
3.5 ± 0.8
42.9 ±8.0
aMean ± SD of three rats. Empty cells represent time points and conditions that were not evaluated; dashes indicate
results were below the limit of detection (SD > 5% of the radioactive counts per minute for the 72 hour sample).
bMetabolism cages were washed with distilled water 96 hours after administration of urea; 1.2% radioactivity was
recovered after 96 hours in the carcasses of rats dosed i.v.
Source: Nomura et al. (2006).
In fasted rats more than 90% of the radiolabel was in the urine, approximately 4% in
exhaled air, and only around 1% in feces, with almost all of the radiolabel excreted during the
first 24 hours. In nonfasted rats much less of the administered dose was recovered in urine;
>70% after iv. dosing and only 54% after oral administration. Fecal excretion did not differ
much between fasted and nonfasted animals. However, in nonfasted animals 20% of the
administered radioactivity was recovered from exhaled air following i.v. dosing, and almost 43%
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following oral administration (Table 3-3). The increased [14C] recovery in expired air and
decreased recovery in urine in i. v.-dosed, compared with p. o.-dosed animals, was attributed to a
higher oral absorption rate. Additional sampling up to 96 hours after dosing increased the
percentage of total [14C] recovered by less than 1% regardless of exposure route or fasting
condition.
Overall, the results from studies of exogenously administered urea presented in this
section illustrate that the route of administration has little effect on the distribution, metabolism,
or excretion of urea. In animal studies, maximum plasma and tissue concentrations were
achieved 30 minutes to one hour after dosing. Excluding the gastrointestinal tract, the kidneys
and urinary bladder tended to show the highest urea concentrations. The uptake of [14C] urea
and distribution from plasma into the lateral ventricular choroid plexus of rats was shown to be
much slower than in skeletal muscle. There was little evidence that exogenously administered
urea undergoes any metabolic transformation in humans or animals other than hydrolysis by
bacteria in the gut. One study reported that approximately 90% absorption of urea administered
into the human colon regardless of the functional condition or location within the colon. Studies
also showed that urea was primarily eliminated via the urine. Although urea administered into
functioning or defunctioning segments of the colon was primarily excreted via the urine, the
percentage of the total dose eliminated was much lower for individuals with defunctioning colon
compared with those with functioning colon.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
Marini et al. (2006) used minimally invasive catheterization protocol to study urea
kinetics in conscious B6C3-derived male mice (n = 6). A single bolus dose of [15N15N]-urea
(160 (ig [2.66 |imol] in 9 |iL pyrogen-free double-distilled water) was injected into the tail vein
via an infusion/sampling catheter and blood samples were collected 5, 10, 15, 20, 25, 30, 40, 50,
and 60 minutes later through the same catheter for analysis of urea content by gas
chromatography-MS (GC-MS). A continuous infusion experiment was also performed in which
six male mice were infused for six hours with [15N15N]-urea (113 (xmol/kg-hour) at a rate of
50 (iL/hour and blood samples were collected from the distal tail vein catheter at 1.5, 3, 4, 5, and
six hours after the start of infusion. Three blood samples were collected prior to infusion to
establish the background urea plasma concentration.
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The plasma enrichment and disappearance of urea was analyzed using a two
compartmental model previously described by Matthews and Downey (1984) and illustrated in
Figure 3-2. In this model, two urea pools are assumed to be present and the flow of urea through
Pool A (the primary pool) represents inflow from hepatic production of urea (Fao) and outflow of
urea via renal excretion and bacterial hydrolysis in the gut (Foa). The urea mass-flow rate (/¦) is
expressed in mmol/kg-hour. Pool B is assumed to be a secondary "blind" pool that does not
have separate inflows or outflows and is connected to Pool A. The urea pool size (Q) in each
compartment is reported as (imol/kg. The fractional rate constant (k) is expressed as hour-1 and
is based on the tracer urea enrichment (mole % excess) at time (t) and the rate constants from the
fitted curve using nonlinear regression analysis. The subscript ba refers to flow through Pool A
to B, ab refers to flow from Pool B to A, ao refers to flow from space outside the system into
Pool A, and oa refers to flow from Pool A into space outside the system.
ao
Pool B
Size = QB
Fba
^ba Fab
kab
Pool A
Size = Qa
oa
oa
Figure 3-2. Two compartmental model of human urea kinetics
Source: Matthews and Downey (1984).
Data from the single bolus injection were fit to 2- or 3-exponential decay curves and
analyzed based on this model. Results showed that the primary urea Pool A exchanged rapidly
(Fab = Fba = 70.65 ± 14.96 mmol/kg-hour) with secondary Pool B. This resulted in the mean pool
size in the secondary compartment B (Qb = 4.54 ± 0.45 (imol/kg) being approximately five times
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that of the primary compartment A (Qa = 0.93 ± 0.28 (xmol/kg). The mean UER, also referred to
as F in the model, is determined from the concentration of the single dose divided by the area
under the best-fitted curve (AUC). The UER reported for Fao which also equals Foa in the single
dose study was 3.36 ± 0.30 mmol/kg-hour. In the continuous infusion study, plasma urea
concentrations reached a plateau at 3.3 ± 0.2 hours, and a mean UER of 3.24 ± 0.23 mmol/kg-
hour was calculated based on the plateau value. This rate did not differ significantly from the
UER of the single dose protocol demonstrating that the two compartmental model can be used to
analyze data obtained from different exposure types (Marini et al., 2006).
Kaplan et al. (1999) described methodologies designed to address limitations of
approaches traditionally used to assess urea kinetics as they relate to dialysis challenges. A two
compartmental model like that described above was used to analyze urea kinetics derived from
concentrations of radiolabeled and nonlabeled urea in blood and urine samples collected from
dialysis patients and normal volunteers. The study protocol was approved by the Northwestern
University Institutional Review Board and details of the model are described in the appendix of
the published study. Five patients (four males and one female, 41-62 years old) who had
received standard hemodialysis for more than one year were recruited for the study. Venous
catheters were inserted into each arm of the participants and [15N15N]-urea (3-4 grams) was
injected over a 5-minute period into one arm and 16 blood samples were collected via the
catheter in the other arm over a period of 5-720 minutes. Dialysis began 24 hours later and nine
arterial and venous blood samples were collected over a period of 15-240 minutes after dialysis
was initiated. Five volunteers who were not dialysis patients (three males and two females, 36-
54 years old) and who had participated in a previous study of urea kinetics based on a three
compartmental model (Odeh et al., 1993; described below), received 2 grams of [15N15N]-urea
and 19 blood samples were collected over a period of 5^480 minutes. Timed urine collections
were also obtained from all participants throughout the study.
[15N15N] - and [15N14N]-urea concentrations in all samples collected were analyzed by
GC-MS. Samples collected during the predialysis period were analyzed as a function of time
using the two compartment model. [15N14N]-Urea concentrations were modeled assuming they
followed the same disposition kinetics as the radiolabeled urea; however, adjustable parameters
for describing the initial concentration of urea in each compartment at time zero and the constant
rate of urea production input into the primary compartment were included. The results showed
that there was no difference in intercompartmental clearance rates (the rate at which the volume
of the central compartment multiplied by its transfer rate constant equals the volume of the
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peripheral compartment multiplied by its transfer rate constant) for the predialysis (1.26 ±0.5
L/minute), intradialysis (1.2 ± 0.5 L/minute), and normal subjects, (1.14 ± 0.31 L/minute).
Likewise, the nonrenal clearance rates for the pre- and intradialysis samples did not differ (5.9 ±
3.6 and 6.5 ± 2.9 mL/minute, respectively). The results from the pharmacokinetic modeling
presented in this study suggest that a two-compartment model satisfies all aspects of urea
distribution and removal; however, the authors noted that the compartments should not be
equated with any specific physiologic spaces (Kaplan et al., 1999).
Inulin (naturally occurring polysaccharides) and [15N15N]-urea kinetics were assessed in
five healthy subjects (three males and two females, 36-54 years old) following simultaneous i.v.
injection (Odeh et al., 1993). Review of the publication did not provide information on the
human subjects research ethics procedures undertaken in this study, but there is no evidence that
the conduct of the research was fundamentally unethical or significantly deficient relative to the
ethical standards prevailing at the time the research was conducted. Blood and urine samples
were collected over an 8-hour period and urea concentrations were measured as described above
for the Kaplan et al. (1999) study. The data were used to describe the physiologic basis of multi-
compartmental systems often used to model drug distribution based on a three-compartment
model that uses intravascular space as the central compartment and splanchnic and somatic
tissues as two peripheral components. The results reported for urea only are discussed here. One
of the unique features of this model is that it includes interstitial and intracellular fluid spaces for
both of the tissue compartments; however, the transfer of urea in these spaces occurs too rapidly
for characterization of the kinetics. Urea from the primary compartment (intravascular space)
distributes rapidly into the splanchnic tissue compartment but slowly into the somatic tissue
compartment. The mean volume of distribution for urea calculated from the three-compartment
model was 0.670 ± 0.143 L/kg which was very similar to the value of 0.68 L/kg previously
reported by Matthews and Downey (1984) using a two-compartmental model. Blood flows and
permeability coefficient-surface area products for the peripheral compartments (i.e., splanchnic
and somatic tissue compartments) were determined. The average sum of compartmental blood
flows was 5.39 ± 0.49 L/minute, a value similar to the results of Doppler measurements of
cardiac output (5.47 ± 0.40 L/minute) (Odeh et al., 1993).
[14C]-Urea concentrations in brain, plasma, and CSF obtained from rats treated with urea
by three different exposure regimens were analyzed by Rapoport et al. (1982) using a four-
compartmental model of the central nervous system to determine the best-fit values for transfer
constants. The four intracranial compartments used in the model were capillary blood plasma
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(compartment 0), brain extracellular fluid (compartment 1), brain intracellular fluid or bound
space (compartment 2), and CSF (compartment 3). Steady-state urea concentrations for plasma,
CSF, and brain tissue of nephrectomized rats dosed by i.p. injection, previously published by
Johanson and Woodbury (1978), were used for comparison (see Section 3.2). Urea
concentrations in samples from male Osborne-Mendel rats (250-350 grams) infused with
[14C]-urea (0.07-3.7 MBq [2-10 mCi]/mM; dose not reported) into the femoral vein at a constant
rate were determined 10, 20, and 40 minutes after start of the infusion. Samples following a
single i.v. bolus were taken at 10, 20, and 30 minutes post injection. The transfer constants (k)
and other parameters derived from all three datasets were found to be consistent among the three
dosing regimens and to agree with published values (when available). The brain/plasma
distribution constant for the Johanson and Woodbury (1978) study was 1.1 x 10 4/second
compared to the constants calculated from the six tissue sites in the infusion (0.6-1.1 x 10"
4/second) and bolus study (0.7-0.9 x 10 4/second). The steady-state concentration ratios
(.ki/lt2 * + k_=, where kj = the transfer constant for the exchange between cerebral capillary plasma
and extracellular brain space, &2* = &2 x kj adjusted for blood flow rate Iki, ki = the transfer
constant for exchange between extracellular brain space and capillary plasma, and ks = the
transfer constant for the exchange between extracellular brain space and CSF) for the three
regimens were 0.19, 0.10-0.17, and 0.09-0.19, respectively, which were comparable to values of
0.15-0.25 reported in the literature. The brain/CSF distribution constant, calculated from the
Johanson and Woodbury (1978) data, was 2.0 x 10 4/second, consistent with the dispersion of a
water-soluble nonelectrolyte like urea that can diffuse through the brain primarily via the
aqueous intercellular matrix. Overall, the results presented in this study indicate that a four-
compartmental model can be used to calculate transfer constants between plasma, brain
intracellular or extracellular fluid, and CSF. All of the equations used for fitting data to a
nonlinear least-squares regression and for calculating transfer constants are described in detail in
the paper (Rapoport et al., 1982).
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY AND CASE REPORTS
4.1.1. Oral Exposure
No epidemiologic studies on oral exposure to urea were identified. However, there are
some volunteer or accidental exposure studies on urea toxicity after oral ingestion. In studies by
Eknoyan et al. (1969), blood from 26 patients with renal disease was analyzed to assess the role
of urea in the pathogenesis of thrombopathy observed in renal failure. The patients were divided
into two groups, 10 who had bleeding complications and 16 who did not. A variety of platelet
function studies were conducted in both groups including platelet counts, bleeding time, clotting
time, prothrombin time, prothrombin consumption, thromboplastin generation, platelet
adhesiveness, and activated partial thromboplastin time. Review of the publication did not
provide information on the human subjects research ethics procedures undertaken in this study,
but there is no evidence that the conduct of the research was fundamentally unethical or
significantly deficient relative to the ethical standards prevailing at the time the research was
conducted.
According to the authors, the most consistent difference observed between the two
groups was a reduction in the platelet adhesiveness. Comparison of the two groups showed that
platelet adhesiveness was significantly lower in patients with bleeding than in those without
bleeding: mean platelet adhesiveness was 4.2 ± 7.4% in the bleeding group compared to 22 ±
17% in the non-bleeding group (p < 0.01) (below 20% was considered abnormal). Compared to
a control value of five minutes, seven of the nine evaluated bleeders had longer bleeding times
(5.5-10 minutes) while only three non-bleeding patients showed longer bleeding times (5.5-
10 minutes). Overall, prothrombin consumption was only decreased in individuals with
bleeding. Evaluation of the relationship between platelet adhesiveness and concentration of
serum urea nitrogen (SUN) concentrations showed an inverse correlation. Platelet adhesiveness
was decreased when SUN concentrations were > 1 mg/mL (p < 0.01). Additionally,
experimental azotemia was induced in 10 normal subjects. The subjects ingested 2-3 g/kg-hour
urea. Serum urea nitrogen concentrations of 0.06-1.2 g/ L were maintained for 24 hours in six
subjects. In the remaining four subjects, similar SUN concentrations were maintained for 8-
10 hours. As observed in the patients with renal disease, platelet adhesiveness was reduced.
Among the group in which SUN concentrations were maintained for 24 hours, 83% (5/6)
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exhibited decreased platelet adhesiveness after urea treatment. Differences in platelet
adhesiveness percent before and after administration ranged from 18 to 38%. Of the group in
which urea concentrations were maintained for 8 to 10 hours, 75% (3/4) also exhibited decreased
platelet adhesiveness after urea treatment. Differences in platelet adhesiveness percent before
and after administration ranged from 2 to 60%. The authors concluded that urea or a urea
metabolite may play a role in development of thrombopathy observed in renal failure.
Bensinger et al. (1972) evaluated the effect of oral administration of urea on erythrocyte
survival in patients with sickle cell disease. Review of the publication does not provide
information on the human subjects research ethics procedures undertaken in this study.Eight
African-American subjects (six males and two females, 19-53 years old) with sickle cell disease
ingested 8 to 40 grams urea two to five times per day (total of 40-120 g/day) for at least
51 32
three weeks. Autologous erythrocyte survival was measured by [ Cr] and/or DF[ P], Studies
indicated that urea administration increased RBC half-life on average by 1.2 days, which was not
significant. The authors noted that isotopic techniques used in this study may not have been
sensitive enough to assess slight changes in hemolysis.
A report by Steyn (1961) described an outbreak of accidental poisoning among 80 farm
workers presumed to be exposed to a fertilizer containing 98% urea. The workers developed
symptoms three to five hours after exposure. The first symptoms were nausea and persistent
vomiting. This was followed by excitement and convulsions accompanied by urination. The
symptoms were similar to those observed with strychnine poisoning. None of the patients died
and all of them completely recovered within a few days. No quantitative data related to urea
concentrations in the patients were provided. The author stated that, in a confirmatory
experiment using rabbits, the suspected fertilizer was approximately three times more toxic than
British Pharmacopoeia-quality urea, but the postmortem symptoms were similar for both agents.
4.1.2. Inhalation Exposure
Studies have been conducted to assess the impact of inhalational exposure to urea, urea-
based formulations and products, and urea-containing mixtures. The following sections discuss
studies and results from retrospective assessments, experimental studies, and case reports.
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4.1.2.1. Cohort Studies
El Far et al. (2006) evaluated the impact of occupational exposure to industrial
environmental chemicals, including urea, on liver and kidney function and on the levels of three
biomarkers of carcinogenesis, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and
prostate-specific antigen (PSA). Male workers that were negative for the hepatitis C virus and
hepatitis B surface antigen were included in the study. One study group consisted of eight
workers exposed to urea for an average length of eight years, while another group consisted of
13 workers exposed to mixed vapors (phenol, formaldehyde, and urea) for an average length of
13.5 years. The average length of exposure was eight hours per day. Fifteen subjects not
exposed to urea were used as controls. Exposure concentrations were not quantified.
As shown in Table 4-1 liver and kidney function tests indicated that urea exposure, alone
or in combination with other industrial chemicals, significantly increased serum aspartate
aminotransferase (AST) and alanine aminotransferase (ALT) levels. Increases in blood levels of
CEA and PSA were also statistically significant. It should be emphasized that all biomarker
levels, even the ones showing significant changes, were still within normal physiological ranges
(Halstead, 1976, Gomella and Haist, 2004).
Table 4-1. Liver and kidney function tests from workers exposed to urea and
urea-containing mixtures
Parameter
Controls
Urea
Urea mixture
Normal physiological ranges
AST (U/mL)
27.86 ±2.2
32.00 ±4.42a
33.07 ± 5.66b
15 - 40 d
ALT (U/mL)
28.53 ± 1.5
33.88 ±6.66"
33.92 ±8.22"
15-35 d
Creatinine (mg%)
0.86 ± 0.12
0.75 ± 0.09a
0.84 ±0.26
<1.2 (males)6
<1.1 (females)e
Serum CEA
0.78 ±0.24
2.13 ± 1.2°
2.15 ± 1.06°
< 3 (nonsmokers)e
(ng/mL)
< 5 (smokers)e
Serum AFP
1.39 ±0.26
1.54 ±0.74
1.71 ±0.75a
< 16 e
(ng/mL)
Serum PSA
0.75 ±0.2
0.43 ±0.23b
0.66 ±0.33
< 4 e
(ng/mL)
Significantly different from control values,/) < 0.05.
bSignificantly different from controls,/? < 0.01.
°Significantly different from controls,/) < 0.001.
d-Halstead (1976)
e Gomella and Haist (2004)
Source: El Far et al. (2006).
Bhat and Ramaswamy (1993) evaluated lung function in workers at a fertilizer chemical
plant. Thirty subjects worked at a urea plant while 68 subjects of comparable body surface area
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and with the same socioeconomic status and gender served as controls. All participants were
nonsmokers and appeared to be in good health. Lung function was measured using a spirometer.
Participants forcibly exhaled into the spirometer, from the standing position, after taking the
deepest breath possible. The parameters evaluated were forced vital capacity (FVC)1, forced
expiratory volume in one second (FEVi)2, and peak expiratory flow (PEF) rate per minute
(PEFR/min)3 (Pagana and Pagana, 2003). Occupational exposure to urea decreased the
PEFR/min when compared with controls (306.9 ± 18.8 L/min vs. 383.3 ± 7.6 L/min; p < 0.001)
but did not affect FVC or FEVi, which are screening markers for obstructive or restrictive
pulmonary effects.
Marsh et al. (2002) evaluated a cohort of 995 workers (93.2% white) whose work
histories included exposure to nitrogen products (specifically, nitric acid, ammonia and urea)
(27,666 person-years). The nitrogen products cohort consisted of workers exposure to nitrogen
products only, nitrogen product followed by acrylonitrile, and potential intermittent exposure to
nitrogen products. Cohort mortality from bladder cancer was compared to bladder cancer deaths
from a local seven-county area to compute expected numbers of deaths. The SMR for bladder
cancer in the nitrogen product-exposed cohort, based on four bladder cancer cases was 3.31
(95% CI: 0.90-8.47). When workers with potential intermittent exposure to nitrogen products
were removed the SMR increased to 3.77 (95% CI: 1.03-9.65) in a reduced subcohort of 820
workers. Effects based on urea exposure alone were not derived. The authors noted that a low
incidence of bladder cancers deaths - four in the full and the reduced cohorts - and the mixed
chemical exposure limited the power of the study, even suggesting that the bladder cancer excess
may be due to occupation exposure prior to employment in the nitrogen products division.
4.1.2.2. Experimental Studies
Lung function in symptom-free asthmatic subjects was evaluated after inhalation of urea
aerosol (urea was tested for asthma-inducing potential) (Cade and Pain, 1972). A total of 56
subjects (32 males and 24 females, 16-78 years, average 42 years) were evaluated. Criteria for
inclusion in the study were (1) clinical episodes of dyspnea with wheeze, (2) responsiveness of
symptoms to bronchodilators, (3) intervals of complete remission, (4) absence of chronic disease,
and (5) absence of complicating factors (e.g., localized disease). Review of the publication does
1 The FVC is the total amount of air that can be forced out of the lungs after taking the deepest breath possible.
2 FEVj is the amount of air that is forced out of the lungs during the first second of the FVC.
3 The PEFR/min is the maximum speed at which air moves out the lungs during forced expiration
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not provide information on the human subjects research ethics procedures undertaken in this
study.Urea aerosol was inhaled as a 4 M solution from a nebulizer for 10 minutes. Spirometric
measurements of vital capacity (VC) and FEVi were made. Peak expiratory flow was measured
using an air flow meter. Additional measurements included functional residual capacity, residual
volume, total lung capacity, tidal volume, and respiratory rate. Measurements (spirometric and
lung volume) were taken before and tow minutes after urea exposure. Overall, urea inhalation
produced mild and variable impairments of VC (decrease 13 ± 17%; p < 0.001) and FEVi
(decrease 12 ± 20%; p < 0.001). The changes in the other evaluated parameters were not
significant and there was no significant correlation between individual initial and postexposure
values of VC and FEVi, respectively.
4.1.3. Dermal Exposure
Disparate results were reported for the skin-irritating effects of urea after dermal
administration. Two of four studies showed that dermal application of urea produced no skin
irritation (Serup, 1992; Gollhausen and Kligman, 1985). In studies by Serup (1992), forearms of
healthy volunteers were treated with two applications of 3% (22 volunteers: 5 males and
17 females) or 10% (23 volunteers: 7 males and 16 females) urea creams daily for three weeks.
The composition of the vehicles for the two creams evaluated is provided in Table 4-2.
Table 4-2. Composition of 3% and 10% urea creams used for assessment of
urea skin-irritating effects
Component
3% Urea cream
10% Urea cream
Lactic acid
1.5%
5%
Betaine
1.5%
5%
Additional
components
Propylene glycol, mineral oil,
polyethylene glycol 5-stearyl stearate,
ethylhexyl ethylhexonate, steareth-21,
cetearyl alcohol, self-emulsifying glyceryl
stearate, tromethamine, fragrance, water
Propylene glycol, cetearyl alcohol,
ethylhexyl ethylhexonate tromethamine,
self-emulsifying glyceryl stearate,
diethanolamine-cetyl phosphate,
demethicon, fragrance, water
Source: Serup (1992).
The total lipid content of the 3 and 10% creams was 14 and 6%, respectively. The pH of both
creams was 3.5. The contralateral side served as the control. Cream dose was controlled by the
users; mean amount of test cream used per application was 0.021 g/cm2. The final application of
the cream was made 12 hours prior to evaluation. Review of the publication does not provide
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information on the human subjects research ethics procedures undertaken in this study, but there
is no evidence that the conduct of the research was fundamentally unethical or significantly
deficient relative to the ethical standards prevailing at the time the research was conducted.
Urea-induced irritation was noted based on visual inspection and effects on barrier function were
assessed by transepidermal water loss. The hydration of skin was assessed by electrical
capacitance and conductance. Urea did not induce skin irritation at the concentrations tested and
it did not produce any changes in transepidermal water loss or induce inflammation.
Gollhausen and Kligman (1985) placed closed chambers on the forearm of four young
adult Caucasian volunteers. Review of the publication does not provide information on the
human subjects research ethics procedures undertaken in this study, but there is no evidence that
the conduct of the research was fundamentally unethical or significantly deficient relative to the
ethical standards prevailing at the time the research was conducted. The chambers were filled
with nonwoven cotton disks and 60% aqueous urea solution and mounted in a fashion such as to
apply pressure to the exposed skin. Controls exposed to urea in the absence of pressure were
also evaluated. Forearms were exposed to urea for three days and were examined 30 minutes
after removal. Urea did not induce skin irritation at the concentrations tested, based on visual
inspection for urea-induced irritation.
Two other studies reported that formulations containing 20% urea produced edema and
skin irritation (Agner, 1992; Fair and Krum, 1979). In Agner (1992), 20% urea in water or
petrolatum was applied to the upper arm of 17 healthy volunteers (13 males and 4 females) using
Finn chambers or Scanpore tape. Chambers with the vehicles (water or petrolatum) served as
controls. The authors noted that informed consent was obtained from all the participants and that
the study was approved by the local medical ethics committee. Chambers that had been attached
to the skin to allow contact with the test material were removed after 24 hours. Test sites were
evaluated prior to patch testing and after 24- and 48-hour exposures. Erythema was visually
scored on a scale of 0-3, inflammatory responses were assessed superficial blood flow by laser
Doppler flowmeter and edema formation by ultrasound A-scan, and barrier function was
assessed by transepidermal water loss. Urea in petrolatum produced visible reactions that were
more pronounced than the reactions observed with urea in water. Erythema scores of 1 or 2 were
observed in 10 of 17 volunteers exposed to urea in petrolatum; comparatively, 3 of 17 volunteers
had a score of 1 or 2 when exposed to urea in water (p < 0.001). Petrolatum and water controls
had no scores of 1 and two scores of 2. Urea in petrolatum increased blood flow and produced
edema after 24-hour exposure compared with pre-application and control values (p < 0.01) [data
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provided in figure in Agner (1992)]. Transepidermal water loss also significantly increased after
24 hours (p < 0.01) [data provided in figure in Agner (1992)]. The changes were transient and
values returned to control levels within 24 hours. Urea in water produced edema after 24-hour
exposure. However, the increase was not significant when compared to controls [data provided
in figure in Agner (1992)].
Fair and Krum (1979) applied 0.3 grams of a 10% urea base and 20% urea cream (with
nonlipid emollients) daily to a paraspinal area of the skin on 16 male volunteers. Substances
were applied for 21 days using a closed patch system. Irritation was evaluated 30 minutes after
each daily exposure period ended. The authors noted that informed consent was obtained from
all the participants. Urea-induced irritation was based on visual inspection (score from 0 to 4).
The 10% urea base produced no irritation (average cumulative irritancy score = 0). By
comparison, 20% urea cream was shown to be one of the most irritating substances tested. The
cumulative irritancy scores for the 20% urea cream ranged from 7.5-43.5. The authors
hypothesized that the increased irritation was due to the greater protein denaturing effect of the
higher percentage of urea under occlusion.
Johnson et al. (1970) used a skin window technique to assess the effects of varying
concentrations of urine and its electrolytic and nonelectrolytic components (saline and urea) on
the phagocytic ability of leukocytes. Isotonic urea solutions were applied to two healthy male
volunteers. The samples were applied to the arm or forearm (no further information on location
provided) using the skin window technique of Rebuck and Crowley (1955). Review of the
publication does not provide information on the human subjects research ethics procedures
undertaken in this study, but there is no evidence that the conduct of the research was
fundamentally unethical or significantly deficient relative to the ethical standards prevailing at
the time the research was conducted. In this method, the skin was abraded with a sterile scalpel
or razor blade to expose the corium. One drop of isotonic urea was applied to the corium that
was then covered by a sterile cover slip. After 24 hours, the solutions were added again along
with a drop of India ink to assess phagocytic ability of the cells in the exudate. Cover slips were
changed at 10 hours and removed after 28 hours. Results were described in a qualitative manner.
Isotonic urea produced mixed effects on the skin of the two volunteers. While one volunteer
showed a normal response, the other showed diminished exudate (with absence of mononuclear
cells) and disruption of multinuclear cells. The hypertonic solution decreased the number of
responding cells in both volunteers and produced toxic changes (changes shown in figure by
Johnson et al., 1970). It could be inferred that the mononuclear cells were affected to a greater
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extent than the multinuclear cells. Hypotonic urea also decreased the number of responding
cells. Overall, exposure to urea seemed to decrease phagocytosis.
4.1.4. Additional Human Studies
The following studies discuss the relationship between (1) genetic variation in the urea
transporter (UT) and blood pressure, (2) plasma urea concentrations and blood pressure, and (3)
cord blood urea concentrations and intrauterine growth retardation (IUGR).
Urea transport in the kidneys, via UTs, is proposed to play a role in the maintenance of
blood pressure. The kidneys reabsorb urea to establish an osmotic gradient that aids in body
fluid volume control, which can affect blood pressure (Ranade et al., 2001). Alterations in urea
transport in the kidneys can thus play a role in modulation of blood pressure. Ranade et al.
(2001) evaluated whether single nucleotide polymorphisms (SNPs) in the human UT-2 gene are
associated with blood pressure variations. The authors noted that the study protocol was
approved by all the institutional review boards of all the participating sites and that all of the
participants provided written informed consent. Of the seven SNPs identified, two (Val227Ile
and Ala357Thr) were shown to be associated with lower blood pressure in men. The authors
noted that Val227 and Ala357 are evolutionarily conserved (Val227 in rats, rabbits, and humans
and Ala 357 in rabbits and humans), suggesting that they play a role in transporter function.
Altered protein folding and transporter function are two proposed mechanisms by which the
identified SNPs may alter transporter activity. The Ile227 and Ala357 alleles were shown to be
associated with low diastolic blood pressure in men (odds ratio [OR] 2.1, CI = 1.5-2.7, p < 0.001
and OR 1.5, CI = 1.2-1.8,p < 0.001, respectively). These alleles were also associated with
lower systolic blood pressure (OR 1.7, CI = 1.2-2.3,p = 0.002 and OR 1.3, CI = 1.1-1.6,
p = 0.007, respectively). The authors noted that if the p values had been corrected for multiple
testing, the hazard ratios would likely have been only marginally significant. This relationship
between Ile227 and Ala357 SNPs and diastolic and systolic blood pressures was not observed in
women; the reason for this is unclear. These studies suggested that urea-induced effects on
blood pressure may be modulated by the presence of SNPs in the UT gene.
Bulpitt and Breckenridge (1976) evaluated the relationship between plasma urea
concentrations and blood pressure levels. In a group of 253 patients with essential hypertension,
the average plasma urea concentrations were significantly higher in (a) men (380 mg/L
[6.35 mmol/L], n = 86) than in women (330 mg/L [5.55 mmol/L], n = 90; p < 0.001), and (b)
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men with secondary hypertension (430 mg/L [7.13 mmol/L], n = 33) compared to those with
essential hypertension (380 mg/L [6.35 mmol/L], n = 86;p < 0.05). A group of 1,217 patients
(614 men and 603 women) was also evaluated to assess the relationship between age, urea
concentration, and blood pressure on presentation. As shown in Table 4-3, the mean plasma urea
concentrations increased with the mean blood pressure for both sexes.
Table 4-3. Relationship between mean blood pressure and plasma urea concentrations
Sex
Mean blood pressure (mm Hg)
<130
<140
<150
<160
<170
<180
>180
Blood urea levels (mmol/L)a
Male
5.65 (86)
6.07 (89)
6.35 (98)
6.57 (100)
7.16 (100)
8.34 (68)
8.55 (73)
Female
5.24 (105)
5.47 (100)
5.28 (102)
6.35 (88)
5.78 (93)
6.18 (67)
6.85 (48)
aValues in parentheses are numbers of patients in the respective blood pressure groups.
Source: Bulpitt and Breckenridge (1976).
McKay and Kilpatrick (1964) evaluated the relationship between plasma urea
concentrations and birth weight. Studies in 106 infants (single births) showed that mean cord
plasma urea concentrations were significantly increased (p < 0.02) in those infants that were
categorized as showing IUGR. Mean cord plasma urea concentration in 16 infants classified as
IUGR was 232 mg/L. By comparison, mean cord plasma concentration in 90 normal infants was
186 mg/L. Further studies showed that when the infants were divided into birth weight groups,
urea concentrations overall were shown to decrease with increasing birth weight (31-71 ounces:
218 mg/L; 72-79 ounces: 207 mg/L; 80-87 ounces: 206 mg/L; 88-103 ounces: 190 mg/L;
104-119 ounces: 145 mg/L; 120-152 ounces: 188 mg/L). Evaluation by analysis of variance
showed that plasma concentrations for the birth weight range of 104-119 ounces were
statistically different from the remaining weight groups (p < 0.05). The urea concentrations
were adjusted to a common gestation length of 40 weeks to control for varying gestation lengths
because it was observed that umbilical cord plasma urea concentrations increased with
gestational duration. Using a multiple range test, a comparison between any two of the six
adjusted means was conducted to assess significance. When controlled for gestation length,
differences in mean urea concentrations between the birth weight groups became highly
significant (31-71 ounces: 251 mg/L; 72-79 ounces: 218 mg/L; 80-87 ounces: 214 mg/L; 88-
103 ounces: 195 mg/L; 104-119 ounces: 148 mg/L; 120-152 ounces: 184 mg/L). Results
showed that birth weight over 88 ounces was associated with mean urea concentrations that were
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significantly lower than urea concentrations associated with birth weight under 72 ounces
(p < 0.05). Additionally, plasma urea concentrations were estimated for 27 pairs of twins.
Comparison of twin birth weights and urea concentrations did not show an association between
weight and urea concentrations (heavier twin: 93.35 ± 20.11 ounces, plasma urea
concentrations: 236 ± 65 mg/L; lighter twin: 80.00 ± 17.13 ounces, plasma urea concentrations:
241 ± 57 mg/L).
Al-Homrany (2001) noted that the serum activities of a variety of enzymes (creatinine
kinase, lactic dehydrogenase, alkaline phosphatase, alanine and aspartate transaminases, and
gamma-glutamyl transpeptidase) are elevated in dialysis patients but it is not known whether this
is a direct effect of blood urea levels or an indicator of toxicity. Serum samples were obtained
from two different groups, 100 hemodialysis patients and 80 control patients with normal renal
function but high serum enzyme levels. Review of the publication does not provide information
on the human subjects research ethics procedures undertaken in this study, but there is no
evidence that the conduct of the research was fundamentally unethical or significantly deficient
relative to the ethical standards prevailing at the time the research was conducted. For the
hemodialysis patients, a portion of the sample was dialyzed against 200 mL phosphate buffered
saline to remove endogenous urea. For the control patients, 10 mL of concentrated urea solution
(10%) was added to a portion of the sample to increase urea concentrations by 3 mg/mL. Studies
showed that removal of urea from hemodialysis patients increased lactic dehydrogenase activity
significantly (7793.71 ± 3482.09 vs. 10696.79 ± 12905.51 units/mg serum protein; p = 0.0007)
but the other enzyme activities were not significantly changed. Urea addition decreased gamma-
glutamyl transaminase activity fivefold (4418.67 ± 9664.20 vs. 871.15 ± 1368.52 units/mg serum
protein after addition; p = 0.04) and increased alkaline phosphatase activity slightly (1792.42 ±
1712.40 vs. 1761.23 ± 1686.31 units/mg serum protein; p = 0.05). The author emphasized the
importance of recording blood urea concentrations when assessing serum enzyme activities for
diagnostic purposes.
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4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INJECTION
4.2.1. Oral Exposure
4.2.1.1. Subchronic Studies
No subchronic exposure studies were identified that addressed the toxic effects of urea in
animals via the oral route.
4.2.1.2. Chronic Studies
A chronic carcinogenicity assay was conducted on urea by the National Cancer Institute
(Fleischman et al., 1980). Urea was administered in ground feed to male and female F344 rats
and C57BL/6 mice 7 days a week for 12 months. Technical grade urea was supplied by Aldrich
Chemical Company, Inc., Milwaukee, WI; chemical purity was not noted in this report. Feed
(Wayne Lab Blox, Allied Mills, Inc., Chicago, IL) in meal form was administered ad libitum
alone or in combination with urea. Male and female rats and mice at six weeks of age were
treated with 0.45, 0.9, and 4.5% urea in the diet (food consumption and dose per kg were not
noted). Using default values provided by the EPA (U.S. EPA, 1988), body weights for F344 rats
in a 1-year study are 0.29 kg for males and 0.175 kg for females. For C57BL/6 mice,
corresponding values are 0.0238 kg for males and 0.0206 kg for females. Food consumption is
calculated by using the formula feed intake (kg/day) = 0.065 x (body weight)0'7919. The resulting
feed intakes are 0.0244 kg/day for male rats, 0.0163 kg/day for female rats, 0.0034 kg/day for
male mice, and 0.0030 kg/day for female mice. Thus, approximate dose levels for the various
groups were 0, 379, 757, or 3,786 mg/kg-day for male F344 rats and 0, 419, 838, or 4,191
mg/kg-day for F344 females; 0, 644, 1,288, or 6,442 mg/kg-day for male C57BL/6 mice and 0,
655, 1,311, or 6,553 mg/kg-day for C57BL/6 females. Dose levels were formulated each week
and stability studies of urea mixed with feed were conducted on day 1 and day 14. Control and
treated group sizes were 50/sex for rats and 100/sex and 50/sex, respectively, for mice.
Study parameters included clinical observations, body weight taken at the start and end of
test, and gross and microscopic pathology on brain, lung, trachea, heart, thymus, pituitary,
thyroid, parathyroid, adrenal, esophagus, stomach, duodenum, jejunum, ileum, colon, liver, gall
bladder (mouse only), pancreas, kidney, bladder, gonads, accessory sex organs, spleen, lymph
nodes, bone, bone marrow, skin, salivary gland, and mammary gland. At necropsy there was no
weight depression observed in either sex of mice or rats at any of the doses. Survival was only
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affected in the 0.9% urea male rat group; these rats experienced decreased survival (89%)
compared with the control group (95%) (statistics were not noted). A statistically significant
increase in malignant lymphomas was noted only in the 0.9% dose group in female mice,
(incidences: 10/92, 7/43, 10/38 (p = 0.008), and 9/50 in the control, 0.45, 0.9, and 4.5% dose
groups, respectively). Fleischman et al. (1980) concluded that this effect was not dose dependent.
(There is a discrepancy in Fleischman et al. (1980) between the table providing the data and the
narrative. The narrative cites the number of lymphomas as shown above. The table cites the
number of lymphomas for the three dose groups as 6, 6, and 6, respectively. Since the total
number of lymphomas is based on a combination of several evaluations, the total number of
animals used for histological evaluation is unknown). Among urea-exposed male rats, there was
a significant linear trend (p = 0.008) and a significant occurrence in tumor incidence (21/50,
27/48, 25/48, and 35/50) at the high dose for interstitial adenomas in the testes (p = 0.004).
(There is a discrepancy in Fleischman et al. (1980) between the table providing the data and the
narrative. The narrative cites the number of adenoma as shown above. The table cites the
number of adenomas for the three dose groups as 25/48, 25/47, and 35/50, respectively.) Since
the change in the incidence of malignant lymphoma occurrence did not show a dose response,
these results were considered by the authors to be of questionable biological significance. The
high incidence of testicular tumors also was of questionable significance since this type of tumor
can occur at a high incidence in controls. Therefore, the authors concluded that urea was not a
carcinogen when given orally in the feed.
Krishna et al. (1990) conducted a feeding study using 0, 0.5, 1.0 and 1.5% urea in the
diet of rabbits (7/group) for up to 180 days. The authors reported no clinical signs of urea
toxicity or changes in body weight in any of the treatment groups. Cellular changes were noted
in all treatment groups but no incidence data or information about food consumption were
provided.
4.2.2. Inhalation
Animal studies using the inhalation route of administration were not identified.
4.2.3. Other routes of exposure
4.2.3.1. Subchronic Studies
No subchronic exposure studies were identified that addressed the toxic effects of urea in
animals via the subcutaneous (s.c.) or i.v. route.
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4.2.3.2. Chronic Studies
A chronic study in mice was conducted by the National Cancer Institute to determine if
s.c. injection of urea causes tumors (Shear and Leiter, 1941). Twenty strain A and 10 C57BL
male mice (3-4 months old) were injected s.c. in the left flank with 10 mg urea. The amount was
progressively increased to 50 mg and repeated injections were given over an 11-month period for
a total of 800 mg. No further details were reported on the injection protocol. A total of 19 mice
survived to 12 months but at the termination of the experiment, 15 months, only five mice
remained. The authors stated that no induced tumors were observed at the injection site.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES
4.3.1. Oral Exposure
Teramoto et al. (1981) screened 11 urea compounds for developmental toxicity using
urea and thiourea as a negative controls and water as a solvent control. The other nine urea
compounds tested were 1-methylurea, 1-methylthiourea, 1-ethylurea, 1-ethylthiourea,
1,3-dimethylurea, 1,3-dimethylthiourea, 1,1,3,3-tetramethylurea, 1,1,3,3-tetramethylthiourea, and
ethylenethiourea. Female Wistar rats (15 weeks old) or ICR mice (8 weeks old) were mated, and
the day of vaginal plug detection was designated as gestational day (GD) 0. A single
2000 mg/kg dose of urea was administered to pregnant rats (n = 4) at GD 12 and pregnant mice
(n = 10) at GD 10. Vehicle control animals (17 mice and 6 rats) were dosed with an equivalent
volume of water. No maternal toxicity was noted. Rats were killed on GD 20 and mice on
GD 18. Number of implants and number of live and dead fetuses were counted. Living fetuses
from each litter were divided into two groups after being weighed individually and examined for
gross abnormalities. Fetuses from the right uterine horn were processed for skeletal examination
and those from the left horn were processed for visceral examination. For statistical
comparisons, the litter was considered the experimental unit. There were no statistical
differences between the vehicle control and the urea-treated rats based on the mean ± SD for
number of implants (13.7 + 1.0 vs.13.8 ± 2.2), number of live fetuses (13.3 ± 0.8 vs. 13.8 ± 2.2),
percent fetal resorptions (2.4% vs. 0%), fetal body weight (3671 ± 197 mg vs. 3626 ±104 mg),
or percent fetuses malformed (0% vs. 1.8%). These endpoints also were unaffected in mice
treated with urea (data not shown).
Seipelt et al. (1969), translated from German, investigated the effect of urea, added to the
dam's diet, on fetal kidney weights. This experiment was based on results from a previous study
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by MacKay et al. (1931) who reported that addition of urea to the diet of adult male rats for 26
and 54 days increased renal weights. In the Seipelt et al. (1969) study, pregnant albino Wistar
rats were divided into test and vehicle control groups (six/group). No additional information on
mating procedure was provided. The test group was dosed, by gavage, with urea for 14 days
starting six days after the last estrus. Urea was dissolved in water and administered in two doses
totaling 50 g/kg-day. No maternal toxicity was reported. Within 48 hours of birth, pups were
sacrificed by decapitation and kidneys removed and weighed; the right kidney was then dried at
105°C and weighed. A total of 39 and 34 pups were delivered in the test and control groups,
respectively. The authors used the total number of fetuses per group for statistical comparison.
The fresh weight for the test group (n = 39) was 7.76± 1.33 g (mean ± SD) compared with 8.01
± 0.88 g for the control group (n = 34). Dry weights, reported as a percentage of the fresh
weights, were 14.4% ± 2.54% (-1.12 g) for the test group compared with 14.7% ± 1.96% (-1.18
g) for the control group. There was no statistical difference in the fresh or dry weight of the
kidneys from the test group compared to the vehicle controls.
High plasma urea nitrogen (PUN) concentrations of urea have been associated with
decreased fertility in dairy cows. This effect was demonstrated in lactating cows and dairy
heifers fed a diet high in crude protein to evaluate elevate PUN (Rhoads et al., 2006). Lactating
Holstein dairy cows (n = 23) between 50 and 120 days in milk were used as donor cows and
were given isoenergetic diets for 30 days, resulting in either moderate (<190 mg/L) or high
(>190 mg/L) PUN. The crude protein content of the two isoenergetic diets was 15.7 and 21.9%,
respectively. Estrus was synchronized in these donor cows with an injection of gonadotropin-
releasing hormone (GnRH) followed by prostaglandin F2« (PGF20;) one week later and then
follicle stimulating hormone (FSH) nine days after estrus. PGF20; was administered
simultaneously with the second to last injection of FSH and an additional dose was given
eight hours later. The donor cows were then artificially inseminated twice with semen from
high-fertility bulls 12 and 24 hours after onset of standing estrus. A third insemination was
given at 36 hours to cows still in standing estrus after the second insemination. Embryos were
then recovered from donor cows on day 7 after estrus, evaluated for quality and stage of
development, and stored in liquid nitrogen until ready for transfer into virgin heifers. Heifers
(n = 122; between 12 and 20 months old) were given either a low protein (9.6% crude protein) or
high protein (24.4% crude protein) isoenergetic diet to yield either low or high PUN
concentrations, respectively, for approximately 30 days. (There was no discussion as to why
different protein concentrations were used in the diets of the donor cows compared to the
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heifers.) As with the donors, an injection of GnRH was administered to synchronize estrus
cycles followed by PGF20; seven days later. An embryo was then nonsurgically transferred on
approximately day 7 after estrus synchronization to each heifer having a corpus luteum on one or
both ovaries (n = 57 for low protein group and n = 37 for high protein group). Pregnancy status
was examined 28 to 40 days after transfer (i.e., 35-47 days of pregnancy). Blood samples were
obtained from donor cows daily from day 0 to 7 after embryo implant and from embryo recipient
heifers daily from day 0 to 7, on day 10, then twice weekly until pregnancy diagnosis.
In donor cows, the moderate protein and high protein isoenergenic diets resulted in PUN
values of 155 ± 7 and 244 ± 10 |ig/mL (p < 0.001), respectively. Recipient heifers had PUN
values of 77 ± 9 and 252 ±15 |ig/mL for the low protein and high protein diets, respectively
(p < 0.001). Grade, stage, and number of embryos collected (n = 41 from moderate donor cows
and n = 55 from high donor cows) were not affected by the PUN level of the donor cows.
Additionally, embryo recovery and quality were similar in the two groups and were not affected
by sire (data not provided). Pregnancy rates were similar between recipient heifers on the low
protein and high protein diets (21 and 23%, respectively). However, the transfer pregnancy rates
from donor cows on a high protein diet (high PUN) were lower (11%) than those from cows on a
low protein diet (moderate PUN) (35%), independent of the recipient heifer's diet (p < 0.02).
Plasma progesterone concentrations were not significantly different between heifers with low or
high PUN (data not provided). The authors concluded that high PUN concentrations alter the
viability of the bovine oocyte or embryo prior to day 7 of pregnancy (Rhoads et al., 2006).
Results from a similar study by Ordonez et al. (2007) showed that high urea
concentrations in the serum of pasture-fed dairy cows did not affect reproductive performance.
Spring-calved Holstein-Friesian cows (20/group) were grazed for 101 days on either five pasture
paddocks without fertilizer (controls) or on four paddocks to which supplementary urea nitrogen
fertilizer (approximately 40-50 kg nitrogen/hectare [1 hectare is approximately 2.5 acres] was
added every 4-6 weeks, 1-3 days after grazing). The amount of fertilizer varied during the study
to maintain a significant difference (p = 0.05) in crude protein content of pastures between
treatments. Control animals (n = 20) were grazed on similar paddocks that had no application of
fertilizer during the same period. Cows were weighed and body condition was assessed weekly;
at the same time, blood samples were collected from the tail vein for urea measurement. Milk
samples were collected every second day before the morning milking throughout the study for
progesterone determination. Ovarian activity was assessed using ultrasound every other morning
(after milking) and categorized into one of three periods: the first to assess follicular dynamics
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during the resumption of ovarian activity, the second to monitor the development of the
dominant follicle that would lead to ovulation, and the third to observe corpus luteum
development and establishment of pregnancy. Serum urea concentrations were significantly
higher in the animals on urea-fertilized pastures than those on control pastures (mean 500 vs. 325
mg/L [8.3 vs. 5.4 mmol/L]; p < 0.001). Change in body weight over time was not significantly
different (p > 0.40) between treated cows and control cows; however, treated cows did show
lower body weights than control animals during weeks 6-9 of the study [data provided in figure
in Ordonez et al. (2007)]. The authors reported no difference between groups for the following
ovarian parameters: intervals between calving and first estrus (25.8 ± 2.5 days for treated cows
vs. 31.9 ± 2.7 days for controls), emergence of first ovulated follicle and its ovulation (7.0 ±
0.8 days vs. 7.0 ± 0.9 days), maximum diameter of first dominant follicle to be ovulated (20.0 ±
0.1 mm vs. 19.0 ±0.1 mm), and emergence of follicle of conception cycle to ovulation (6.0 ±
0.8 days vs. 5.8 ± 0.7 days). The maximum diameter of dominant follicles resulting in
conception, however, was higher in control cows compared with cows grazing on pastures
fertilized with urea (20.2 ± 0.1 mm for controls vs. 17.1 ± 0.1 mm for treated cows; p = 0.02).
The numbers of luteal phases <10 days and >10 days during the study were identical for both
groups of cows. Additionally, milk progesterone concentrations did not differ between the two
groups (178.6 ± 17.5 ng/mL for treated cows vs. 155.4 ± 7.5 ng/mL for controls). Overall, the
authors showed that there were no negative effects on the reproductive performance of dairy
cows that ate in pastures supplemented with urea nitrogen fertilizer compared with control cows.
Treated and control cows exhibited similar intervals from calving to second estrus (29.5 ± 3.6 vs.
25.6 ± 3.2 days), from calving to first insemination (82.8 ± 2.5 vs. 85.1 ± 1.8 days), and from
calving to conception (87.2 ±1.9 vs. 88.3 ±1.9 days, respectively). The period from calving to
first estrus, however, was higher in control animals compared to treated cows (58.7 ± 5.4 and
54.3 ±3.7 days, respectively). In addition, services per conception were not significantly
different (1.35 ± 0.1 for treated cows vs. 1.19 ±0.1 for controls).
4.3.2. Intrauterine, Intraperitoneal, or Intravenous Exposure
Conner et al. (1976) tested the efficacy of urea as a contragestational agent in rats.
Female Sprague-Dawley rats (200-250 grams) were mated at the supplier's facility and
conception monitored by the presence of a vaginal plug (day 1 of pregnancy). Animals were
shipped to the laboratory on day 2. Dams were injected on GD 3 or GD 7 with 0.9% NaCl into
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one uterine horn (0.05 mL) and either 29 (58 mg/kg) or 58% (116 mg/kg) urea (w/v) into the
other horn (0.05 mL; n = 4-6/group). Implantation sites were counted before injection on GD 7
of pregnancy. Rats were killed on GD 15 and corpora lutea, resorptions, and conceptuses
counted. The authors reported that the animals injected on GD 3 with 58% urea had
approximately 1% viable fetuses, while animals injected with 29% urea had approximately 40%
viable fetuses. By comparison, 80-85% fetus viability was observed when 0.9% NaCl was
injected into uterine horn (statistics not reported; numbers estimated from figures in report).
However, when injected on GD 7, the effect of urea injection was comparable to that of 0.9%
NaCl.
Blake et al. (1976) investigated the abortifacient (abortion-inducing) effect of
intraamniotic, i.v., and i.p. injections of urea into adult rhesus monkeys. Nonpregnant and
pregnant rhesus monkeys (no control animals were noted) were placed under halothane
anesthesia during the administration of the chemical. Four pregnant monkeys were administered
a single intraamniotic injection of urea (2.2, 2.3, 2.5, or 12.5 g/kg) via a transabdominal catheter.
Two pregnant monkeys were administered urea (1.6 and 1.8 g/kg) through a catheter placed into
the peritoneal cavity. A single pregnant monkey was administered urea (1.8 g/kg) by injection
into a superficial leg vein. Oxytocin, at a human abortifacient dose, was administered
immediately after all urea injections. Two nonpregnant monkeys were included in each of the
i.v. and i.p. injection groups. The sole pregnant monkey in the i.v. injection group was
euthanized four hours after injection due to nonsterile procedures. One monkey (receiving 1.8 g
urea/kg) died approximately 24 hours after i.p. injection. The authors attributed the death to
hemorrhage caused by an incomplete abortion. Death (no clinical signs described) occurred
20 hours after intraamniotic dosing of the single animal that received 12.5 g urea/kg; SUN
concentration was 14 mg/mL in this animal. There were "no serious side effects" noted by the
authors. The remaining monkeys in the intraamniotic injection group resorbed their fetus and
survived. The remaining monkey in the i.p. injection group spontaneously aborted the fetus and
survived. The nonpregnant monkeys all survived.
4.3.3. Other Studies
Urea may be transported via UTs. In mammals, two genes have been identified as UTs:
UT-A and UT-B (additional information on these transporters can be found in Section 4.5).
Previous studies have shown that UT-A5 and UT-B are localized in the testis; UT-A5 is
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expressed in the outer cell layer of seminiferous tubules and UT-B is expressed in Sertoli cells of
seminiferous tubules (Fenton et al., 2000; Tsukaguchi et al., 1997).
Guo et al. (2007) used UT-B null male mice to investigate the effect of the lack of this
protein on male fertility. Brain, liver, and testis from transgenic knockout mice (CD-I
background) deficient in UT-B protein and wild-type CD-I mice were homogenized and the urea
concentrations in the resulting centrifugation supernatants and in serum were measured by
colorimetry. Furthermore, histological examination of one testis from each animal was
performed at selected ages from 10-84 days. Urea concentrations (as measured in 84-day-old
mice) were statistically significantly higher {p < 0.01) in serum and testis from UT-B null mice
(9.3 ± 0.6 mM and 57.5 ± 2.6 mmol/kg tissue weight, respectively) than from wild-type mice
(7.6 ± 0.1 mM and 46.9 mmol/kg tissue weight, respectively), but not in the brain and liver.
Total testis urea content was 335.4 ± 43.8 |ig in UT-B null mice and 196.3 ± 18.2 |ig in wild-
type mice (p < 0.01). Testis weights in UT-B null mice were statistically significantly higher
(p < 0.05) than in wild-type animals from postnatal day (PND) 17 throughout the experimental
period. On PND 84 testis weights were 103.7 ± 6.9 mg in UT-B null males compared with 80.3 ±
6.7 mg in controls (p value not provided); testis-to-body weight ratios were 0.31 ± 0.02 and 0.22
± 0.03% (p < 0.05), respectively. Water content in testes was similar in both groups of mice.
Histological examination showed there was no difference in the features or distribution of stages
of spermatogenesis between the two groups. Additionally, no differences were observed in
cellular integrity of the epithelium, sperm numbers in caudal epididymes, or sperm morphology.
Elongating spermatids were detected in all the null animals by PND 28, but they were not
detected in the wild-type animals until PND 36. Other than the detection of elongated
spermatids, there were no other differences in testicular morphology noted between the two
groups.
UT-B null and wild-type mice were also used in competing mating studies (Guo et al.,
2007). One UT-B null male and one wild-type male (35 days old) from the same litter were
mated with one 70-day-old wild-type female. In the control groups, two wild-type males were
mated with one wild-type female. The litter size and gender of the pups were noted and all pups
were genotyped. The time to first litter in null mouse groups (n = 7) was 69 ± 3 days, which was
notably earlier than 11 ±2 days in control groups (n = 7; statistics not reported). All pups in the
competing groups were UT-B heterozygotes, suggesting that they were sired by the UT-B null
male and not by the wild-type male. The number and gender of pups in the test and control
groups were similar. The authors estimated the average mating age of males, based upon time to
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first litter, as 48 ± 3 days and 56 ± 2 days for test groups and control groups, respectively. These
data supported the conclusion that testicular development occurred earlier in UT-B null mice. In
addition, earlier Sertoli cell development was observed in UT-B null mice, as indicated by the
occurrence of significantly higher FSH receptor and androgen binding protein mRNA expression
levels at 10 days in null males compared with 17 days after birth in wild-type males {p < 0.01)
[data provided in figure in Guo et al. (2007)]. At 10 days of age, testis urea concentration was
significantly higher (p < 0.05) in UT-B null mice (34.3 ±1.6 mM) than in wild-type mice (31.4
±0.5 mM); the difference became more significant with age. Serum urea concentrations were
also observed to be significantly higher in UT-B null mice compared with wild-type mice at all
ages (10 to <45 days old; p < 0.05) [data provided in figure in Guo et al. (2007)]. Guo et al.
(2007) concluded that UT-B deletion resulted in urea accumulation in the testis and early
maturation of the male reproductive system.
4.4. OTHER ENDPOINT- SPECIFIC STUDIES
4.4.1. Acute Studies
A pilot study was initiated prior to the screening of 11 urea compounds for
developmental toxicity to evaluate acute toxicity in female mice and rats and establish dose
ranges for testing (Teramoto et al., 1981). ICR mice (8 weeks old) and Wistar rats (15 weeks
old) were dosed with urea by gavage. Mice (n = 3) received single 1000 or 2000 mg/kg doses of
urea. Rats (n = 4) received a single 1000 mg/kg dose. Animals were observed for clinical signs
of toxicity (e.g., diarrhea) for one week. None of the animals displayed any sign of toxicity, and
there were no deaths.
Blake et al. (1976) investigated the effect of intraamniotic, i.v., and i.p. injection of urea
into pregnant and nonpregnant adult rhesus monkeys (reproductive and developmental outcomes
discussed in Section 4.3). Monkeys (no control animals were noted) were placed under
halothane anesthesia during the administration of the chemical. Urine was collected through a
Foley catheter and the animals received a continuous i.v. infusion of 5% dextrose in 0.45%
saline. A 58% (w/v) urea solution (approximately 2 g/kg) was administered over 3.5 minutes via
intraamniotic (three pregnant monkeys), i.v. (one pregnant and two nonpregnant monkeys), or
i.p. (two pregnant and two nonpregnant monkeys) injection. An additional pregnant monkey
received an intraamniotic injection of urea solution at a dose of 12.5 g/kg. Immediately after
urea injection, 24 mU/minute of oxytocin (often used as an augmenting agent chemically) was
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added to the dextrose/saline infusion. Urea absorption and elimination were recorded by adding
radioactive urea (0.92 MBq [25 |iCi] of [14C] urea) to the injection solution. Arterial pressure,
CSF pressure, heart rate, and respiratory rate were monitored. Physiologic monitoring was
continued for four hours, after which all incisions were sutured and the animals were allowed to
recover from the anesthesia. Total voided urine was collected for seven days. Venous blood
was collected prior to the start of the experiment and at 1, 4, and 7 days after urea injection.
Hematocrit, total white blood cells, sodium, potassium, chloride, bicarbonate, glucose, and urea
nitrogen were also monitored. Animals were observed for 3-6 months after injection.
Three monkeys died during the course of the study. One pregnant monkey treated with
1.8 g urea/kg had to be euthanized four hours after i.v. dosing because of complications
following a nonsterile procedure. Another pregnant monkey died approximately 24 hours after
intraamniotic administration of 1.8 g urea/kg under signs of excessive bleeding from an
incomplete abortion. The authors considered the two deaths non-treatment related. Death (no
clinical signs described) occurred 20 hours after intraamniotic dosing of the single (pregnant)
monkey with urea solution at 12.5 g/kg; SUN concentration was 14 mg/mL shortly after death in
this animal. In comparison, average SUN concentrations shortly after intraamniotic exposure in
the remaining animals were less than 1 mg/mL and near baseline levels one day after exposure
[data provided in figure in Blake et al. (1976)].
The authors reported a rapid fall in arterial pressure after i.v. injection of hyperosmolar
urea, followed by a rise above preexposure levels, then a return to normal within 30 minutes;
consistent changes in heart rate were not noted during the same time period. Intraperitoneal
injection increased systolic and diastolic pressures during the injection procedure peaking
approximately 1.5 minutes after completion of the injection; both pressures returned to
preinjection levels within another 10 minutes [data provided in figure in Blake et al. (1976)].
Decreases of 5-15 mm Hg in CSF pressures at two hours, followed by a gradual rise to baseline
values were noted after i.v. and i.p. injections [data provided in figure in Blake et al. (1976)].
Intraamniotic injections decreased the CSF pressure by an average of 5 mm Hg at one hour,
returning to baseline values by four hours after injection. The rate of spontaneous respiration
was not affected by any of the dosing regimens used. Based on the graphs provided, urea
administration by i.v. or i.p produced a persistent decrease in serum potassium and glucose.
(The results for i.v. and i.p. injection were combined by the authors for comparison to
intraamniotic injection since absorption rate and maximal SUN concentrations after i.v. and i.p.
injections were similar.) Based on the graphs presented, intraamniotic administration decreased
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serum chloride concentrations. Hematological studies indicated that the hematocrit decreased in
all animals between days 1 and 4 (which was likely due to blood withdrawal for studies) and was
approaching preexposure levels by day 7. White blood cell counts increased (50-200% above
preexposure level) in all animals within 24 hours. White blood cell counts remained increased in
animals administered urea via i.p or i.v. injection [data provided in figure in Blake et al. (1976)].
As mentioned previously, no control animals were noted thus interpretation of treatment-related
effects is limited.
Thurston et al. (1986) evaluated the effects of acute hyperosmolar urea injection in
normal suckling/weanling mice. Sixty-five mice (17 to 23 days old, strain and sex not provided)
were treated with equimolar solutions of either 2 M urea (calculated as 7.2 g/kg) or 1 M sodium
chloride (NaCl) in two concurrent 30 mL/kg doses, one s.c. to the back and another via i.p.
injection. Weight-matched controls received equivalent volumes of 0.9% NaCl. Mice were
killed by decapitation at selected time points and blood was collected. Soon after injection, the
following behaviors were observed in the urea-treated mice: staggering, hopping, running in
circles, head shaking, walking on toes, and hypersensitivity to touch. Improvement occurred
rapidly; one hour after dosing the behavior of treated animals was indistinguishable from
controls. Treated animals lost 10% of their body weight by six hours after urea injection
compared with 5% observed in control animals; the authors noted that the initial injections had
added 6% to each animal's body weight. Hemorrhagic encephalopathy, similar to what was
observed in NaCl-loaded animals, was noted by gross evaluation of the brain one hour after urea
administration. Histological evaluations were not made.
Urea-treated mice had approximately 14% lower plasma sodium concentration within
15 minutes after injection, as compared with time zero concentrations. Concentrations began to
recover quickly, reaching ~ 7% above normal by three hours after dosing (data estimated from
figure). By comparison, plasma potassium levels were not affected during the experiment (data
not provided). Plasma osmolality was calculated based on the concentrations of sodium,
potassium, glucose, and urea. After a rapid initial increase in osmolality from 312 at 0 hours to
412 at 15 minutes to 427 at one hour, there was a steady decline to 352 mOsm/kg H20 at
six hours after injection. The osmolality measurements at 2, 3, and 6 hours after dosing were
statistically significantly lower (p < 0.01) than the peak value at one hour. Brain dehydration and
decreased brain sodium concentrations were observed in the treated animals. However, all
effects were reversed six hours after injection [data provided in figure in Thurston et al. (1986)].
Additionally, mice exhibited no changes in brain potassium concentrations (data not provided).
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The half-life (ti/2) of urea, calculated by assuming elimination by first-order kinetics, in the brain
(ti/2 = 4.7 hours) was approximately two times longer than in plasma (ti/2 = 2.2 hours). Urea
concentrations in the brain were in equilibrium with those in plasma at about 2.5 hours after
injection. As illustrated in Table 4-4, urea affected several metabolic energy-related parameters
in the brain 10 and 45-60 minutes after injection.
Table 4-4. Early and late effects of urea injection on plasma and brain
metabolite concentrations
Measurement
Early effects (10 min after injection)
Late effects (45-60 min after injection)
0.9% NaCl
(mmol/kg) (n = 6)
2 M urea
(mmol/kg) (n = 4)
0.9% NaCl
(mmol/kg) (n = 11)
2 M urea
(mmol/kg) (n = 3)
Lactate
2.37 + 0.19
3.21 +0.03a
2.60 + 0.27
1.81+0.11
a-Ketoglutarate
0.044 + 0.003
0.059 + 0.0033
0.095 +0.012
0.085 + 0.005
Malate
0.596 + 0.019
0.722+ 0.0123
0.574 + 0.030
0.530 + 0.016
Adenosine 5'-triphosphate
2.49 + 0.03
2.77 + 0.03b
2.48 + 0.03
2.81 +0.02b
Glycogen
1.42 + 0.05
1.67 + 0.05
1.53+0.08
0.96 + 0.03b
Aspartate
3.77 + 0.19
4.15 + 0.05
3.25+0.08
4.44 + 0.07b
Glutamate
12.31+0.40
14.13+0.11
11.74 + 0.13
13.45 + 0.13b
Phosphocreatine
2.88 + 0.07
3.15 + 0.10
2.40 + 0.09
3.34 + 0.02a
"Significantly different from 0.9% NaCl control,/? < 0.05.
bSignificantly different from 0.9% NaCl control,/) < 0.01.
Source: Thurston et al. (1986).
4.4.2. Short-Term Studies
Safety concerns about the consumption of urea-adulterated milk in India resulted in the
study of the possible genotoxic and toxic effects of urea (Kommadath et al., 2001). Swiss
Albino male mice (12 animals/group, 3-4 months old,) were administered urea-adulterated
cow's milk by gavage in three treatment groups: 0.73, 0.365, and 0.1825 mg urea/day for
28 days. Based on an average body weight of 25 grams, the calculated doses per treatment group
were 29.2, 14.6, and 7.3 mg/kg-day. A control group was dosed with cow's milk. Animals were
killed on treatment days 7 and 28, and kidney and liver samples were taken from three animals in
each group. Samples were fixed with 40% formaldehyde, paraffin-embedded, and stained with
hemotoxylin and eosin. Incidence data were not reported. Pathological changes were noted in
the liver and kidney of all treated animals on day 7, with an increase in severity on day 28. No
pathological changes were noted in the control group. In the liver, degenerative and necrotic
changes in hepatocytes, focal areas of necrosis, and initiation of lymphoid follicle formation was
reported. In the kidney, fatty changes in the perirenal tissue, mild necrosis, mild glomerulitis,
mild to moderate congestion, leukocytic infiltration of interstitial tissue, and lymphoid cell
aggregates in perivascular tissue were described for the animals treated with the high dose of
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urea. The severity of the renal lesions decreased with decreasing dose; animals treated with 7.3
mg/kg-day did not exhibit fatty changes and congestion was limited to larger blood vessels. The
authors concluded that urea was hepatotoxic and nephrotoxic in adulterated milk at the
concentrations tested. As the authors reported that pathological changes were noted in all treated
animal, the lowest dose of 7.3 mg/kg-day could be interpreted as a LOAEL.
Levine and Saltzman (2001) investigated whether urea is a major toxin in renal failure in
Lewis rats. Male rats (age not provided) had their right kidney surgically removed. Laboratory
Rodent Diet 5001, given ad libitum, was replaced with sucrose cubes saturated with olive oil.
One week after the first surgery, the left kidney was surgically removed and three days before
the second surgery, 120 mg neomycin and 50 mg dihydrostreptomycin were added to 100 mL
drinking water. For three days after the second surgery, treatment groups received an i.p.
injection of 2 mL/100 grams of either water (n = 8), 1.5 M urea (1,800 mg/kg) (n = 12), or
0.033 M, 0.067 M, or 0.1 M (100, 200, or 300 mg/kg) creatinine (n = 8/dose). Four animals per
group were necropsied on the fourth day and serum was obtained. Spleen and thymus were fixed
in Bouins fixative, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Urea
treatment did not affect creatinine serum concentration but creatinine treatment increased urea
serum concentration above control values (1.98 and 2.48 mg/mL with 100 and 200 mg/kg,
respectively, vs. 1.87 mg/mL with water; p < 0.01). Serum osmolality was significantly
increased after urea injection compared with water-injected controls (472 vs. 368 mOsm/kg;
p < 0.05); creatinine injections (364 and 379 mOsm/kg with 100 and 200 mg/kg, respectively;
p < 0.05) were also significantly increased. The i.p. injection of urea in nephrectomized rats also
decreased survival time (5.7 days with urea vs. 8.3 days with water and 7.0 days with no
injections), and increased atrophy of thymus and spleen (i.e., more severe atrophy of the thymic
cortex and splenic lymphoid follicles) when compared with control nephrectomized animals.
The authors stated that the elevation in serum osmolality with urea injections may be important
in uremic toxicity and in decreased survival time.
Finlayson and Baumann (1956) investigated the effect on rats fed urea mixed with a diet
given ad libitum as compared with a spaced feeding of two hours per day. Holtzman male albino
rats (21 days old) were fed a casein/corn oil diet containing vitamins and mineral supplements ad
libitum for 10 days and then habituated to a two hour per day feeding schedule for two weeks.
Five rats per group received a diet containing 0, 5, or 10% urea for two hours per day or a diet
containing 0, 20, and 30% urea ad libitum. Rats were weighed weekly and daily food
consumption was determined. After five weeks, weight gain was decreased with increased urea
concentration for both diets. A 5% urea diet administered with the spaced feeding and a 30%
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urea fed ad libitum diet decreased the growth to similar extents, even though the daily urea
intake was 12-fold greater in the latter group (0.4 g/day vs. 4.8 g/day). In a subsequent
experiment, blood urea nitrogen concentrations were compared for these two groups. Adult rats
(n = 3/group) were fed 5% urea two hours per day or 30% urea ad libitum for six days. After six
days, blood of animals fed the 5% urea diet was taken by cardiac puncture two hours after eating
and blood urea nitrogen determined. Blood urea nitrogen values were comparable between the
two treatment groups. The authors concluded that growth depression was related to the rate of
urea ingestion.
The role of urea in uremia was studied in 12 mongrel dogs (Balestri et al., 1971).
Animals had a kidney surgically removed and were allowed to recover for 10-15 days (control
period). A 10% urea solution (at a dose of 3-4 g/kg) was injected s.c. every eight hours for 30-
45 days. In four of the dogs, spontaneous movements were continuously recorded for three days
during the control period and three days during the test period. Permanent electrodes were
implanted for electroencephalogram recording in two additional dogs. Hematocrit and platelet
counts were performed in five dogs at intervals of five days. All animals were necropsied at the
end of the experiment. Gross pathology was negative and the liver, heart, kidney, stomach, and
duodenum had normal histology. Plasma urea concentrations ranged from 6-7 mg/mL at 20-
30 minutes after injection to 2-3 mg/mL just prior to the next injection. Concentrations during
the control period were not provided. Following urea injection, the only symptoms noted were
mild drowsiness and a reduction of spontaneous movements. During the test period diuresis was
increased and the animals drank more water but had no obvious gastrointestinal disturbances and
ate normally. Weights, hematocrit values, platelet counts, and bleeding times did not change
over the course of study. The authors concluded that urea does not induce severe toxicity in dogs
at blood concentrations up to 7 g/L.
In an earlier study of the role of urea in canine uremia, Grollman and Grollman (1959)
removed both kidneys from six dogs with an interval of 7-14 days recovery between each kidney
removal. One liter of commercially available sterile peritoneal lavage solution was administered
i.p. and exchanged twice a day. Urea (5-30 g/L) was added to this solution. Animals were
maintained for 4-9 days. Anorexia, weakness, diarrhea, and vomiting were early symptoms
followed by hemorrhage from the bowel, coma, and death. The authors attributed these clinical
signs to urea toxicity.
Urea toxicity in 10-week-old male cross-Landrace piglets was assessed over a 15-day
exposure period (Button et al., 1982). A single piglet was used per dose group (no control
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animals noted). Acute oral doses of urea (1-4 g/kg) were administered with a dosing gun on day
one. Two piglets received meal containing doses up to 10% urea in feed over a 15-day period.
One piglet received meal containing doses up to 5% urea in feed over a 10-day period followed
by two additional doses of urea (8 and 16 g urea/kg) by mouth on days 12 and 14. The authors
reported no diarrhea or symptoms of urea intoxication for any of the study animals.
Das et al. (1997) fed urea to day old chicks for eight weeks. Chicks (30/group) were
divided into eight treatment groups 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5% urea. Decreased body
weight was noted in chicks fed with diet containing 2 and 2.5% urea. The authors also noted
enlarged and mottled kidneys and livers. However, no incidence data is provided, nor is there
information with regard to food consumption.
4.4.3. Cardiotoxicity
Studies have evaluated the effects of urea on the cardiovascular system. Overall, the
studies indicate that urea and its metabolites have various cardiotoxic effects including protein
carbamylation, nitric oxide synthase inhibition, and mechanical and electrical alterations.
Urea may carbamylate proteins, which in turn produces a variety of toxic effects. During
carbamylation, urea is spontaneously degraded to cyanate. The active form of cyanate, isocyanic
acid, may then react with nonprotonated amino groups of proteins. The carbamylation of these
proteins can lead to altered protein structure and activity. Ok et al. (2005) evaluated whether
protein carbamylation was related to atherosclerosis. Human coronary artery endothelial cells
and human coronary artery smooth muscle cells were treated in vitro with carbamylated low-
density lipoprotein (cLDL) or native low-density lipoprotein (nLDL). Studies showed that cLDL
produces morphological changes in human coronary artery endothelial cells at concentrations
ranging from 50-400 (ig/mL after exposure for 24 hours. Many of the treated cells decreased in
size and detached from the plate. Additionally, cellular debris was observed. cLDL increased
the release of lactate dehydrogenase (marker for cytotoxicity) in a dose- and time-dependent
manner; cytotoxicity was shown to increase linearly [data provided in figure in Ok et al. (2005)].
At a concentration of 200 (ig/mL, cytotoxicity as measured by trypan blue exclusion in several
experiments was 10-20% (expressed as the ratio of lactate dehydrogenase (LDH) released by
treated cells into medium to the total LDH) with cLDL vs. 0-7% with nLDL. Endothelial cells
exposed to cLDL exhibited a higher percentage of apoptosis (as measured by annexin V binding)
than nLDL-exposed cells (24 ± 4 vs. 14 ± 3%; p 0.01) but there was no significant change in
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the percentage of necrotic cells. Further studies demonstrated that cLDL induced proliferation
(as measured by bromodeoxyuridine incorporation) of human coronary artery smooth muscle
cells. Bromodeoxyuridine incorporation showed that cLDL induced cellular proliferation at
concentrations ranging from 0-200 (ig/mL in a dose-dependent manner.
Based on prior studies that indicated that chronic kidney disease was a risk factor for
cardiovascular disease, the authors evaluated total protein carbamylation and cLDL levels in
patients with renal disease (Ok et al., 2005). Total plasma protein carbamylation and plasma
cLDL levels were measured by homocitrulline assay and ELISA, respectively. Hemodialysis
patients with advanced renal disease (n = 13) exhibited elevated levels of protein carbamylation
when compared with controls (n = 11) (42 ± 4 vs. 12 ± 3 nmol homocitrulline/mg protein;
p < 0.01). cLDL levels also were increased 267% in hemodialysis patients when compared with
controls (p < 0.001; no biological values given). Combined, the studies suggest that cLDL,
which may be formed by high concentrations of urea, produces a variety of biological effects that
may be relevant to the development of arthrosclerosis.
Studies by Moeslinger and Spieckermann (2001) showed that urea induced a dose-
dependent inhibition of inducible nitric oxide synthase (iNOS) in stimulated mouse macrophages
without any impact on cell viability. The monocyte/macrophage RAW264.7 cell line was
incubated with 0- 9g/L (0-150 mmol/L) urea for 48 hours. Cellular proliferation was assessed
by cell counting, incorporation of [ H]-thymidine, protein expression (as assessed by Western
blots), and apoptosis. Increasing concentrations of urea were associated with a dose-dependent
decrease in iNOS production in RAW264.7 cells, with decreases becoming statistically
significant at 0.36-7.2 g/L (60-120 mmol/L; p < 0.05) [data provided in figure in Moeslinger
and Spieckermann (2001)]. A concomitant decrease in cell viability was not observed (>95%
cell viability at concentrations up to 9 g/L [150 mmol/L]). While iNOS protein levels were
decreased by urea at concentrations up to 7.2 g/L (120 mmol/L) [data provided in figure in
Moeslinger and Spieckermann (2001)], mRNA levels were not affected (data not provided).
Additionally, urea was shown to significantly stimulate macrophage proliferation at
concentrations ranging from 0.36-9 g/L (60-150 mmol/L) after incubation for 48 hours (control:
2.09 ± 0.12 x 105 cells/well; 9 g/L (150 mmol/L): 3.36 ± 0.38 x 105 cells/well; p < 0.05). The
authors noted that the in vitro concentration of 360 mg/L (60 mmol/L) corresponded to a blood
urea nitrogen concentration of approximately 3.6 g/L. The authors suggested that the inhibition
of iNOS decreases nitric oxide-induced apoptosis, and may contribute to the development of
atherosclerosis due to the increased cellular proliferation of macrophages.
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A study in apolipoprotein E knockout mice (Apo E mice), which have delayed
lipoprotein clearance, was conducted to assess the impact of the uremic state on development of
atherosclerosis (Massy et al., 2005). At eight weeks of age, mice were divided into two groups,
uremic (8 males and 14 females) and non-uremic control (6 males and 19 females). Chronic
renal failure (CRF) was initiated to induce uremia by cauterizing the right kidney cortical region
of mice in the uremic group and then performing a total nephrectomy of the left kidney two
weeks later. Control animals underwent sham operation (decapsulation of both kidneys). Mice
were killed at six weeks after surgery. The authors noted that all procedures were conducted in
accordance with National Institutes of Health guidelines for the care and use of experimental
animals. Plaque area was significantly increased in the thoracic aorta (p < 0.04) [data provided
in figure in Massy et al. (2005)], but not the aortic root, six weeks after uremia was induced
when compared with nonuremic animals. Areas of athermanous and medial calcification in
uremic animals were larger than in nonuremic animals. Assessment of the composition of the
lesions in uremic animals indicated increases in collagen (control mice: 12.5 ± 1.0%; CRF mice:
18.9 ± 2.3%; p 0.012), calcite, and hydroxyapatite content [data provided in figure in Massy et
al. (2005)].
Urea was shown to produce mechanical and electrical alterations in isolated papillary
muscles and in Langendorff perfused rat hearts (Abaurre et al., 1992). EPM strain of Wistar rats
(both sexes) were placed into one of three groups. The left ventricle papillary muscles were
obtained from one group while isolated rat hearts were obtained from the other two groups and
perfused according to the Langendorff technique at a constant pressure of 75 mm Hg. A balloon
mounted on the tip of a plastic tube was placed in the left ventricle and used to modify the
diastolic pressure. Papillary muscles were exposed to test media containingl7 mM urea because
it is similar to the concentration of plasma urea (1 g/L) in patients with severe renal
insufficiency. Contraction recordings in papillary muscles were taken before exposure, during
exposure (30 minutes), and after washout of the test chemical. Urea reduced the isometric force
(77% of control; p < 0.05) and rate of force development (85% of control; p < 0.05) in the
papillary muscles. Additionally, urea was shown to decrease isovolumic systolic pressure (33%
of control; p < 0.05), but not heart rate, as measured in perfused hearts. Electrocardiographic
studies showed that 17 mM urea reduced the total QRS amplitude by 4.16 ± 1.58 mm (p < 0.05),
increased QRS duration, decreased P wave amplitude, and elevated the ST segment in a majority
of the samples evaluated. (The authors noted that the changes could not be quantified since the
changes were not uniform.) Evaluation of changes in ventricular isovolumic systolic pressure as
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a function of diastolic pressure showed that exposure to urea produced a depressant effect [data
provided in figure in Abaurre et al. (1992)]. Biochemical studies indicated that urea reduces
calcium binding to the glycocalyx outside the sarcolemma, which is proposed to decrease
contraction force.
An additional foreign language article, which had an accompanying English summary,
discussed cardiotoxic effects of exogenous urea (Cuparencu et al., 1961). This summary
reported that i.v. injection of urea (0.17-0.5 g/kg) in dogs induced a short-lasting hypertension
followed by a longer-lasting pronounced hypotension. The authors suggested that the
hypertension was induced by a vasoconstrictor reflex of the veins while the hypotension might
have been caused by an exocrine vasoactive substance. Intraarterial injection of urea at the same
doses produced an increase in blood pressure that was occasionally followed by hypotension
caused by a vasodilator reflex from the arteries. Both administration methods were shown to
induce tachyphylaxis. The authors also hypothesized that urea affected the vagal control center
in the CNS resulting in a stimulation of vasomotor and respiratory control centers, an effect that
may be masked by a urea-induced inhibitory action of the sinus caroticus.
4.4.4. Pituitary Effects
Okada and Kobayashi (1989) showed that short-term administration of urea produced
alterations in intermediate cells of the mouse pituitary. Jcl/ICR male mice (numbers not
provided) were given urea mixed in food at concentrations of 6, 12, and 24% for 14 days. Urea
produced significant (p value not provided), dose-dependent increases in protein synthesis
(287.5% of control levels at the highest dose) and decreased density of secretory granules (44%
of control levels at the highest dose).
4.4.5. Dermal Toxicity
A 24-hour dermal exposure study on skin penetration enhancers evaluated their potential
to induce skin irritation (Lashmar et al., 1989). Male MF 1 h nude mice (4 weeks old) were
exposed to a 10% w/v urea solution in water (n = 3) or control vehicle (1% w/w Carbopol 940
neutralized with sodium hydroxide). Test chemical was filled into a polyvinyl chloride cup and
fastened to the dorsum of the animal using surgical tape and Superglue then left in contact with
the skin for 24 hours. Animals were sacrificed and specimens of the exposed area and an
adjacent untreated skin area were taken for histological examination. Tissues were fixed in
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formalin, paraffin-embedded, and stained with hemotoxylin and eosin. Treated skin samples
from each animal were randomly selected and microscopically examined and scored using a
standard scoring system. The authors reported that urea did not cause any significant change in
skin histology after the 24-hour exposure period.
4.4.6. Intracranial and Intraocular Effects
Intravenous urea (30% concentration) has been used to lower the intracranial and
intraocular pressure in humans (Javid and Anderson, 1958). Javid and Anderson (1958)
investigated these effects in adult female rhesus monkeys to determine the optimal rate of
administration of a lower urea concentration and the effect of s.c. injections. Animals (average
body weight 4.5 kg) were under Nembutal anesthesia during the study period. In the first set of
experiments, CSF pressure was measured; urea doses of 1 g/kg were injected i.v. as 2.5, 5, 10,
and 30% solutions at varying rates (15-120 minutes). Decreases in CSF pressure were found to
be proportional to the rate of urea administration, but not the dose [data provided in figure in
Javid and Anderson (1958)]. In another set of experiments, intraocular pressure measurements
on three monkeys were taken every 30 minutes with a McLean tonometer, and in one animal
CSF was also measured. In the experiments, 10 and 30% urea solutions (dose of 1.5 g/kg) were
administered s.c. to the monkeys. Urea was effective in lowering intraocular and CSF pressure
when given by the s.c. route, but CSF pressure was not lowered to the extent observed when urea
was given by the i.v. route [data provided in figure in Javid and Anderson (1958)].
4.4.7. Urea Toxicity in Ruminants and Non-Laboratory Animals
Case reports of probable urea toxicity are usually reported in ruminants, although other
species can be affected. Urea readily degrades to ammonia in water and so urea or urea-based
fertilizers mixed with water can be a biological hazard (Raidal and Jaensch, 2006; Zarnke and
Taylor, 1982).
Ruminants are more sensitive to oral urea toxicity than monogastric animals. This is
because the rumen contains microbes that can hydrolyze urea to ammonia. Urease produced by
bacteria in the rumen converts urea to ammonia which then combines with ketoacids formed by
the bacteria to produce amino acids. If too much urea is present, ammonia will still be formed,
but there will be insufficient amounts of ketoacids to prevent the absorption of ammonia
(Decker, 1996). Ammonia in the blood can be detoxified by the liver and excreted as urea.
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However, if that safeguard is overwhelmed, acute ammonia toxicity results (Ortolani et al., 2000;
Word et al., 1969).
In simple stomach animals, such as humans, nonhuman primates, rodents, and pigs,
ingested urea is primarily absorbed into the blood in the upper gastrointestinal tract and excreted
by the kidneys. Some nonruminant mammals, such as rabbits, guinea pigs, and horses, have a
sizable fermentation sac, the cecum (which does not have that function in humans), which
digests roughage such as grasses. Most ingested urea is absorbed before reaching the cecum and
any ammonia generated would readily enter the portal vein and be detoxified by the liver. Diets
containing 1-2% urea and supplemented with amino acids have been shown to increase the rate
of weight gain in growing pigs (Kornegay et al., 1970). Horses fed up to 5% urea in the grain
ration or up to 0.44 kg/day over four weeks had increased weight and improvement of physical
condition (Rusoff et al., 1965). In this study, no signs of urea toxicity were observed.
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION
4.5.1. Mechanistic Data from In Vivo and In Vitro Studies
4.5.1.1. Neurological Effects
Neurological complications of uremia can include seizures, lethargy, jerking movements,
and stimulus-sensitive myoclonus. Chung et al. (1985) investigated whether the mechanism by
which urea produces myoclonus was similar to strychnine, which inhibits glycinergic
neurotransmission in the medulla. Sprague-Dawley rats (n = 6 for highest dose group, number of
rats for lower dose groups not provided; sex not specified) were administered 0.5 to 2.0 g/kg of
urea (33% urea in 10% invert sugar) via i.p. injection every 15 minutes for a total of 4 to 14
injections. Animals were monitored for stimulus-induced (auditory, tactile, and air puffs) and
spontaneous behavioral changes. Minimal observed neurological changes (exaggerated
sensitivity to stimuli) were reported in animals that received 10 injections of 0.5 g/kg. At a dose
rate of 1.0 g/kg, for a total dose of 4-6 g/kg, stimulus-induced and some spontaneous myoclonus
was observed. Three injections of 2 g/kg (total of 6 g/kg) produced jerking movement and
spontaneous myoclonus 1 and 1.5 hours after the initial injection, respectively. The behaviors
were gone 3-4 hours after the first injection. Four injections of 2 g urea/kg produced
spontaneous moderate intensity myoclonus that lasted for 30-50 minutes. Then the rats appeared
cyanotic and exhibited decreased locomotor activity before convulsions occurred and death
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followed. No information was provided on animal behavior when more than four injections
were given at any of the noted doses.
Brain and plasma urea concentrations were evaluated at the peak of myoclonus in rats
given four i.p. injections of 2 g urea/kg every 15 minutes. Six rats each in the control and treated
group were sacrificed by decapitation 45 minutes after the last injection. Cervical blood, the
medulla, frontal cortex, and spinal cord were evaluated for urea concentrations. Studies showed
that urea concentrations in the brain tissues were five- to eightfold higher than control values and
plasma urea concentrations were 18-fold greater than control values (p < 0.0001 for brain tissues
and plasma; see Table 4-5).
Table 4-5. Effect of exogenous urea on brain and plasma urea concentration
Tissue source
Urea concentration (mM)
Controls
Treated animals
Medulla
10.2 ±0.8
68.7 ± 10.5
Spinal cord
13.3 ±0.7
69.2 ±4.7
Frontal cortex
9.7 ± 1.0
78.7 ±5.3
Plasma
14.0 ±0.7
248.0 ±21.0
Source: Chung et al. (1985).
Control and urea-treated rats exhibited similar glycine levels in whole or crude
synaptosomal fractions of the medulla. Additionally, 0.1-100 mM urea had no effect on uptake
of [3H]-glycine into prepared synaptosomes (0.32 M sucrose homogenates centrifuged at
1000 x g for 10 minutes) (Chung et al., 1985).
In a separate set of experiments, the affinity of urea for a variety of receptors in rat
medulla and spinal cord membrane preparations was evaluated (Chung et al., 1985). Urea
(0.1-100 mM) and mannitol (n = 4/concentration of chemical tested) were tested for their
potency to inhibit membrane binding of [3H]-strychnine, [3H]-gamma-aminobutyric acid,
[3H]-quinuclidinyl benzilate, [3H]-diazepam, or [3H]-glutamate. Mannitol was used as a positive
control in order to determine if inhibitory effect of urea was due to its hyperosmotic action. Urea
3 3
inhibited [ H]-strychnine and [ H]-diazepam binding to the spinal cord and medulla at 10-
100 mM, which is comparable to blood concentrations of urea that induced myoclonus in the rat.
To assess whether urea binding was due to its hyperosmotic action, the authors evaluated the
3 3
effect of mannitol on [ H]-strychnine and [ H]-diazepam binding. At concentrations up to
100 mM, mannitol did not alter [3H]-strychnine binding. However, 10-100 mM mannitol
significantly decreased [ H]-diazepam binding (p < 0.05). The authors proposed that the
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inhibition of [ H]-strychnine binding is not likely related to the osmotic action of urea, while it
may play a role in its affinity for [3H]-diazepam binding sites. In vivo studies, similar to those
described above, with 6 g mannitol/kg (equimolar concentration to 2 g urea/kg) injected i.p.
every 15 minutes for four doses induced seizures in the rats, then death. Myocolonus was not
observed. Comparison of the effects produced by 50 jj.g strychnine and 40-800 |ig urea after
stereotaxic injection into the nucleus gigantocellularis showed that while strychnine produced
moderately intense generalized myoclonus, urea produced tremors that were induced by stimuli
or voluntary movements.
Maddock and Westenfelder (1996) evaluated the effect of urea on human neuroblastoma
cells (SK-N-SH). Cells were exposed to 0.2-2 g/L urea and regulation of heat shock response
was evaluated. Studies showed that clinically relevant concentrations (0.4-2 g/L corresponding
to blood urea nitrogen concentrations of 1.9 to 9.5 g/L) induced production of Hsp72. The
increase in production of Hsp72 plateaued at a concentration of 1.5 g/L. Time-course studies
indicated that the protein is present 30 minutes after addition of urea and the response was
maximal after 10 hours (9.8-fold over the 30-minute value). The response then returned to
baseline 48 hours after addition of urea. Similar responses were not observed with other
chemicals tested (mannitol, NaCl, or glycerol) at equivalent osmolalities. In addition to
upregulation of Hsp72, urea induced carbamylation of proteins in a time-dependent manner. The
authors suggested that urea induces cellular stress via its ability to produce cyanate, which may
carbamylate cellular proteins. It was proposed that this carbamylation may induce the observed
heat shock response. Since the cells seemed to recover after approximately 10 hours, the authors
further proposed that these cells may be able to adapt to the effects of urea.
The potential neuroexcitatory effect of 17 candidate neurotoxins associated with uremia
was studied in dissociated mouse spinal cord neurons (D'Hooge et al., 2003). Whole cell
recordings were made using a single-patch pipette with a resistance of 3-5 Ohms. Urea
(300 mg/L [5 mmol/L]) was used as a reference and at this concentration did not produce an
effect.
Together, these studies suggest that urea may produce some of the observed neurological
effects (e.g., altered locomotion) through interaction with strychnine-sensitive glycine receptors,
which are ligand-gated ion channels (D'Hooge et al., 2003; Maddock and Westenfelder, 1996;
Chung et al., 1985). Binding and modulation of these ion channels by urea may affect
neurotransmission. Additionally, modification of proteins through carbamylation likely plays a
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general role in the effects produced by urea. Protein carbamylation may induce production of
heat shock proteins.
4.5.1.2. Effects on the Renal System
P-glycoprotein (P-gp) is an adenosine 5'-triphosphate-dependent transporter found in the
apical portion of the proximal tubule. The putative function of P-gp is to pump hydrophobic
drugs out of cells, decreasing their intracellular concentrations and their toxicity. In mice, there
are two genes encoding drug-transporting P-gps, mdrla and mdrlb, whereas in humans a similar
function is filled by MDR1.
Miryata et al. (2002) investigated the effect of hyperosmotic urea on Na+/H+ exchange
(NHE) in mouse proximal tubules and whether P-gp is involved. NHE was measured in isolated
mouse proximal tubule S2 segments incubated in bicarbonate-free HEPES media. Na+-
dependent acid extrusion rate (Jh) was assessed using a pH-sensitive fluorescent dye after an
acid load with ammonium chloride prepulse. Hyperosmotic urea (500 mOsm/kg H2O) induced
NHE activation in wild-type (p < 0.05, n = 13) and in mdrla and mdrlb knockout mice (p < 0.05,
n = 8). Genistein (10 |iM), a tyrosine kinase inhibitor, inhibited NHE activation by
hyperosmotic urea (n = 7). Hyperosmotic mannitol (500 mOsm/kg H2O) induced NHE
activation in knockout mice (p < 0.05, n = 13) but had no effect on NHE activation in wild-type
mice (n = 16). The authors concluded that NHE activation by hyperosmotic urea is mediated by
tyrosine kinase and is independent of P-gp.
Zhang et al. (2004) investigated the effect of oxidative stress on DNA and protein in renal
murine inner medullary collecting duct (mIMCD3) cells. The level of reactive oxygen species
(ROS) was measured by fluorescence of dichlorodihydrofluorescein diacetate. Oxidative
damage to protein was measured by detection of protein carbonyl content after urea exposure (0-
300 mM). Increasing the osmolality of cells to 600 mOsm/kg by urea addition increased the
levels of ROS approximately 2.6-fold when compared to the basic 300 mOsm/kg (mean relative
fluorescence values: 638 ± 112 vs. 243 ±31;/; 0.05). Protein carbonylation peaked at 20 mM
urea (p < 0.05 vs. 300 mOsm/kg, n = 3) and decreased slightly at higher concentrations but
remained above control levels at concentrations up to 300 mM [data provided in figure in Zhang
et al. (2004)]. The authors noted that plasma urea concentrations observed during uremia range
from 20-80 mM (normal plasma concentration are 5 mM). Time course studies showed that
protein carbonylation occurred rapidly (300 mM increased protein carbonylation within
5 minutes) [data provided in figure in Zhang et al. (2004)]. The authors propose that
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carbonylation at higher urea concentrations may decrease because available carbonyl groups may
oxidize to carboxylic acids. Urea did not cause protein carbonylation directly as evidenced by
the lack of protein carbonylation when 300 mM urea were added for 15 minutes to cell
homogenates (data not provided). Furthermore, carbonylation was not an effect secondary to
protein carbamylation because direct addition of cyanate, which is formed from urea under
physiological conditions and causes carbamylation, did not cause carbonylation ([data provided
in figure in Zhang et al. (2004)]. Extensive protein carbonylation was also detected in the inner
medulla of the normal mouse kidney but not in the renal cortex [data provided in figure in Zhang
et al. (2004)]. These results suggest that hyperosmolality as a result of urea exposure can cause
oxidative stress in renal medullary cells in vitro and in vivo via protein carbonylation.
Esaian et al. (1997), translated from Russian, evaluated the role of increasing plasma urea
concentrations in renal failure progression in Wistar rats. Wistar rats (200 grams, n = 18) were
subjected to surgical removal of 2/3 of one kidney under diethyl ether anesthesia. Afteroneweek,
the contralateral kidney was removed. Urine was collected noncontinuously every week in a
metabolic chamber under water deprivation conditions from the first stage of the subtotal
nephrectomy. A blood sample was taken from a tail vein when urine was collected. Urea,
creatinine, and electrolytes were determined in the serum. The authors also evaluated proteinuria
and clearance of endogenous creatinine. The animals were divided into three groups (n = 6):
Group 1 rats received a diet with at least 40% protein, Group 2 rats received a diet with 4-5%
protein and 0.01 g urea/kg, Group 3 (control animals), received a diet with 4-5% protein. The
rats were sacrificed by ether narcosis 1.5 months after the second surgery. The remaining kidney
was fixed in formalin; paraffin sections were prepared and stained with hemotoxylin and eosin,
chromotrope, and periodic acid-Schiff stain. Histological changes were observed in the
glomerulus, renal tubules, and interstitium. Serum urea concentrations were significantly lower
in control animals (Group 3) when compared to Group 1 and Group 2 rats; serum urea
concentrations did not differ between Group 1 and Group 2 rats (results provided in figure). A
similar pattern was observed in the evaluation of the proteinuria index and serum creatinine
concentrations. At day 60, Group 1 and Group 2 proteinuria index values (0.83 ± 0.27 g/L and
0.74 ± 0.26 g/L, respectively) were significantly higher than those of the control group (0.36 ±
0.042 g/L) (p value not provided). Control values of serum creatinine concentrations were also
lower than those of Groups 1 and 2 (control: 0.091 ± 0.007 mmol/L; Group 1: 0.125 ±
0.009 mmol/L; Group 2: 0.121 ± 0.01 mmol/L). (There is a discrepancy between the text and
figure provided. The text states serum concentrations while the figure states plasma
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concentrations.) Histological evaluation showed that expressed renal structural changes were
less severe in the control group compared with the test groups. Observable protein cylinders in
the lumen of the proximal tubules were absent in control and Group 2 rats, but were marked in
Group 1 rats. Markers of intraglomular hypertension were associated with increased size of the
glomerulus, proliferation of mesangium, and an increase in mesangial matrix in the test rats and
scarcely noted in the control rats.
Cohen and Gullans (1993a) evaluated a proposed growth-promoting effect of urea on
renal epithelial cells. In these studies, a variety of confluent, growth suppressed cell types were
exposed for 24 hours to urea at concentrations ranging from 0 to 300 mM (concentrations that
are typically found in the renal medulla), and [ H]-thymidine uptake was evaluated. The cell
types included two lines of renal epithelial cells, Madin-Darby canine kidney (MDCK) and LLC-
PKi cells; renal, nonepithelial rat mesangial (RME) cells; nonrenal, epithelial T84 human colon
carcinoma cells; and nonrenal, nonepithelial bovine aortic endothelial (BAE) cells. Urea
addition increased [ H]-thymidine uptake incorporation up to 2.5-fold in MDCK cells (p < 0.05
compared with urea-free culture media). The half-maximal effect occurred at approximately
100 mM. By comparison, 100 mM NaCl inhibited [ H]-thymidine uptake by 57%, glycerol
produced no effect, and 10% serum increased [ H]-thymidine uptake by 34%. Similar to MDCK
cells, renal epithelial LLC-PKi cells also increased [3H]-thymidine uptake in response to urea
exposure (p value not provided). On the other hand, [ H]-thymidine uptake was not increased by
urea in RME, T84 human colon carcinoma, or BAE cells. The incorporation may be unique to
cells of renal epithelial origin. No increase in thymidine transport or cellular proliferation (cell
number, total protein content, or cell cycle distribution) and no induction of aneuploidy or
polyploidy were observed; however, a 15% increase in total DNA content was seen in MDCK
cells treated with urea compared with controls (p < 0.05). In this system, urea was able to
increase DNA synthesis without increasing cellular proliferation or inducing polyploidy or
aneuploidy, potentially through a novel mechanism.
In summary, urea may produce a variety of effects in the renal system. Urea has been
shown to modulate NHE and induce the formation of ROS (Zhang et al., 2004; Mirayata et al.,
2002). The formation of ROS is proposed to lead to the carbonylation of proteins, which may
lead to protein denaturation and altered enzyme and protein activities (Zhang et al., 2004).
Furthermore, urea may produce unique effects in specific cell types, such as increased DNA
synthesis in cells of renal epithelial origin (Cohen and Gullans, 1993a).
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4.5.1.3. Hematological Effects
Uremia leads to impaired RBC survival and function (Wardle, 1970). Normal human
RBCs from a single donor were incubated with various reagents including urea (2.5 g/L
incubation volume) under a variety of culture conditions and assayed from two hours to
overnight. Endpoints evaluated included urea effects on pyruvate kinase and glutathione
reductase activities, reduced glutathione concentration, uptake of [32P]-orthophosphate,
methemoglobin concentration, Heinz body formation, [42K] uptake, and autohemolysis.
Incubation of urea for two hours at pH 7.8 increased reduced glutathione concentration in the
cells by one standard deviation (control value: 600 ±105 mg/L RBC at pH 7.8; no other data
provided), but urea had no effect after incubation for two hours and overnight at lower pHs (6.8
and 7.4). Urea also increased [32P]-orthophosphate uptake after a four hour incubation
(7053 counts/300 seconds vs. 6563 ±180 counts/300 seconds for controls; SD = +2) but not after
two hours (4677 counts/300 seconds vs. 4478 ± 280 counts/300 seconds). An increase in [42K]
uptake by RBCs was observed during the first two hours but not thereafter, resulting in an
average potassium uptake similar to controls (1.50 mEq/L cells-hour for treated cells vs.
1.47 mEq/L cells-hour for controls). Urea impaired pyruvate kinase activity (1.9 U/1010 RBCs
vs. 3.3-4.0 U/1010 RBCs for controls) and glutathione reductase activity (37 U/min-mL RBCs
vs. 50 U/min-mL RBCs for controls). Urea did not trigger methhemoglobin production and had
no effect on the osmotic fragility of the cells, but it induced ring forms with abnormally crenated
and distorted cells after two hours. No Heinz bodies were observed. While urea did produce
some effects (e.g., increased potassium influx, inhibited pyruvate kinase activity), the author
concluded that overall it did not cause significant toxicity in RBCs (Wardle, 1970).
4.5.2. Role of Urea Transporters
Urea can permeate cell membranes by passive diffusion or urea transport proteins. Two
mammalian UT genes have been identified, UT-A and UT-B (for a review, see Sands, 2003). In
the kidney, splice variants of UT-A are expressed in the inner and outer medullary collecting
ducts and thin and long descending limbs of the loop of Henle (Fenton et al., 2002) while UT-B
is expressed in the descending vasa recta of the renal medulla (Lucien et al., 2005). UT-B was
identified in the ureter and urinary bladder of the dog and rat and found to have a role in the
regulation of urea excretion (Spector et al., 2007).
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Both transporters are also expressed in a number of extrarenal tissues and in RBCs. For
example, UT-A and UT-B proteins are expressed in the colon, heart, liver, brain, and testis
(Doran et al., 2006; Lucien et al., 2005; Stewart et al., 2004; Sands, 2003; Fenton et al., 2002,
2000). Transporter presence in extrarenal tissues suggests that they play a role in accelerating
efflux of urea after ureagenesis, which may occur normally (as it does in the liver) or as a by-
product of polyamine synthesis (Sands, 2003). Additionally microorganisms that are present in
the colon have high urease activity. It is proposed that colon UTs transport urea to the
microorganisms for breakdown into carbon dioxide and ammonia and play a role in
gastrointestinal health (Bagnasco, 2005; Stewart et al., 2004). A study using human volunteers
(Wolpert et al., 1971) provided early evidence that transport of urea from plasma into the colon
occurred and that urea was hydrolyzed by bacteria in the colon.
4.5.2.1. In Vivo Studies in Rats
Hu et al. (2000) studied the effect of CRF on expression of UTs in the kidney. Male
Sprague-Dawley rats had two thirds of the right kidney removed. One week later, the left kidney
was completely removed. Control rats underwent sham surgery and kidney decapsulation with
both kidneys left intact. Animals had free access to water and received 20 g/day standard rat diet
with 25% protein. mRNA expression of UTs was studied one week and five weeks after surgery.
Five days prior to sacrifice, animals were placed in metabolic cages and urine collected. Blood,
the remaining kidney, brain, and one testis were collected. Tissues were weighed and medulla
dissected from the kidney. Hematocrit osmolality and osmolality and urea concentration in urine
and plasma were measured. RNA was isolated from the renal medulla, brain, and testis and
Northern blot analysis was performed. cDNA probes for UT-A1/2 and UT-B1 were used with
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a reference for RNA loading;
abundance of UT mRNA was expressed relative to G3PDH. Protein expression by Western blot
analysis was measured in tissues from three rats with CRF and three control rats.
One week after surgery, urine and blood clinical parameters were indicative of renal
failure in the animals undergoing partial nephrectomy as evidenced by increased dilute urine
flow (control: 14.9 ± 62.4 mL/day, nephrectomy: 48.16 ±3.8 mL/day; p < 0.001), decreased
urea excretion (control: 14.6 ± 0.6 mmol/day, nephrectomy: 9.5 ±1.1 mmol/day; p < 0.05), and
increased plasma creatinine and urea concentrations (2.8- and 4.1-fold increases, respectively;
p < 0.001). Additionally, UT-A1 mRNA expression in the renal medulla had decreased to 29%
of control values (p < 0.01, n = 6) after only one week and to 3% of control values (p < 0.001,
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n = 6) after five weeks. UT-A2 and UT-B1 mRNA expressions were also significantly decreased
in the renal medulla by five weeks (p< 0.001) [data provided in figure in Hu et al. (2000)]. In
the brain, UT-B1 mRNA expression was decreased to approximately one third of control
(p < 0.01) by five weeks, but there was no change in UT-B1 transcript expression in the testes.
As determined by Western blotting, isoforms of UT-A1 in CRF animals were undetectable in
renal medullary extracts and UT-A2 and UT-B1 bands were reduced at five weeks, compared
with sham-operated animals [data provided in figure in Hu et al. (2000)]. Thus, renal failure
reduced the expression of UTs in the kidney and to a lesser extent in the brain, but not in the
testes (Hu et al., 2000).
Inoue et al. (2005) examined the expression of UT-B in the renal medulla, colon, and
ileum of male Sprague-Dawley rats (125-200 grams; four to six rats/group). Animals were fed a
14% protein diet (low protein diet) or a 23% protein (standard) diet supplemented with 20%
urea. All animals were fed for 12 days. Control rats received the standard 23% protein diet. All
animals had unrestricted access to water. Urine was collected over 24 hours and urea
concentration was measured in urine and blood. In another group, uremia was induced by a 7/8
unilateral nephrectomy and then feeding a 43% protein diet for two weeks; control rats were
sham-operated and fed a 43% protein diet. Both groups had free access to water containing 25%
normal saline to avoid salt depletion. At the end of treatments animals were sacrificed, kidneys
removed and dissected into cortex, outer medulla, and inner medulla, and the internal mucosal
layers of the ileum and colon were harvested. UT-B protein was identified by
immunohistochemistry using an antibody to the carboxyl terminus of the rat UT-B. In rats fed a
14% protein diet or a 20% urea-supplemented diet, UT-B protein levels increased in the outer
medulla (25 and 60%, respectively; p < 0.05), but did not change in the inner medulla. In rats
fed a 43% protein diet, UT-B protein levels decreased significantly in both the outer and inner
renal medulla (50 and 54%, respectively; p < 0.05, n = 4). UT-B mRNA levels in the outer
medulla were significantly increased (approximately two-fold) in urea-supplemented rats [data
provided in figure in Inoue et al. (2005)] and in rats fed a high protein diet (, p < 0.05, n = 4).
Levels in the inner medulla were only increased in rats fed the low protein diet (p < 0.05, n = 4)
[data provided in figure in Inoue et al. (2005)]. UT-B protein levels were decreased in the colon
mucosa of rats fed the 14% protein or 20% urea-supplemented diets (34 and 37%, respectively;
p < 0.05) [data provided in figure in Inoue et al. (2005)]. Overall, the study by Inoue et al.
(2005) suggested that UT mRNA and protein levels are differentially regulated by urea
concentrations depending on the tissue under observation and the origin of elevated urea levels.
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Kim et al. (2005) investigated UT abundance during osmotic diuresis induced by high
glucose, NaCl, or urea loads in male Sprague-Dawley rats (125-200 grams; six rats/group).
Diuresis was induced by feeding standard diets augmented with 2.5 or 4% NaCl or 5 or 20%
urea. Glucose diuresis was induced by streptozotocin and monitored by detection of glucose in
spot urine samples. Additional groups of glucose diuresis and 4% NaCl diuresis rats were fed a
diet supplemented with 10 and 3% urea, respectively, to keep the percentage of urea in total
urinary solute at the control level. Animals were sacrificed after 15-20 days, kidneys removed,
and medulla dissected. One kidney per rat was processed to determine interstitial urea
concentration and the contralateral kidney was processed for Western blot analysis using
antibodies to UT-A1, UT-A2, and UT-B. (Detailed specificities of the antibodies were not
provided.) UT-A1 levels in both portions of the inner medulla were significantly increased in
diabetic rats (255% of control in inner medullary base, 171% of control in inner medullary tip;
p < 0.05) and in rats under 4% NaCl diuresis for 15 days (166% of control in inner medullary
base, 162% of control in inner medullary tip; p < 0.05). Rats under 4% NaCl diuresis for five
days exhibited significantly increased levels of UT-A1 protein only in the inner medullary base
(p < 0.05) [data provided in figure in Kim et al. (2005)]. By comparison, UT-A2 levels in the
outer medulla were significantly (p < 0.05) decreased in diabetic rats (66% of control) and in rats
with 2.5% NaCl diuresis (72% of control). UT-B levels were unchanged in both the diabetic rats
and the 4% NaCl diuresis rats. By comparison, UT-A1, UT-A2, and UT-B levels were not
different from control levels in diabetic rats and 4% NaCl diuretic rats fed a urea-supplemented
diet. UT-A1 levels in the inner medullary base or tip were not different from control animals in
rats fed a urea-supplemented diet. However, UT-A2 and UT-B levels were significantly
(p < 0.05) increased in both urea-supplemented dose groups in the outer medulla (5% urea group:
163 and 150% of controls, respectively; 20% urea group: 155 and 130% of controls,
respectively). The authors concluded that during osmotic diuresis, UT-A1 expression increases
with decreasing urea concentration in the urine and/or percentage of urea in total urinary solute
and UT-A2 and UT-B expressions increase when urea concentration in the medullary
interstitium is high. The increase in UT-A1 protein level is proposed to increase the transport of
urea from the inner medullary collecting duct lumen to the inner medullary interstitium, while
the increase in UT-A2 and UT-B abundance is proposed to increase intrarenal urea recycling
(Kim et al., 2005).
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4.5.2.2. In Vivo Studies in Mice
Lucien et al. (2005) examined the role of dehydration on UT-B1 transporter protein
regulation. Adult male C57BL/6 mice (n = 4-5) were given free access to food and water. Water
was then withdrawn for 48 hours to induce dehydration as determined by weight loss and
increased plasma osmolality. Animals were killed and urinary bladder, ureter, kidney medulla,
colon, testis, and brain were evaluated for altered protein levels. Immunoblot analysis showed
that dehydration significantly decreased UT-B1 protein levels in the urinary bladder and ureter
by 43 and 38%, respectively (p < 0.01). Protein levels in kidney medulla, colon, testis, and brain
were unaffected by dehydration.
Overall, UTs play a critical role in the movement of urea and are important in the
maintenance of normal physiological function in a variety of tissues. Additionally, these studies
show that urea transport expression in different tissues is differentially regulated by urea
concentration (Inoue et al., 2005; Kim et al., 2005; Lucien et al., 2005; Hu et al., 2000). Altered
urea concentrations may lead to decreased levels of the transporter. This could then lead to a
variety of toxicological effects including oxidative stress, disruption of protein structure, and
altered protein function.
4.5.3. Gene Expression Studies
Urea exposure results in many changes in gene expression in renal medullary cells. How
urea activates or depresses these signaling pathways is an area of active investigation (reviewed
by Burg et al., 2007; Cohen, 1999). Transcriptional expression and translation of two immediate
early genes (IEGs), Egr-l and c-fos, were identified in response to hyperosmotic urea (Cohen
and Gullans, 1993b). The effect was found to be specific to cells of renal epithelial origin. (See
Cohen and Gullans [1993a] in Section 4.5.1.2 for descriptions of the cell types evaluated.) In
confluent, growth-suppressed MDCK cells, urea (200 mM) increased Egr-l mRNA level by
almost threefold at 30 minutes and fourfold at two hours and c-fos mRNA level by
approximately fourfold at 30 minutes and threefold at two hours; the changes were time and dose
dependent (0-300 mM urea tested for 30 minutes). The changes occurred in the absence of
cytotoxicity or inhibition of protein synthesis, both potential nonspecific inducers of IEG
expression. Control treatment (NaCl) had no effect in the cells. LLC-PKi cells exhibited a
response to urea comparable to the one observed in MDCK cells. However, urea did not
increase Egr-l mRNA levels in RME, C(, rat glioma, or T84 human colon carcinoma cells. Urea
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induced Egr-l expression via transcriptional activation rather than increased message stability
because, in the presence of the transcription inhibitor actinomycin D, urea-induced Egr-l mRNA
half-life was similar to that following treatment with! 2-O-tetradecanoylphorbol- l 3-acetate
[TP A], a known transcriptional activator of Egr-l. Overall, the authors concluded that renal
epithelial cells can increase expression of c-fos and Egr-l through transcriptional activation in
response to hyperosmotic urea.
Cohen et al. (1996) evaluated the mechanism by which urea induces IEG transcription.
mIMCD3 cells were transiently transfected with a luciferase reporter plasmid linked to the
murine Egr-l 5' flanking sequence. Urea was found to induce Egr-l expression through a
protein kinase C (PKC)-dependent mechanism (evidenced by abrogation of urea-inducible
reporter gene activity by the PKC inhibitors staurosporine and calphostin C or downregulation of
PKC through chronic treatment with TPA (Cohen et al., 1996) [data provided in figures in Cohen
et al. (1996)]. In growth-suppressed mIMCD3 cells, urea (200 mOsm) increased inositol
1,4,5-triphosphate (IP3) release threefold within five minutes of exposure (IP3 formation was
measured using a radioreceptor binding assay). In lysates from mIMCD3 cell monolayers
treated with urea (200 mM for 10 minutes), the degree of phosphorylation of the receptor
tyrosine kinase-specific phospholipase C (PLC) isoform, PLC-y, was upregulated (quantitative
data not provided). The authors stated that these data suggest that urea induced IEG expression
(specifically Egr-l) via a cell surface or cytoplasmic tyrosine kinase, which leads to,
sequentially, activation of PLC-y, IP3 release, and PKC activation.
Urea treatment has been shown to be associated with increased oxidative stress and the
stress-responsive transcription factor Gaddl53 (Zhang et al., 1999). In mIMCD3 cells, urea
(200 mM) markedly increased Gaddl53 mRNA (greater than 10-fold) and protein levels but did
not increase protein levels of the molecular chaperone grp78 [data provided in figures in Zhang
et al. (1999)]. Urea-induced increase in Gaddl 53 mRNA levels was shown not to be associated
with an increase in RNA stability. Furthermore, urea-induced Gaddl 53 mRNA and protein
expression was found to be antioxidant sensitive; expression was inhibited by pretreatment with
the antioxidants, N-acetylcysteine and dimethylthiourea. Furthermore, urea-induced
transcription of Egr-l (transiently transfected in mIMCD3 cells) was decreased by 55% in renal
cells pretreated with N-acetylcysteine. Intracellular reduced glutathione content, a biochemical
indicator of oxidative stress, was decreased in urea-treated cells within 15 minutes of exposure.
The role of Ras protein in urea signaling and induction of IEG expression was also
investigated in mIMCD3 cells (Tian et al., 2000). Compared to basal conditions (where
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approximately 5% of the immunoprecipitable Ras was activated), urea (12 g/L [200 mM])
increased Ras activation to 15.3% of the immunoprecipitable material (p < 0.05) [data provided
in figure in Tian et al. (2000)] within two minutes of treatment. Urea had no effect on Ras in the
nonrenal 3T3 cell line (data not provided). A stably transfected cell line with an expression
plasmid containing a dominant-negative N17Ras mutation was used to further characterize the
intracellular signaling pathway (defined as N17Ras-B7 cells). NITRas induction inhibited urea-
inducible Egr-1 and Gaddl53 transcription, indicating a role for wild-type Ras signaling in
response to urea (data not provided). However, N17Ras overexpression only partially inhibited
urea-induced extracellular signal-regulated kinase (ERK) phosphorylation (38% at 15 minutes
and no effect at 5 minutes), indicating that activation of ERK may involve other signaling
pathways. Effects on other mitogen-activated protein kinases (MAPKs) (i.e., p-38 and SAPK)
were not observed in N17Ras-B7 cells treated with urea. Overexpression of N17Ras also had no
effect on urea-inducible apoptosis (400 mM urea) [data provided in figure in Tian et al. (2000)]
or phosphorylation of Akt, which is associated with urea-induced apoptosis (data not provided).
Finally, urea treatment induced recruitment of SOS, a guanine nucleotide exchange factor and
Ras activator, to the cell membrane (SOS levels increased by approximately 100%), suggesting
that SOS may mediate Ras activation by urea. Together, these studies suggest that Ras signaling
may play a role in renal epithelial cell responses to urea-induced oxidative stress.
Zhang et al. (2000a) evaluated the role of phosphatidylinositol-3 kinase (PI3K) in the
urea signaling pathway in mIMCD3 cells. Urea (200 mOsm/kg) increased PI3K activity
(assessed using immunoprecipitation) 3.2-fold within one minute of treatment and 2.5-fold after
five minutes of treatment in confluent, serum-deprived mIMCD3 cells. PI3K activity returned to
control levels by 15 minutes. Urea was shown to increase PI3K activity at up to the highest
concentration tested, 800 mM, with a peak at 600 mM. PI3K activation was not involved in
induction of Egr-1 transcription as evidenced by the fact that PI3K inhibitors wortmannin and
LY-294002 did not block urea-induced transcription of Egr-1 (data provided in figure). Urea
(200 mM) significantly increased p70 S6 kinase activity by 75% (p < 0.05) within five minutes
of treatment. The observed effect was inhibited by wortmannin (10 nM) and LY-294002
(10 |iM) pretreatment. Urea treatment (200 mM) also increased Akt phosphorylation and
wortmannin (10 and 100 nM) and LY-294002 (10 |iM and 30 |iM) inhibited the effect. She
activation and recruitment of Grb2 (as assessed by immunoblots) were also observed after urea
treatment (200 mM) [data provided in figure in Zhang et al. (2000a)]. The authors noted that
400 mM, but not 200 mM, urea increased caspase-3 activity (data not provided) and that this
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effect was increased by 266% after pretreatment of wortmannin (100 nM). Urea induced
annexin V binding (a biochemical marker of apoptosis) increased 178% (compared to control)
after pretreatment of wortmannin. These studies suggest that activation of PI3K may play a role
in renal cell responses to urea.
Pretreatment with urea can protect renal medullary cells, but not 3T3 cells, from the
proapoptotic effect of NaCl (Zhang et al., 2000b). This was exemplified by using two
biochemical indices of apoptosis, caspase-3 activation and annexin V binding. In mIMCD3
cells, urea (200 mM) did not exert a proapoptotic effect (i.e., increase caspase-3 activity) in
accordance with the results from Zhang et al. (2000a). However, when urea was applied before
NaCl treatment a 61% inhibition of the NaCl-induced caspase-3 activation and a 63% inhibition
of the NaCl-induced annexin V binding were observed (p < 0.05 when compared to NaCl alone).
Urea also was shown to block the proapoptotic effects of mannitol (data not provided). Urea
treatment by itself decreased annexin V binding by 18%, which was statistically not significant.
The proapoptotic effect (as evaluated by caspase-3 activation) was not observed when
fibroblastic 3T3 cells were used [data provided in figure in Zhang et al. (2000b)]. The protective
effect of urea against NaCl-induced apoptosis was found to be similar to that of the mitogens
epidermal growth factor (EGF) and insulin-like growth factor (IGF). When applied
simultaneously with IGF, a potentiation in effect was observed (p < 0.05 when compared to urea
pretreatment alone). Urea, however, failed to protect mIMCD3 and 3T3 cells from another
proapoptotic stressor, ultraviolet B irradiation, suggesting that the protective effects of urea are
cell type- and stimulus-specific.
In a study of gene expression using microarrays, exposure of mIMCD3 cells to high urea
concentration (200 mM) was compared to exposure to EGF, NaCl, and mannitol (Tian and
Cohen, 2002). Urea exposure resulted in downregulation of approximately 6% of 12,000 genes
on the Murine Genome U74A GeneChip (Affymetrix) array, whereas 0.8% were upregulated.
Of the upregulated genes only 21 were upregulated significantly (threefold or more) in response
to urea. Most notable was a 27-fold increase in activating transcription factor 3 (ATF3) mRNA.
Expression of ATF3 protein in mIMCD3 cells also increased as evidenced by Western blotting
[data provided in figure in Tian and Cohen (2002)]. In contrast, NaCl (100 mM) upregulated
approximately 4% of the genes evaluated; 71 genes were upregulated sevenfold or more.
Additionally, NaCl downregulated expression of approximately 12% of the 12,000 transcripts
studied. These data supported earlier speculation that hyperosmotic urea and NaCl have
different signaling mechanisms (Cohen and Gullans, 1993b). However, the profile of IEG
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expression in response to urea stress was more similar to treatment of cells by EGF (100 nM)
than to hypertonic stress induced by mannitol (200 mM). Urea pretreatment for 30 minutes
partially restored genes affected by hypertonic NaCl stress to basal levels of expression. The
authors suggested that urea may play a cytoprotective role in response to hypertonicity induced
by other substances (e.g., NaCl).
4.5.4. Genotoxicity
The genotoxic effects of urea have been studied in a variety of short-term test systems, in
vitro (bacteria and mammalian cells) and in vivo (mouse bone marrow). A summary of the
results from these genotoxicity assays of urea discussed in the following section is presented in
Table 4-6.
Table 4-6. Genotoxicity and mutagenicity data from in vitro and in
vivo assays of urea
Species/
cell line
Test system
(strain/species)
Exposure
Metabolic
activation
Reference
-S9 +S9
In vitro
Bacterial systems
Salmonella
typhimurium
Reverse mutation
(TA98, TA100,
TA1535, TA1537)
0-10,000
Hg/plate
Mortelmans et al.
(1986)
Reverse mutation
(TA98, TA100,
TA1535, TA1537,
TA1538)
0-5000 (ig/plate
Shimizu et al. (1985)
Reverse mutation
(TA98, TA100,
TA1537)
NAa
NDb
Ishidate et al. (1981)
Escherichia coli
Differential DNA
repair test (K-
12/343/113)
Up to 375 mM
ND
Hellmer and
Bolcsfoldi (1992)
Mammalian cells - rodent
Mouse renal inner
medullary collecting
duct (mIMCD3)
cells
Alkaline comet assay
(single strand breaks)
300 and 600
mOsm/kg
+
ND
Zhang et al. (2004)
Neutral comet assay
(double strand breaks)
600 mOsmol/kg
-
ND
Zhang et al. (2004)
Neutral comet assay
(double strand breaks)
600 mOsm/kg
-
ND
Kultz and
Chakravarty (2001)
Mouse lymphoma
cells L5178Y TK+/~
Micronucleus assay
500-2000 ng/mL
-
-
Nesslany and Marzin
(1999)
Alkaline unwinding
0-0.718 mol/1
+°
ND
Garberg et al. (1988)
Forward mutation
0-0.662 mol/1
+°
ND
Wangenheim and
Bolcsfoldi (1988)
Rat hepatocytes
Alkaline elution
0.03-3.0 mM
-
ND
Sina et al. (1983)
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Table 4-6. Genotoxicity and mutagenicity data from in vitro and in
vivo assays of urea
Species/
cell line
Test system
(strain/species)
Exposure
Metabolic
activation
Reference
-S9
+S9
Chinese hamster
lung
Chromosomal
aberrations
NA
+
+
Ishidate and
Yoshikawa (1980)
NA
+
ND
Ishidate et al. (1981)
<16 mg/mL
(266 mM)
+°
ND
Ishidate and
Odashima (1977)
Intercellular
communication
0-5 mg/mL
+°
Umeda et al. (1980)
Epithelioid C3H
mouse embryo cells
Multinucleated cells
1-10 |ig/mL
-
ND
De Brabander et al.
(1976)
Mammalian cells - human
Lymphocyte
Chromosomal
aberrations
0.01-1.0 mg/mL
-
ND
Zhurkov (1975)
0.06 and 3.0
mg/mL
(1 and 50 mM)
+d
ND
Oppenheim and
Fishbein (1965)
In vivo
Mammalian - rodent
Bone marrow
Chromosomal
aberrations (male
mice)
0.1825,0.3650,
or 0.7300
mg/day
Kommadath et al.
(2001)
Chromosomal
aberrations (male and
female mice)
500 mg/day
+
Chaurasia (1991)
Chromosomal
aberrations (mice; sex
not provided)
500 mg/day
+
Chaurasia and Sinha
(1987)
Sperm
Sperm head
abnormalities (male
CBAxBALB/c mice)
250-2000
mg/kg-day, i.p.
Topham (1980)
aNot available.
bNot detected.
°Cytotoxic concentrations achieved in the test.
d Effect observed only at highest dose tested.
Data from mutagenicity and genotoxicity tests in bacteria show that urea does not cause
mutations in^. typhimurium strains TA98, TA100, TA1535, TA1537, or TA1538, in the
presence or absence of an exogenous metabolic activation system (S9), at doses up to
1000 (ig/plate under the conditions stated (Mortelmans et al., 1986; Shimizu et al., 1985; Ishidate
et al., 1981). Likewise, urea did not induce damage in E. coli in the absence of an S9 fraction
using the differential DNA repair test. This assay evaluates response differences to chemical
exposures using DNA repair proficient (avrB I reck ) compared to deficient (nvrB free A )
strains (Hellmer and Bolcsfoldi, 1992).
Studies have been conducted in different mammalian cells using various genotoxicity
assays. Both DNA single strand breaks (measured with the alkaline comet assay) and double
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strand breaks (measured using the neutral comet assay) were studied in mouse renal inner
medullary (mIMCD3) cells exposed to high levels of urea (300 - 600 mOsmol/kg) for 15
minutes and one hour. Urea caused single strand breaks (18 and 26%) at concentrations of 300
and 600 mOsmol/kg, respectively (Zhang et al., 2004). Comparatively, Garberg et al. (1988)
showed that with the relative fraction of DNA single strand breaks to relative toxicity, urea
produced 9.2 and 17.3% fraction of single strand breaks at the two highest doses tested (0.628
and 0.718 mol/L) and not at lower doses. Furthermore, primary rat hepatocytes exposed to 0.03 -
3.0 mM urea for three hours did not induce DNA single strand breaks in the alkaline elution
assay (Sina et al., 1993). No double strand breaks were observed when cells were exposed to
600 mOsmol/kg of urea (Zhang et al., 2004). Results from a similar exposure to urea (600
mOsmol/kg for 1 hour) in mIMCD3 also did not induce DNA double strand breaks (Kultz and
Chakravarty, 2001). Based on the above DNA strand break studies, urea, at the high
concentrations tested, may have the potential to produce single strand breaks in some systems,
but not double strand breaks. It is possible that urea is forming reactive oxygen species (ROS)
resulting in single strand breaks.
Forward mutations in mouse lymphoma L5178Y thymidine kinase locus was determined
in mouse lymphoma L5178Y TK cells exposed to several different compounds, including urea,
for four hours (Wangenheim and Bolcsfoldi, 1988). In this study, mutation frequency was
significantly increased; however, no dose response relationship was observed except for the two
highest doses tested (0.53 mol/L and 0.662 mol/L) in the absence of S9. It should be noted that
the total growth (suspension growth x clonal efficiency) was only 24 and 8% compared to
control in the highest two doses tested. Nesslany and Marzin (1999) studied micronucleus
formation as a result of exposure to urea, in which mouse lymphoma L5178Y TK cells were
exposed to 500, 1000 or 2000 ub/ml of urea for four hours in both the presence and absence of
S9. Increase in micronuclei frequency was not observed at any of the concentrations tested.
In vitro studies were conducted (Ishidate et al.,1981; Ishidate and Yoshikawa, 1980;
Ishidate and Odashima, 1977) to examine chromosomal aberrations (CA) in Chinese hamster
lung cells after 24 or 48 hour exposure to urea concentrations up to 266 mM. In the first study
(Ishidate and Odashima, 1977), at a maximum effective dose of 16 mg/ml, 37 percent of cells
showed CA of one or more types including gaps, breaks, translocations and fragmentation. In a
second study (Ishidate and Yoshikawa, 1980), although details are not provided about the
experimental design, the authors state that urea (among several other compounds) was positive in
the CA test. In a follow up study (Ishidate et al., 1981), urea was judged to be positive (a
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determination of "positive" was defined by the authors as when the incidence of polyploidy cells
or cells with structural aberrations exceeded 10%, since the incidence in both untreated and
solvent-treated control cells was usually less than 5%) based on the number of CA. Urea at a
concentration of 13 mg/ml showed more than 20% of the metaphases had CA.
Similarly, Oppenheim and Fishbein (1965) reported that exposure of cultured normal
human leukocytes to 50 mM urea for 72 hours increased the incidence of CA two-fold over
controls. Along with chromosome fragmentations, there were other signs of cell toxicity such as
an increased proportion of damaged cells and metaphase breakage occurring concurrently.
Furthermore, Umeda et al. (1980) reported that urea (5 mg/mL) inhibited intercellular
communication between wild-type Chinese hamster V79 lung cells and a 6-thioguanine-resistant
clone in vitro. However, it was noted that toxic effects were also observed at this dose.
Chaurasia (1991) and Chaurasia and Sinha (1987) conducted in vivo experiments in 7- to
10-week-old male Swiss albino mice fed with urea (500 mg/kg-day) for 5-7 days. Bone marrow
samples were collected seven days after the last treatment and a minimum of 100 metaphases
were examined. Both studies showed that urea was capable of inducing CA. Among several
types of CA found, chromatid breaks were the most frequent. The authors concluded that urea
may be a potent clastogen. On the contrary, urea exposure of 3- to 4-month-old male Swiss
albino mice to 0.1825-0.7300 mg/day via food for up to 28 days did not show an increase in
bone marrow CA (Kommadath et al., 2001). The lack of consistency between the two groups of
studies is likely due to the difference in the dose. Additionally, urea did not induce sperm head
abnormalities in five male (CBA BALB/c) Fi mice that were assayed five weeks after receiving
five daily i.p. injections of urea (up to 2000 mg/kg-day) (Topham, 1980).
In summary, data from various genotoxicity assays show that urea produces both positive
and negative results in several test systems. It should be noted that in many of these studies, urea
is one of the several compounds tested with the objective of either comparing different types or
the sensitivity of different genotoxicity assays, hence specific study details were not reported in
these studies. Urea does not induce mutations in bacterial or E. coli systems. Based on results of
specific assays that detect DNA strand breaks, urea, at high concentrations, may have the
potential to produce single strand breaks in some systems, but not double strand breaks. It is
possible that urea forms reactive oxygen species (ROS) resulting in single strand breaks. In vitro
and in vivo chromosomal aberration studies have demonstrated mixed results with some studies
showing chromosomal aberrations while others show no indication of chromosomal damage.
This is particularly true among the in vivo studies. It is likely that this lack of consistency is due
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to the difference in dose. Some of the positive responses that have been reported are at high
doses and test concentrations. As noted above, one of the constraints in making specific
conclusions is the lack of study details, especially in studies that evaluated the genotoxicity of
multiple chemicals. Therefore, although there is inadequate information to consider urea to act
specifically through a mutagenic mode of action, based on the induction of chromosomal
aberrations in certain mammalian test systems, the role of genotoxicity in the process of urea-
induced carcinogenicity cannot be eliminated.
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
4.6.1. Oral Exposure
There are limited studies that evaluated the possible association between oral exposure to
urea and noncancer effects in humans. The human-based literature includes two studies that
investigated the potential relationship between urea exposure and altered hematological
endpoints. Eknoyan et al. (1969) showed that ingestion of urea by patients with experimentally
induced azotemia decreased platelet adhesiveness. Additionally, the reduction in platelet
adhesiveness was greater in subjects where high SUN concentrations were maintained for
24 hours compared to subjects where SUN concentrations were maintained for 8-10 hours.
Bensinger et al. (1972) reported that ingestion of urea by patients with sickle cell disease did not
produce a statistically significant effect on RBC survival. In addition to these studies, a single
report on an accidental urea poisoning stated that symptoms resembling strychnine poisoning
(loss of appetite, nausea, vomiting, extreme excitement, severe general convulsions) developed
relatively soon (3-5 hours after ingestion); however, none of the patients died and all recovered
within a few days (Steyn, 1961). Review of all three publications does not provide information
on human subjects research ethics procedures undertaken in these studies. Studies in pigs, rats,
and mice showed that urea did not induce general toxic effects (e.g., death or decreased body
weight) at a variety of doses and dosing regimens. Studies by Button et al. (1982), Teramoto et
al. (1981), Fleischman et al. (1980), and Seipelt et al. (1969) showed that oral administration of
urea, via gavage or feed, did not produce toxicity, decreased body weight, or other symptoms of
urea intoxication in tested animal models. These studies are in contrast to Finlayson and
Baumann (1956) who showed that urea-induced decreases in body weight depended upon the
dose as well as the rate of administration.
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There are conflicting results from the reproductive and developmental studies that have
been conducted to date. Teramoto et al. (1981) showed that a single, high dose of urea did not
affect the number of implants, number of live fetuses, percent fetal resorptions, mean fetal body
weight, or percent of malformed fetuses in mice and rats. However, studies by Seipelt et al.
(1969) suggest that maternal exposure may decrease the number of pups/litter.
There are also conflicting results reported in studies on the effect of urea on reproductive
performance in cows maintained on feeding regimens that increased blood urea concentrations.
Rhoads et al. (2006) modulated plasma urea concentrations by using different protein-enriched
diets and found that a high protein diet altered the viability of the bovine oocyte or embryo.
Ordonez et al. (2007) evaluated cows that were grazed on pastures to which supplementary urea
nitrogen fertilizer was applied. Evaluation of several ovarian parameters, the number of luteal
phases, and milk progesterone concentrations indicated no difference between control cows and
urea-grazed cows. An explanation for the different results could be due to the difference in urea
sources between the studies.
Limited data suggest that the liver, kidney, and/or pituitary could be targets of urea
toxicity. One study by Kommadath et al. (2001) indicated that urea may induce liver and kidney
toxicity. Mice given urea-adulterated milk orally (dose range 0.1825-0.73 mg/kg-day) exhibited
degenerative and necrotic changes in hepatocytes and lymphoid follicle formation of the liver.
Additionally, fatty changes in the perirenal tissue, mild necrosis, glomerulitis, and leukocytic
infiltration were observed in the kidney. Furthermore, studies showed that urea increased protein
synthesis and decreased the density of secretory granules in pituitary intermediate cells after urea
administration via food (Okada and Kobayashi, 1989).
Overall, the available studies provide a limited indication of the effects of urea after oral
exposure.
4.6.2. Inhalation Exposure
While there are several studies that have evaluated the toxicological effects of urea-
containing mixtures, few studies have specifically correlated the effects of urea inhalation to
observed toxicological effects. Similar to experimental outcomes from oral studies, the hepatic
system may be a target of inhaled urea. An occupational exposure study by El Far et al. (2006)
showed that exposure to urea increased AST and ALT activities 15 and 19%, respectively, and
decreased creatinine concentrations by 13%. The same authors also reported statistically
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significant urea-induced increases in the blood levels of CEA and PSA; however, it should be
noted that all biomarker changes, despite statistical significance, were well within the normal
physiological range (Gomella and Haist, 2004).
Studies assessing the impact of urea exposure on lung function indicate that the effects
are minimal. A retrospective cohort study (Bhat and Ramaswamy, 1993) and a therapeutic study
(Cade and Pain, 1972) showed that urea inhalation caused mild impairment of lung function.
Occupational exposure seemed to decrease only PEFR/min (approximately 20%) (Bhat and
Ramaswamy, 1993). The therapeutic study in asthmatics showed that a single exposure to urea
only produced variable effects on VC and FEVi (Cade and Pain, 1972).
4.6.3. Dermal Exposure
There are limited human and animal studies on the effects of urea after dermal exposure.
The majority of the human studies suggest that dermal exposure to urea at concentrations up to
60% does not produce skin irritation. However, two studies showed that a 20% urea formulation
did produce edema and skin irritation (Agner, 1992; Fair and Krum, 1979). Interestingly, in
those cases where urea was shown to produce skin irritation, petrolatum was present in the
formulation. Agner (1992) also noted that previous studies had indicated that penetration of urea
into human skin strongly depends on the vehicle used. Furthermore, Johnson et al. (1970)
showed that hypotonic, hypertonic, and isotonic urea solutions all produced different effects in
the abraded skin of two healthy male volunteers. While isotonic urea produced mixed effects,
hypertonic and hypotonic solutions decreased the number of dermal macrophages in both
volunteers.
A single short-term study in mice by Lashmar et al. (1989) indicated that dermal urea
exposure did not produce changes in skin histology or irritation, as determined by visual
inspection, after a 24-hour occluded exposure to a 10% aqueous urea solution.
Based on the limited information available, studies suggest that dermatotoxic effects of
urea are greatly dependent upon the vehicle used and manifest primarily as skin irritation;
however, further studies are needed to corroborate the results (Agner, 1992; Fair and Krum,
1979; Johnson etal., 1970).
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4.6.4. Additional Studies
In vitro and in vivo studies have shown that the cardiovascular system may be a target for
urea. Induction of a uremic state in Apo Emice was shown to increase aortic plaque area.
Compositional analysis of these lesions showed an increase in collagen, calcite, and
hydroxyapatite when compared with control animals (Massy et al., 2005). Urea also produced
electromechanical alterations and hypotension when applied to rat hearts in vitro (Abaurre et al.,
1992) and induced changes in blood pressure and heart rate in dogs in vivo (Cuparencu et al.,
1961). Abaurre et al. (1992) showed that at a concentration of 17 mM, urea reduced the
isometric force and rate of force development in papillary muscles. Urea also decreased
isovolumic systolic pressure, as measured in Langendorff perfused hearts. Electrocardiographic
studies showed that urea reduced the total QRS amplitude, increased QRS duration, decreased P
wave amplitude, and elevated the ST segment in a majority of the samples evaluated. Cuparencu
et al. (1961) showed that i.v. or intraarterial injection of urea in dogs caused opposite effects on
blood pressure that were caused by vascular reflexes, endocrine vasoactive substances, and/or
nervous regulation. Both i.v. and intraarterial administration methods were shown to induce
tachyphylaxis.
4.6.5. Mode of Action
Urea has been shown to target a variety of organ systems including cardiovascular, renal,
hepatic, nervous, and pituitary. The spectrum of effects produced within these systems suggests
the urea may produce effects through a variety of molecular mechanisms.
Structural modification of proteins, either through protein carbamylation or protein
carbonylation, is one proposed mode of action for urea. Urea breakdown leads to the formation
of cyanate and ammonia. The active form of cyanate, isocyanic acid, may be formed and then
react with nonprotonated amino groups of proteins. The carbamylation and carbonylation of
these proteins may then lead to altered protein structure and protein activity. Protein
carbamylation has been implicated in the development of atherosclerosis (Ok et al., 2005) and in
induction of heat shock proteins in a neuronal cell line (Maddock and Westenfelder, 1996).
Further, Zhang et al. (2004) showed that protein carbonylation may occur in renal cells. The
mechanism (i.e., enzymatic vs. nonenzymatic) by which protein carbonylation and carbamylation
occur is currently unclear. Zhang et al. (2004) showed that direct application of urea to
homogenized cellular preparations did not induce protein carbonylation, suggesting that either
the levels of ROS are insufficient to induce carbonylation or that the necessary enzymatic
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cofactors to induce carbonylation are diluted in the preparation. However, there is no additional
information on the mechanism(s) of action for urea carbonylation and carbamylation.
Uremia complications include seizures, lethargy, and locomotor alterations, suggesting
that urea may induce these effects through the nervous system. As noted above, urea has been
implicated in the induction of heat shock proteins, which play a role in protein folding and
intracellular trafficking (Maddock and Westenfelder, 1996). Specific interactions within the
glycinergic pathway is another proposed mechanism by which urea may produce
neurotoxicological effects. In the CNS, glycine has been shown to act as an inhibitory
neurotransmitter (i.e., induces hyperpolarization of neurons) and inhibitory activity has been
shown to occur between spinal interneurons and motoneurons (Bloom, 1990). Glycine-induced
hyperpolarization occurs through increased chloride conductance (Bloom, 1990), which is
antagonized by strychnine. Chung et al. (1985) showed that urea can specifically inhibit binding
of strychnine to glycine receptors and that this effect is reversible. Studies by Chung et al.
(1985) suggested that the observed binding inhibition is not due to osmotic effects of urea.
Furthermore, urea was shown to not inhibit glycine uptake suggesting that urea-induced
alteration of glycinergic neurotransmission occurs through direct modulation of the anion
channel (Chung et al., 1985). Disinhibition of the glycinergic pathway by urea may play a role
in the altered locomotor effects observed after urea administration.
The effects of urea may also occur through modulation of IEGs, such as c-fos and Egr-1.
Increased expression of IEGs is proposed to occur through two pathways, (a) a PKC-dependent
mechanism and (b) She protein (Tian and Cohen, 2002; Tian et al., 2000; Zhang et al., 2000a;
Cohen et al., 1996; Cohen and Gullans, 1993b). Activation of the PKC-dependent pathway is
proposed to lead to increased activation of MAPKs, which then induce transcription of IEGs.
Activation of She protein, via phosporylation, is proposed to lead to recruitment of Grb2. This
would lead to downstream activation of Ras, via recruitment of the guanine nucleotide exchange
factor SOS, and increased transcription of IEGs. The full effect of the upregulation of these
genes and their roles in producing the observed toxicological effects continue to be under
evaluation.
Urea has also been shown to modulate transcription of the oxidative stress response
factor Gaddl53 (Zhang et al., 1999). This increase was shown to occur at concentrations where
oxidative stress was induced in cells. The effect of urea on gene transcription was shown to be
sensitive to the presence of antioxidants, suggesting that reactive oxygen intermediates may play
a role in the signaling mechanism. Zhang et al. (1999) also showed that a component of Egr-1
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transcriptional activation is sensitive to antioxidants. While the effect was studied only in renal
cells, it may occur in other cell types. As discussed with the IEGs, the role that this mechanism
may play in producing the observed toxicological effects needs further evaluation.
Urea has been shown to differentially modulate levels of UTs depending on the tissue
evaluated (Inoue et al., 2005; Kim et al., 2005; Lucien et al., 2005; Hu et al., 2000). Altered
transporter levels may significantly alter the osmotic balance present in tissues and lead to the
development of oxidative stress. As discussed previously in this section, oxidative stress can
produce a variety of effects including disruption of protein structure and upregulation of
oxidative stress factors.
4.7 EVALUATION OF CARCINOGENICITY
4.7.1. Summary of Overall Weight of Evidence
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is inadequate
information to access the carcinogenic potential of urea. Fleishman et al. (1980) observed a
significant increase in malignant lymphomas in the mid-dose group of female mice (incidences:
10/92, 7/43, 10/38 (p = 0.008), and 9/50 in the control, 0.45, 0.9, and 4.5% dose groups,
respectively) in a 12-month feeding study (Fleishman et al., 1980). In addition, there was a
statistically significant linear trend (p = 0.008) and a statistically significant occurrence in tumor
incidence at the high dose for interstitial adenomas in the testes in male rats (incidences: 21/50,
27/48, 25/48, and 35/50 (p = 0.004) in the control, 0.45, 0.9, and 4.5% dose groups,
respectively). As discussed in Section 4.2.1.2, there were reporting problems with this study such
that the exact number of animals used for histopathological evaluation is unknown. A chronic
study (11 month treatment period with follow-up to 15 months) by Shear and Leiter (1941)
showed no treatment related increase in tumors following s.c. administration in mice.
Epidemiologic studies of humans chronically exposed to urea alone or urea-containing mixtures
are limited. One occupational study showed that exposure to urea increased levels of
carcinogenic biomarkers (e.g., CEA and PSA), but these changes were within the normal
physiologic range (El Far et al., 2006). Urea has been tested for its genotoxic potential and has
showed little capacity to produce genotoxic effects in bacterial test strains. Results from in vitro
and in vivo studies in mammalian systems were mixed.
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4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
The human carcinogenicity potential of urea and urea-containing mixtures has been
evaluated in a limited number of studies. While some of the results from studies that evaluated
urea-containing mixtures indicated that urea exposure may have contributed to the occurrence of
tumor development or increased sister chromatid exchange and CA frequency, its role in
producing the observed effects was not clearly established. Therefore, the available data do not
permit a conclusion about human carcinogenicity potential from exposure to urea alone.
El Far et al. (2006) evaluated the effect of occupational inhalational exposure to urea or
urea mixed with other vapors (phenol and formaldehyde) on the levels of three carcinogenic
biomarkers. Their studies showed that serum concentrations of CEA were significantly
increased in both exposure groups while AFP concentrations were increased in the group
exposed to a mixture of vapors including urea. PSA concentrations were decreased in the group
exposed to urea alone. All biomarker effects reported by El Far et al. (2006) were within
physiologically normal ranges and exposure levels were not estimated in the study. These results
provide no relevant evidence that urea may play a role in tumorigenesis.
Two chronic studies in laboratory animals have evaluated the carcinogenic potential of
urea (Fleischman et al., 1980; Shear and Leiter, 1941). Fleischman et al. (1980) conducted a
chronic study in which male and female F344 rats and C57BL/6 mice were exposed to urea
(0.45-4.5%) in feed for 12 months. Female mice in the middle dose group only exhibited a
significant increase in malignant lymphomas. A statistically significant increase in interstitial
adenomas in the testes was also observed in the high dose group in male rats.There were
reporting problems with this study. Shear and Leiter (1941) evaluated the carcinogenic potential
of urea when administered by s.c. injection (<20 doses over 11 months) to strain A and C57BL
male mice. The authors stated that no tumors at the injection site were observed. Neither study
treated or observed the animals for the usual 24-month period.
Genotoxicity and mutagenicity studies in bacterial strains indicate that urea may not be
mutagenic in S. typhimurium (with or without metabolic activation) or E. coli (Hellmer and
Bolcsfoldi, 1992; Mortelmans et al., 1986; Shimizu et al., 1985; Ishidate et al., 1981). Based on
the results of specific assays that detect DNA strand breaks, urea, at high concentrations, may
have the potential to produce single strand breaks in some test systems, but not double strand
breaks. It is possible that urea forms reactive oxygen species (ROS) resulting in single strand
breaks (Zhang et al., 2004; Kultz and Chakravarty, 2001; Garberg et al., 1988). Urea produced
CA in different mammalian cell types and test systems (e.g., mouse lymphoma forward mutation
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assay and mouse renal inner medullary collecting duct cells evaluated using the alkaline comet
assay), generally at high concentrations (approximately 5-38 mg/ml) (Zhang et al., 2004;
Garberg et al., 1988; Wangenheim and Bolcsfoldi, 1988, Ishidate et al., 1981; Ishidate and
Yoshikawa, 1980; Umeda et al., 1980; Ishidate and Odashima, 1977). However, several of the
studies observed effects that were accompanied by a concomitant decrease in cell viability
(Garberg et al., 1988; Wangenheim and Bolcsfoldi, 1988; Umeda et al., 1980) or occurred at
high concentrations (e.g., 50 mM; Oppenheim and Fishbein, 1965). In vivo, urea produced CA
in bone marrow cells of Swiss albino mice fed high doses of urea (500 mg/kg-day for 5-7 days)
but not in mice fed doses of 7.3, 14.6, and 29.2 mg/kg-day for up to 28 days (Kommadath et al.,
2001; Chaurasia, 1991; Chaurasia and Sinha, 1987). Additionally, urea did not induce sperm
head abnormalities in male mice that received five daily i.p. injections of urea (up to
2000 mg/kg-day) (Topham, 1980).
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
The reproductive and developmental studies of urea that have been conducted in animals
to date (described in Section 4.3) present limited and conflicting data that are insufficient to
determine if urea is a teratogen or a developmental toxicant. No human studies directly relating
to susceptible populations and life stages are available. However, studies on patients with renal
disease and asthma have been identified that may be informative for identification of susceptible
populations. For example, Eknoyan et al. (1969) showed that patients with experimentally
induced azotemia or uremia displayed decreased platelet adhesiveness. An additional study by
Cade and Pain (1972) showed that inhaled urea caused mild hypoxemia, impairment of gas
transfer, and ventilation-blood-flow inequality in symptom-free asthmatic patients. Importantly,
Cade and Pain (1972) noted that these bronchoactive effects of urea were only observed in
asthmatics and were not observed in normal subjects.
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
The RfD is an estimate (with uncertainty spanning perhaps an order of magnitude) of a
daily oral exposure to the human population (including sensitive subgroups) that is likely to be
without an appreciable risk of deleterious effects during a lifetime. It can be derived from a no-
observed-adverse-effect level (NOAEL), lowest-observed-adverse-effect level (LOAEL), or
benchmark dose (BMD), with uncertainty factors generally applied to reflect limitations of the
data used.
5.1.1. Choice of Principal Studies and Critical Effect with Rationale and Justification
Information regarding the potential toxicity of oral exposure to exogenous urea in
humans is limited to accounts of accidental exposure (Steyn, 1961), studies on volunteers with
renal disease (Eknoyan et al., 1969), and studies where therapeutic uses of urea were employed
(Bensinger et al., 1972). These studies are of limited value in developing a chronic RfD due to
the acute nature of exposure to urea, evaluation of high doses, the lack of observed toxicity,
limited study design, and insufficient exposure characterization (Besinger et al., 1972; Eknoyan
et al., 1969; Steyn, 1961).
Two human studies investigated the potential relationship between urea exposure and
altered hematological endpoints. Eknoyan et al. (1969) showed that ingestion of urea by patients
with experimentally induced azotemia decreased platelet adhesiveness. Additionally, the
reduction in platelet adhesiveness was greater in subjects where SUN concentrations were
maintained for 24 hours compared to subjects where SUN concentrations were maintained for 8-
10 hours. Ingestion of urea by patients with sickle cell disease did not produce a statistically
significant effect on RBC survival (Bensinger et al., 1972). In addition to these studies, a single
report on an accidental urea poisoning stated that symptoms resembling strychnine poisoning
developed relatively soon (3-5 hours after ingestion); however, all patients recovered within a
brief period of time (Steyn, 1961).
Limited data suggest that the liver and kidney could be potential target organs of urea
toxicity (Kommadath et al., 2001; Das et al., 1997; Krishna et al., 1990), as well as data
indicating decreased weight gain following urea exposure (Finlayson and Baumann, 1956).
Exposure to urea via food caused an increase in protein synthesis and decreased the density of
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secretory granules in pituitary intermediate cells (Okada and Kobayashi, 1989). Button et al.
(1982), Teramoto et al. (1981), Fleischman et al. (1980), and Seipelt et al. (1969) showed no
effects from oral administration of urea, via gavage or feed, in tested animal models. These
studies are in contrast to Finlayson and Baumann (1956) who compared the effect of feeding rats
urea mixed with a diet given ad libitum to a spaced feeding scheduled of two hours per day. For
both feeding schedules, a decrease in weight gain was observed with increased urea doses.
However, no information was provided to assess whether this observation could be associated
with decreased food consumption due to palatability.
A 28-day study by Kommadath et al. (2001) showed degenerative and necrotic changes
in hepatocytes and lymphoid follicle formation of the liver in mice at the lowest tested dose of
7.3 mg/kg-day. Fatty changes in the perirenal tissue, mild necrosis, glomerulitis, and
leukocytic infiltration were also observed in the kidney. However, Kommadath et al. (2001) did
not report incidence data for these effects.
Overall, the available studies provide limited information on the potential toxicity of urea
following oral exposure. The studies identify the liver and kidney as potential target organs for
the toxicity of urea; however, the available information is either from short-term studies (e.g., 28
day exposure), or is insufficient to characterize a dose-response relationship due to a lack of
incidence reporting. The 28-day study conducted by Kommadath et al. (2001) is the only study
that provides an effect level that could be used for the derivation of an RfD, i.e., a LOAEL of
7.3 mg/kg-day based on degenerative effects in the liver and kidney in male mice. The use of this
LOAEL for the derivation of an RfD is confounded by, among other things, the significant
uncertainty associated with the short duration of the study, extrapolation of this dose to humans,
accounting for susceptible populations and the limited database of studies. Therefore,
information on the oral toxicity of urea is considered insufficient for the derivation of an RfD.
5.1.2. Previous RfD Assessment
An IRIS assessment does not currently exist on the IRIS database.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
The inhalation RfC is an estimate (with uncertainty spanning perhaps an order of
magnitude) of a continuous inhalation exposure to the human general population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects over a
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lifetime. It can be derived from a NOAEL, a LOAEL, or a benchmark concentration (BMC),
with UFs generally applied to reflect uncertainties and/or limitations in the data used.
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Limited information is available regarding the inhalation toxicity of exogenous urea. Four
studies (three occupational and one therapeutic) have been identified and are discussed in
Section 4.1.2. Briefly, El Far et al. (2006) compared liver and kidney function as well as
carcinogenicity biomarkers in eight workers exposed to urea for an average of eight years to 15
non-exposed subjects. This study reported elevated AST, ALT and prostate-specific antigen
levels among exposed workers as compared to controls; however, all results were within the
normal physiological range. Bhat and Ramaswamy (1993) evaluated lung function in 30 workers
at a fertilizer chemical plant. Compared to the 68 controls, exposed workers had decreased
PEFR/minute rates, and no change in FVC or FEVi was observed. For both studies (El Far et al.
(2006), Bhat and Ramaswamy (1993)), no quantitative exposure levels were provided. Marsh et
al. (2002) observed a low incidence of bladder cancers deaths - four in a cohort of 995 workers -
among workers at a nitrogen products plant. The mixed chemical exposure limits the specificity
and power of the study. The authors stated that the bladder cancer excess may be due to
occupational exposure prior to employment in the nitrogen products division. Cade and Pain
(1972) investigated the impact of inhaled urea aerosol (4 M solution from a nebulizer for 10
minutes) on lung function in symptom-free asthmatics. The study authors reported that urea
produced mild and variable impairments in vital capacity and FEVi. However, correlation
between individual initial and post-exposure for vital capacity and FEVi was not noted.
In summary, no studies of inhaled urea in experimental animals were identified and
human studies involving possible inhalation exposure to urea are limited and inconclusive.
Therefore, information on the inhalation toxicity of urea is insufficient to derive an RfC.
5.2.2. Previous RfC Assessment
An IRIS assessment for urea does not currently exist on the IRIS database.
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5.3. CANCER ASSESSMENT
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is
inadequate information to access the carcinogenic potential of urea, (see Section 4.7).
Epidemiologic studies of humans exposed to urea alone or urea-containing mixtures are limited.
A single study showed that occupational exposure to urea increased levels of potential
carcinogenic biomarkers (e.g., CEA and PSA), but these increases were within the normal
physiologic range (El Far et al., 2006). One additional study indicated that urea was a possible
risk factor in bladder cancer deaths (Marsh et al., 2002). However, the low incidence of bladder
cancers deaths and the possibility of coexposure to other chemicals (nitric acid and acrylonitrile)
limited the power of the study.
Two chronic studies in laboratory animals have evaluated the carcinogenic potential of
urea (Fleischman et al., 1980; Shear and Leiter, 1941). Fleischman et al. (1980) reported a non-
dose related statistically significant increase in the incidence of malignant lymphomas among the
mid-dose female mice after 12 months of exposure and a statistically significant increase in the
incidence of interstitial cell adenomas of the testes in high-dose male rats. Additionally,
discrepancies in data reporting were noted. Shear and Leiter (1941) reported no treatment
related tumors in mice that were administered urea via s.c. injection for 12 months. Genotoxicity
assays show that urea did not induce mutations in bacterial or E. coli systems. Urea may have
the potential to induce single strand breaks in some systems, but not double strand breaks. In
vitro and in vivo chromosomal aberration studies have demonstrated mixed results with some
studies showing chromosomal aberrations while others indicate no chromosomal damage. In
several studies, specific details of the results were not provided
5.3.1. Choice of Study/Data - with Rationale and Justification
The limitations of the data available to assess the carcinogenic potential of urea preclude the
derivation of an oral cancer slope factor or inhalation unit risk.
5.3.2. Previous Cancer Assessment
An assessment fro urea does not currently exist on the IRIS database.
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
Urea (CAS No. 57-13-6), also known as carbamide, is an endogenous product of protein
and amino acid catabolism. It can also be produced synthetically by combining ammonia, carbon
monoxide, and sulfur in methanol. It is used in a variety of applications including fertilizers,
animal feed, plastics, flame-proofing agents, diesel-selective catalytic reduction, flavoring agent
in foods, and in the manufacture of consumer goods such as liquid soaps, detergents, and
household cleaning products.
In the occupational setting, the most notable routes of exposure are inhalation and
dermal, while the general population might be exposed to urea through consumption of food and
drinking water and through dermal contact with urea-containing products.
There is limited information on the absorption, distribution, excretion and metabolism of
exogenous urea. There are limited studies that evaluate the possible association between oral
exposure to urea and noncancer effects in humans There is limited information to suggest that the
liver, kidney, and pituitary could be targets of urea toxicity. Results from reproductive and
developmental studies have been inconclusive. There have been few studies that have evaluated
the effects of urea via inhalation. The available studies suggest that the impact of urea exposure
on lung function is minimal. With regard to dermal effects, the available studies showed that
there is a dependence on the vehicle used and effects are typically manifested in the form of skin
irritations.
The human carcinogenic potential of urea and urea-containing mixtures has been
evaluated in a limited number of studies. Some studies that evaluated urea-containing mixtures
indicate that urea exposure may have contributed to the occurrence of tumor development, or
increased sister chromatid exchange and chromosomal aberration (CA) frequency, but its role in
producing the observed effects was not clearly established. One occupational study showed that
exposure to urea increased levels of potential carcinogenic biomarkers (e.g., CEA and PSA), but
these increases were within the normal physiologic range (El Far et al., 2006). Two chronic
studies in rats and mice showed no treatment-related increase in tumors following either oral or
s.c. administration (Fleischman et al., 1980; Shear and Leiter, 1941).
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is
inadequate information to access the carcinogenic potential of urea. Epidemiologic studies of
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humans chronically exposed to urea alone or urea-containing mixtures are limited. Urea has
been tested for genotoxic potential and has shown no mutagenic effects in bacterial systems;
however, chromosomal aberrations have been noted in certain mammalian test systems and
hence the role of genotoxicity in the process of urea-induced carcinogenicity cannot be
eliminated.
6.2. DOSE RESPONSE
6.2.1. Noncancer / Oral
Oral exposure studies of urea were not adequate for the determination of an RfD. The
available animal studies identify the liver and kidney as a potential target organs for the toxicity
of urea; however, the available information is insufficient to fully characterize toxicity outcomes
or dose-response relationships.
6.2.2. Noncancer / Inhalation
Inhalation data were inadequate for the determination of a reference concentration. The
occupational data lacked quantitative exposure measurements. The cited therapeutic study on
lung function was based on acute exposure and had limited information on which to derive an
RfC. No studies of inhaled urea in experimental animals were identified.
6.2.3. Cancer / Oral
One oral cancer bioassay is available for consideration for the derivation of an oral slope
factor for urea. However, the limitations of the study data preclude the derivation of an oral
cancer slope factor.
6.2.4. Cancer / Inhalation
Inhalation studies for urea were not adequate for the determination of an inhalation unit
risk value. Route extrapolation from oral bioassay data was not performed due to the lack of oral
data and suitable kinetic data.
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7. REFERENCES
Abaurre, PF; Stefanon, I; Mill, JO; et al. (1992) Electromechanical effects of urea on the isolated rat heart. Braz J
Med Biol Res 25(7):717-726.
Agner, T. (1992) An experimental study of irritant effects of urea in different vehicles. Acta Derm Venereol Suppl
(Stockh) 177:44-46.
AIHA (American Industrial Hygiene Association). (1999) The AIHA 1999 Emergency Response Planning
Guidelines and Workplace Environmental Exposure Level Guides Handbook. American Industrial Hygiene
Association. Fairfax, VA.
Al-Homrany, M. (2001) In vitro effect of urea on serum enzymes levels. Biomed Res 12(2):87-89.
Bagnasco, SM. (2005) Role and regulation of urea transporters. Pflugers Arch - Eur J Physiol 450(4):217-226.
Balestri, PL; Rindi, P; Biagini, M. (1971) Chronic urea intoxication in dogs. Experientia 27(7):811 —812.
Bensinger, TA; Mahmood, L; Conrad, ME; et al. (1972) The effect of oral urea administration on red cell survival in
sickle cell disease. Am J Med Sci 264(4):283-287.
Bhat, MR; Ramaswamy, C. (1993) Effect of ammonia, urea and diammonium phosphate (DAP) on lung functions in
fertilizer plant workers. Indian J Physiol Pharmacol 37(3):221-224.
Blake, DA; Burnett, LS; Miyasaki, BC; et al. (1976) Comparative effects of hyperosmolar urea administered by
intra-amniotic, intravenous, and intraperitoneal routes in rhesus monkeys. Am J Obstet Gynecol 124(3):239-244.
Bloom, FE. (1990) Neurohumoral transmission and the central nervous system. In: Oilman, AG; Rail, TW; Nies,
AS; et al.; eds. Goodman's and Oilman's the pharmacological basis of therapeutics. 8th edition. Elmsford, NY:
Pergamon Press, Inc.; pp. 244-268.
Bulpitt, CJ; Breckenridge, A. (1976) Plasma urea in hypertensive patients. Br Heart J 38(7):689-694.
Burg, MB; Ferraris, JD; Dmitrieva, NI. (2007) Cellular response to hyperosmotic stresses. Physiol Rev 87(4): 1441—
1474.
Button, C; Joubert, JP; Maartens, BP. (1982) Absence of urea toxicity in young pigs. J S Afr Vet Assoc 53(1):67-
68.
Cade, JF; Pain, MC. (1972) Lung function in provoked asthma: responses to inhaled urea, methacholine and
isoprenaline. Clin Sci 43(6):759-769.
Chaurasia, OP. (1991) Randomness of chromosome breaks in bone marrow cells of fertilizer-fed mice,Mus
musculus. Cytobios 67(268):7-12.
Chaurasia, OP; Sinha, SP. (1987) Effects of urea on mitotic chromosomes of mice and onion. Cytologia 52(4):877-
882.
Chung, E; Yocca, F; Van Woert, MH. (1985) Urea-induced myoclonus: medullary glycine antagonism as
mechanism of action. Life Sci 36(11): 1051-1058.
Cohen, DM. (1999) Signalling and gene regulation by urea and NaCl in the renal medulla. Clin Exp Pharmacol
Physiol 26(l):69-73.
Cohen, DM; Gullans, SR. (1993a) Urea selectively induces DNA synthesis in renal epithelial cells. Am J Physiol 264(4 Pt
2):F601-F607.
84 DRAFT - DO NOT CITE OR QUOTE
-------
Cohen, DM; Gullans, SR. (1993b) Urea induces EGR-1 and c-fos expression in renal epithelial cells. Am J Physiol
264(4 Pt 2):F593-F600.
Cohen, DM; Gullans, SR; Chin, WW. (1996) Urea signaling in cultured murine inner medullary collecting duct
(mIMCD3) cells involves protein kinase c, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase.
J Clin Invest 97(8): 1884-1889.
Conner, EA; Blake, DA; Parmley, TH; et al. (1976) Efficacy of various locally applied chemicals as
contragestational agents in rats. Contraception 13(5):571—582.
Cuparencu B; Grosu L; Tomus L; et al. (1961) Research on the pharmacodynamic effects of urea on the
cardiovascular system. Acta Biol Med Ger 6:123-140.
Dawes, C. (2006) Absorption of urea through the oral mucosa and estimation of the percentage of secreted whole
saliva inadvertently swallowed during saliva collection. Arch Oral Biol 51 (2): 111—116.
De Brabander, M; Van de Veire, R; Aerts, F; et al. (1976) A new culture model facilitating rapid quantitative testing
of mitotic spindle inhibition in mammalian cells. J Natl Cancer Inst 56(2):357-363.
Decker, D. (1996) Ammonia (urea) toxicosis in ruminants. Indiana Animal Disease Diagnostic Laboratory, West
Lafayette, IN. Available online at http://www.addl.purdue.edu/newsletters/1996/winter/ammonia.shtml. Accessed
February 2008.
D'Hooge, R; Van de Vijver, G; Van Bogaert, P-P; et al. (2003) Involvement of voltage- and ligand-gated Ca2+
channels in the neuroexcitatory and synergistic effects of putative uremic neurotoxins. Kidney Int 63(5): 1764-1775.
Doran, JJ; Klein, JD; Kim, YH; et al. (2006) Tissue distribution of UT-A and UT-B mRNA and protein in rat. Am J
Physiol Regul Integr Comp Physiol 290(5):R1446-459.
Eknoyan, G; Wacksman, SJ; Glueck HI; et al. (1969) Platelet function in renal failure. N Engl J Med 280(13):677-
681.
El Far, M; El Naggar, M; Elkhawaga, OA; et al. (2006) Carcinoembryonic antigen, alpha-fetoprotein, and prostate-
specific antigen in the sera of industrial workers exposed to phenol, formaldehyde, urea, and mixed vapors. Inhal
Toxicol 18(13): 1041—1046.
Esaian, AM; Titova, VA; Shishkina, LI; et al. (1997) Effect of urea on the course of experimental uremia in
subtotally nephrectomized rats. Patol Fiziol Eksp Ter 2:39-41.
Fair, RH; Krum, RJ. (1979) Double blind irritation study of a new stabilized base hydrocortisone cream. Curr Ther
Res Clin Exp 26(5):611-614.
Fenton, RA; Howorth, A; Cooper, GJ; et al. (2000) Molecular characterization of a novel UT-A urea transporter
isoform (UT-A5) in testis. Am J Physiol Cell Physiol 279(5):C1425-C1431.
Fenton, RA; Stewart, GS; Carpenter, B; et al. (2002) Characterization of mouse urea transporters UT-A1 and
UT-A2. Am J Physiol Renal Physiol 283: F817-F825.
Finlayson, JS; Baumann, CA. (1956) Responses of rats to urea and related substances. The use of a spaced-feeding
technique. JNutr 59(2):211-221.
Fleischman, RW; Baker, JR; Hagopian, M; et al. (1980) Carcinogenesis bioassay of acetamide, hexanamide,
adipamide, urea and p-tolylurea in mice and rats. J Environ Pathol Toxicol 3:149-170.
Forsythe, SJ; Parker, DS. (1985) Urea turnover and transfer to the digestive tract in the rabbit. Br JNutr 53(1): 183—
190.
85
DRAFT - DO NOT CITE OR QUOTE
-------
Garberg, P; Akerblom, E; Bolcsfoldi, G. (1988) Evaluation of a genotoxicity test measuring DNA-strand breaks in
mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution. Mutat Res 203(3): 155-176. - From §4.4
of LSP
Gollhausen, R; Kligman, AM. (1985) Effects of pressure on contact dermatitis. Am J Ind Med 8(4-5):323-328.
Gomella, LG; Haist, SA. (2004) Clinician's Pocket Reference. McGraw-Hill, New York, NY.
Grollman, EF; Grollman, A. (1959) Toxicity of urea and its role in the pathogenesis of uremia. J Clin Invest
38(5):749-754.
Guo, L; Zhao, D; Song, Y; et al. (2007) Reduced urea flux across the blood-testis barrier and early maturation in the
male reproductive system in UT-B-null mice. Am J Physiol Cell Physiol 293(1):C305-C312.
Halsted, JA (1976) The Laboratory in Clinical Medicine. W.B. Saunders Company, Philadelphia, PA.
Hardy, RN; Lowe, KC; McNaughton, DC (1983) Acute responses during blood substitution in the conscious rat. J
Physiol, 338:451-461.
Hellmer, L; Bolcsfoldi, G. (1992) An evaluation of the E. coli k-12 UVRB/RECA DNA repair host-mediated assay.
I. In vitro sensitivity of the bacteria to 61 compounds. Mutat Res 272(2): 145-160.
Hu, M-C; Bankir, L; Michelet, S; et al. (2000) Massive reduction of urea transporters in remnant kidney and brain of
uremic rats. Kidney Int 58(3): 1202-1210.
Inoue, H; Kozlowski, SD; Klein, JD; et al. (2005) Regulated expression of renal and intestinal UT-B urea transporter
in response to varying urea load. Am J Physiol Renal Physiol 289(2):F451-F458.
Ishidate, M, Jr; Odashima, S. (1977) Chromosome tests with 134 compounds on Chinese hamster cells in vitro—a
screening for chemical carcinogens. Mutat Res 48(3-4):337-353.
Ishidate, M, Jr; Yoshikawa, K. (1980) Chromosome aberration tests with Chinese hamster cells in vitro with and
without metabolic activation—a comparative study on mutagens and carcinogens. Arch Toxicol Suppl 4:41-44. -
From §4.4 of LSP
Ishidate, M; Sofuni, T; Yoshikawa, K. (1981) Chromosomal aberration tests in vitro as a primary screening tool for
environmental mutagens and/or carcinogens. GANN Monogr Cancer Res 27:95-108.
Javid, M; Anderson, J. (1958) Observations on the use of urea in rhesus monkeys. Surg Forum 9:686-690.
Johanson, CE; Woodbury, DM. (1978) Uptake of [14C]urea by the in vivo choroid plexus—cerebrospinal fluid-
brain system: identification of sites of molecular sieving. J Physiol 275:167-176.
Johnson, AJ; Rebuck, JW; Knoll, BF. (1970) The effect of autogenous urine on leukocytic defenses in man. II. The
response to varied concentrations of saline, urea, and urine. Invest Urol 8(2):224-230.
Kamm, DE; Wu, L; Kuchmy, BL (1987), Contribution of the urea appearance rate to diuretic—induced
azotemia in the rat. Kid Int, 32:47-56.
Kaplan, B; Wang, Z; Siddhom, O; et al. (1999) Evaluation of urea kinetics utilizing stable isotope urea and
pharmacokinetic modeling. Artif Organs 23(l):44-50.
Kim, D; Klein, JD; Racine, S; et al. (2005) Urea may regulate urea transporter protein abundance during osmotic
diuresis. Am J Physiol Renal Physiol 288(1):F188-F197.
Kloppenburg, WD; Wolthers, BG; Stellaard, F; et al. (1997) Determination of urea kinetics by isotope dilution with
[13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis. Clin Sci 93(1):73—80.
86
DRAFT - DO NOT CITE OR QUOTE
-------
Kommadath, A; Sharma, A; Jakhar, KK. (2001) Hepatotoxic, nephrotoxic and genotoxic effects in mice fed urea
adulterated milk. Indian JDairy Sci 54(6):316—321.
Kornegay, ET; Mosanghini, V; Snee, RD. (1970) Urea and amino acid supplementation of swine diets. JNutr
100(3):330-340.
Kultz, D; Chakravarty, D. (2001) Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in
murine kidney cells. Proc Natl Acad Sci USA 98(4): 1999-2004.
Lashmar, UT; Hadgraft, J; Thomas, N. (1989) Topical application of penetration enhancers to the skin of nude mice:
a histopathological study. J Pharm Pharmacol 41(2): 118-122.
Levine, S; Saltzman, A. (2001) Are urea and creatinine uremic toxins in the rat? Ren Fail 23(1):53—59.
Lucien, N; Bruneval, P; Lasbennes, F; et al. (2005) UT-B1 urea transporter is expressed along the urinary and
gastrointestinal tracts of the mouse. Am J Physiol Regul Integr Comp Physiol 288(4):R1046-R1056.
MacKay, L; MacKay, E; Addis, T. (1931) Factors which determine renal weight. XII. The nitrogen intake as varied
by the addition of urea to the diet. J Nutr 4(3):379-383.
Maddock, AL; Westenfelder, C. (1996) Urea induces the heat shock response in human neuroblastoma cells. J Am
Soc Nephrol 7(2):275-282.
Marini, JC; Lee, B; Garlick, PJ. (2006) In vivo urea kinetic studies in conscious mice. J Nutr 136:202-206.
Marsh, GM; Gula, MJ; Youk, AO; et al. (2002) Bladder cancer among chemical workers exposed to nitrogen
products and other substances. Am J Ind Med 42(4):286-295.
Massy, ZA; Ivanovski, O; Nguyen-Khoa, T; et al. (2005) Uremia accelerates both atherosclerosis and arterial
calcification in apolipoprotein e knockout mice. J Am Soc Nephrol 16(1): 109-116.
Matthews, DE; Downey, RS. (1984) Measurement of urea kinetics in humans: a validation of stable isotope tracer
methods. Am J Physiol 246(6 Pt 1):E519-E527.
McKay, E; Kilpatrick, SJ. (1964) Maternal and infant plasma urea at delivery. J Obst Gynaecol Brit Comm 71:449-
452.
Miryata, Y; Asano, Y; Muto, S. (2002) Hyperosmotic urea activates basolateral NHE in proximal tubule from P-GP
null and wild-type mice. Am J Physiol Renal Physiol 283(4):F771-F783.
Moeslinger, T; Spieckermann, PG. (2001) Urea-induced inducible nitric oxide synthase inhibition and macrophage
proliferation. Kidney Int Suppl 78:S2-8.
Moran, BJ; Jackson, AA. (1990a) 15N-urea metabolism in the functioning human colon: luminal hydrolysis and
mucosal permeability. Gut 31(4):454-457.
Moran, BJ; Jackson, AA. (1990b) Metabolism of 15N-labelled urea in the functioning and defunctioned human
colon. Clin Sci 79(3):253-258.
Mortelmans, K; Haworth, S; Lawlor, T; et al. (1986) Salmonella mutagenicity tests: II. Results from the testing of
270 chemicals. Environ Mutagen 8 (Suppl 7): 1-119.
National Research Council. (1983) Risk assessment in the federal government: managing the process. Washington,
DC: National Academy Press.
Nesslany, F; Marzin, D. (1999) A micromethod for the in vitro micronucleus assay. Mutagenesis 14(4):403-410.
87
DRAFT - DO NOT CITE OR QUOTE
-------
NLM (National Library of Medicine). (2008a) Urea. HSDB (Hazardous Substances Data Bank). National Institutes
of Health, U.S. Department of Health and Human Services, Bethesda, MD. Available online at
http://toxnet.nlm.nih.gOv/cgi-bin/sis/search/f7./temp/~9kTlQB: 1. Accessed February 2008.
NLM. (2008b) Urea. ChemlDplus Advanced. National Institutes of Health, U.S. Department of Health and Human
Services, Bethesda, MD. Available online at
http://chem.sis.nlm.nih. gov/chemidplus/ProxyServlet?objectHandle=Search&actionHandle=getA113DMViewFiles&
nextPage=jsp%2Fcommon%2FChemFull.jsp%3FcalledFrom%3D&chemid=000057136&formatType=_3D.
Accessed February 2008.
NLM. (2008c) Urea. PubChem. National Institutes of Health, U.S. Department of Health and Human Services,
Bethesda, MD. Available online at http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=l 176. Accessed
February 2008.
Nomura, N; Matsumoto, S; Nishimura, Y; et al. (2006) Disposition of exogenous urea and effects of diet in rats.
Arzneimittelforschung 56(3):258-266.
NTP (National Toxicology Program) (2009) Oral,Feed - Testes, Ademomas. NTP Historical Controls for NTP-2000
Diet. Updated July 2009 (http://ntp.mehs.mh.gov/?objectid=92E705C7-FlF6-975E-72D23026B1645EB9#9).
Accessed December 2009.
Odeh, YK; Wang, Z; Ruo, TI; et al. (1993) Simultaneous analysis of inulin and 15N2-urea kinetics in humans. Clin
Pharmacol Ther 53(4):419-425.
OECD SIDS (Organisation for Economic Co-operation and Development Screening Information Data Set). (2008)
Urea. CASN0: 57-13-6. Available online at http://www.inchem.org/documents/sids/sids/57136.pdf. Accessed
February 2008.
Ok, E; Basnakian, AG; Apostolov, EO; et al. (2005) Carbamylated low-density lipoprotein induces death of
endothelial cells: a link to atherosclerosis in patients with kidney disease. Kidney Int 68(1): 173-178.
Okada, M; Kobayashi, Y. (1989) Fine structural alterations in mouse pituitary intermediate lobe cells following oral
urea administration. Zoolog Sci 6(6):4-6.
O'Neil, MJ; Heckelman, PE; Koch, CB; et al.; eds. (2006) The Merck index: an encyclopedia of chemicals, drugs,
and biologicals. 14th edition. Whitehouse Station, NJ: Merck and Co., Inc.; p. 9867.
Oppenheim, JJ; Fishbein, WN. (1965) Induction of chromosome breaks in cultured normal human leukocytes by
potassium arsenite, hydroxyurea and related compounds. Cancer Res 25(7):980-985.
Ordonez, A; Parkinson, TJ; Matthew, C; et al. (2007) Effects of application in spring of urea fertiliser on aspects of
reproductive performance of pasture-fed dairy cows. N Z Vet J 55(2):69-76.
Ortolani, EL; Mori, CS; Rodrigues Filho, JA. (2000) Ammonia toxicity from urea in a Brazilian dairy goat flock.
Vet Hum Toxicol 42(2):87-89.
Pagana, KD; Pagana, TJ. (2003) Mosby's diagnostic and laboratory test reference. 6th edition. St. Louis, MO:
Mosby, Inc.; pp. 738-741.
Raidal, SR; Jaensch, SM. (2006) Acute poisoning of silver gulls (Larus novaehollandiae) following urea fertilizer
spillage. Avian Pathol 3 5 (1): 3 8-41.
Ranade, K; Wu, KD; Hwu, CM; et al. (2001) Genetic variation in the human urea transporter-2 is associated with
variation in blood pressure. Hum Mol Genet 10(19):2157—2164.
Rapoport, SI; Fitzhugh, R; Pettigrew, KD; et al. (1982) Drug entry into and distribution within brain and
cerebrospinal fluid: [14C]urea pharmacokinetics. Am J Physiol 242(3):R339-R348.
88
DRAFT - DO NOT CITE OR QUOTE
-------
Rebuck, JW; Crowley, JH. (1955) A method of studying leukocytic functions in vivo. Ann NY Acad Sci
59(5):757-805.
Registry. (2008) RN 57-13-6. Database produced by Chemical Abstract Service (CAS) and available on STN
International.
Rhoads, ML; Rhoads, RP; Gilbert, RO; et al. (2006) Detrimental effects of high plasma urea nitrogen levels on
viability of embryos from lactating dairy cows. AnimReprod Sci 91 (1—2): 1—10.
Rusoff, LL; Lank, RB; Spillman, TE; et al. (1965) Non-toxicity of urea feeding to horses. Vet Med Small Animal
Clin 60(11): 1123—1126.
Sahin, S; Rowland, M. (2007) Influence of erythrocytes on the hepatic distribution kinetics of urea and thiourea.
Eur J Pharm Sci 31(3-4): 180-189.
Sands, JM. (2003) Mammalian urea transporters. Annu Rev Physiol 65:543-566.
Seipelt, H; Zoellner, K; Hilgenfeld, E; et al. (1969) Untersuchungen an nieren neugeborener ratten nach chronischer
harnstoffapplikation beim muttertier [Studies on kidneys of newborn rats after chronic urea administration to the
mother], Z Urol Nephrol 62(8):623-627.
Serup, J. (1992) A double-blind comparison of two creams containing urea as the active ingredient. Assessment of
efficacy and side-effects by non-invasive techniques and a clinical scoring scheme. Acta Derm Venereol Suppl
(Stockh) 177:34-43.
Shear, MJ; Leiter, J. (1941) Studies in carcinogenesis. XVI. Production of subcutaneous tumors in mice by
miscellaneous polycyclic compounds. J Natl Cancer Inst 2:241-258.
Shimizu, H; Suzuki, Y; Takemura, N; et al. (1985) The results of microbial mutation test for forty-three industrial
chemicals. Sangyo Igaku 27(6):400-419.
Sina, IF; Bean, CL; Dysart, GR; et al. (1983) Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of
carcinogenic/mutagenic potential. Mutat Res 113(5):357—391.
Spector, DA; Yang, Q; Wade, JB. (2007) High urea and creatinine concentrations and urea transporter B in
mammalian urinary tract tissues. Am J Physiol Renal Physiol 292(1):F467-F474.
Stewart, GS; Fenton, RA; Thevenod, F; et al. (2004) Urea movement across mouse colonic plasma membranes is
mediated by UT-A urea transporters. Gastroenterology 126(3):765-773.
Steyn, DG. (1961) An outbreak of urea poisoning among Bantu farm laborers in the Potgietersrust District,
Transvaal. S African Med J 35(35):721-722.
Teramoto, S; Kaneda, M; Aoyama, H; et al. (1981) Correlation between the molecular structure of N-alkylureas and
N-alkylthioureas and their teratogenic properties. Teratology 23(3):335—342.
Thurston, J; Hauhart, R; Dirgo, J; et al. (1986) Effects of acute hyperosmolar NaCl or urea on brain H20, Na+, K+,
carbohydrate, and amino acid metabolism in weanling mice: NaCl induces insulin secretion and hypoglycemia.
Metab Bram Dis 1(2): 129-146.
Tian, W; Cohen, DM. (2002) Urea stress is more akin to EGF exposure than to hypertonic stress in renal medullary
cells. Am J Physiol Renal Physiol 283(3):F388-F398.
Tian, W; Boss, GR; Cohen, DM. (2000) RAS signaling in the inner medullary cell response to urea and NaCl. Am J
Physiol Cell Physiol 278(2):C372-C380.
Topham, JC. (1980) Do induced sperm-head abnormalities in mice specifically identify mammalian mutagens rather
than carcinogens? Mutat Res 74(5):379-387.
89
DRAFT - DO NOT CITE OR QUOTE
-------
Tsukaguchi, H; Shayakul, C; Berger, UV; et al. (1997) Cloning and characterization of the urea transporter UT3:
localization in rat kidney and testis. J Clin Invest 99:1506-1515.
Umeda, M; Noda, K; Ono, T. (1980) Inhibition of metabolic cooperation in Chinese hamster cells by various
chemical sincluding tumor promoters. Jpn J Cancer Res 71:614-620.
U.S. EPA (Environmental Protection Agency). (1986a) Guidelines for the health risk assessment of chemical
mixtures. Federal Register 51 (185):34014—34025. Available online at http://www.epa.gov/iris/backgr-d.htm.
Accessed February 2008.
U.S. EPA. (1986b) Guidelines for mutagenicity risk assessment. Federal Register 51(185):34006-34012. Available
online at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk assessment. Prepared
by the Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Cincinnati,
OH for the Office of Solid Waste and Emergency Response, Washington, DC; EPA 600/6-87/008. Available online
at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (1991) Guidelines for developmental toxicity risk assessment. Federal Register 56(234):63798-63826.
Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity studies.
Federal Register 59(206):53799. Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed February
2008.
U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation
dosimetry. Office of Research and Development, Washington, DC; EPA/600/8-90/066F. Available online at
http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/R-94/007. Available online at
http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=42601. Accessed February 2008.
U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessment. Federal Register 61(212):56274-56322.
Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (1998) Guidelines for neurotoxicity risk assessment. Federal Register 63(93):26926-26954. Available
online at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (2000a) Science policy council handbook: risk characterization. Office of Science Policy, Office of
Research and Development, Washington, DC; EPA 100-B-00-002. Available online at
http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (2000b) Benchmark dose technical guidance document [external review draft]. Risk Assessment Forum,
Washington, DC; EPA/630/R-00/001. Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed February
2008.
U.S. EPA. (2000c) Supplementary guidance for conducting for health risk assessment of chemical mixtures. Risk
Assessment Forum, Washington, DC; EPA/630/R-00/002. Available online at
http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (2002) A review of the reference dose and reference concentration processes. Risk Assessment Forum,
Washington, DC; EPA/630/P-02/0002F. Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed
February 2008.
U.S. EPA. (2005a) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC;
EPA/630/P-03/001B. Available online at http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
90
DRAFT - DO NOT CITE OR QUOTE
-------
U.S. EPA. (2005b) Supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. Risk
Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available online at http://www.epa.gov/iris/backgr-
d.htm. Accessed February 2008.
U.S. EPA. (2006a) Science policy council handbook: peer review. Third edition. Office of Science Policy, Office of
Research and Development, Washington, DC; EPA/100/B-06/002. Available online at
http://www.epa.gov/iris/backgr-d.htm. Accessed February 2008.
U.S. EPA. (2006b) A framework for assessing health risk of environmental exposures to children. National Center
for Environmental Assessment, Washington, DC, EPA/600/R-05/093F. Available online at
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=158363. Accessed February 2008.
Walser, M; Bodenlos, LJ (1959) Urea metabolism in man. J Clin Invest 38:1617-26.
Wangenheim, J; Bolcsfoldi, G. (1988) Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds.
Mutagenesis 3(3): 193-205.
Wardle, EN. (1970) A study of the effects of possible toxic metabolites of uraemia on red cell metabolism. Acta
Haematol 43(3): 129-143.
Wolpert, E; Phillips, SF; Summerskill, WH. (1971) Transport of urea and ammonia production in the human colon.
Lancet 2(7739): 1387-1390.
Word, JD; Martin, LC; Williams, DL; et al. (1969) Urea toxicity studies in the bovine. J Anim Sci 29(5):786-791.
Zarnke, RL; Taylor, WP, Jr. (1982) Urea poisoning in free-ranging Alaskan bison. J Am Vet Med Assoc
181(11): 1417.
Zhang, Z; Yang, X-Y; Cohen, DM. (1999) Urea-associated oxidative stress and GADD153/CF10P induction. Am J
Physiol 276(5, Pt. 2):F786-F793.
Zhang, Z; Yang, XY; Soltoff, SP; et al. (2000a) PI3K signaling in the murine kidney inner medullary cell response
to urea. Am J Physiol Renal Physiol 278(1):F155-F164.
Zhang, Z; Tian, W; Cohen, DM. (2000b) Urea protects from the proapoptotic effect of NaCl in renal medullary
cells. Am J Physiol Renal Physiol 279(2):F345-F352.
Zhang, Z; Dmitrieva, NI; Park, JH; et al. (2004) Fligh urea and NaCl carbonylate proteins in renal cells in culture
and in vivo, and high urea causes 8-oxoguanine lesions in their DNA. Proc Natl Acad Sci USA 101 (25):9491—9496.
Zhurkov, VS. (1975) Investigation of the mutagenic activity of drug preparations and food additives in a culture of
human lymphocytes. Sov Genet 11:528—530.
91
DRAFT - DO NOT CITE OR QUOTE
------- |