United States
Environmental Protection
1=1 m m Agency
EPA/690/R-14/007F
Final
3-19-2014
Provisional Peer-Reviewed Toxicity Values for
Guanidine Compounds
(CASRN 113-00-8 Guanidine)
(CASRN 506-93-4 Guanidine Nitrate)
(CASRN 50-01-1 Guanidine Chloride)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Dan D. Petersen, PhD., DABT
National Center for Environmental Assessment, Cincinnati, OH
CONTRIBUTOR
Scott C. Wesselkamper, PhD
National Center for Environmental Assessment, Cincinnati, OH
PRIMARY INTERNAL REVIEWERS
Zheng (Jenny) Li, PhD, DABT
National Center for Environmental Assessment, Washington, DC
Anuradha Mudipalli, MSc, PhD
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).

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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	ii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVs	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	6
CASE REPORT SUMMARIES	11
Lambert (1966)	11
Jimenez-Pabon (1968)	11
Vroom and Engel (1969)	12
Nakano and Tyler (1970)	12
Oh (1972)	12
Oh and Kim (1973)	12
Cherington (1976)	13
Henriksson et al. (1977)	13
Blumhardt et al. (1977)	13
Streib (1979)	13
Jenkyn et al. (1980)	14
Strieb and Rothner (1981)	14
Dau and Denys (1982)	14
Jablecki (1984)	14
Bady et al. (1987)	15
Silbert et al. (1990)	15
Oh et al. (1997, 1998)	15
Tim et al. (1998, 2000)	16
GENOTOXICITY DATA	16
CATEGORICAL REGRESSION ANALYSIS	16
DERIVATION OF PROVISIONAL REFERENCE VALUES	20
DERIVATION OF ORAL REFERENCE DOSES	22
Derivation of Subchronic Provisional RfD (Subchronic p-RfD) for Guanidine
Chloride	22
Derivation of Chronic Provisional RfD (chronic p-RfD) Guanidine Chloride	24
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	25
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	25
CANCER WEIGHT OF EVIDENCE (WOE) DESCRIPTOR	25
APPENDIX A. PROVISIONAL SCREENING VALUES	27
CALCULATION OF SCREENING PROVISIONAL ORAL REFERENCES DOSES	27
Screening Subchronic Provisional RfD Calculations for Guanidine Compounds	27
Screening Chronic Provisional RfD Calculations for Guanidine Compounds	28
APPENDIX B. ALTERNATIVE METHODS	29
APPENDIX C. CATREG OUTPUT FILES	30
APPENDIX D. REFERENCES	33
l

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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower confidence limit
BMD
benchmark dose
BMDL
benchmark dose lower confidence limit
ERC
effective response concentration
ERCL
effective response concentration lower confidence limit
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
UFa
interspecies uncertainty factor
UFC
composite uncertainty factor
UFd
database uncertainty factor
UFh
intraspecies uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
GUANIDINE COMPOUNDS
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and sub chronic-duration exposures to substances of concern, to
present the major conclusions reached in the hazard identification and derivation of the PPRTVs,
and to characterize the overall confidence in these conclusions and toxicity values. It is not
intended to be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database (http://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.gov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
Several salts of guanidine are used commercially, pharmaceutically, in a variety of
laboratory applications, and for other purposes as identified below. Guanidine (see Figure 1 for
structure) is a protonated organic base under physiological conditions, sometimes termed
guanidinium. Several salts of guanidine (nitrate, sulfate, chloride, acetate, and carbonate) are
also commercially available. For example, guanidine nitrate (CASRN 506-93-4) is a high
production volume chemical (U.S. EPA, 1990) that is a strong oxidizer and chemically unstable
(see Table 1 for chemical properties). It is explosive from friction, heat, or shock, can ignite
organic materials on contact, and can emit toxic vapors including nitric acid and nitrogen oxide if
heated to decomposition. The chemical is used in the manufacture of explosives, disinfectants,
and photographic materials, and as a monopropellant for some model airplane engines. It is
important to note that while guanidine nitrate and nitroguanidine are both explosives, they are
different chemicals that are at times incorrectly assumed to be the same in some cases in the
literature. The physicochemical properties of the most common guanidine compounds are
shown in Table 1. Other guanidine salts have differing properties and uses. For example,
guanidine hydrochloride, also known as guanidine chloride (CASRN 50-01-1), is used
therapeutically to treat several rare neurologic diseases and in the laboratory as a protein
denaturant. It is known to have immunosuppressive and other side effects above therapeutic
doses.
Guanidine nitrate was considered "slightly" toxic in mice with an oral LD50 of
approximately 1,100 mg/kg (Brown et al., 1988). Other sources listed in the Hazardous
Substances Data Bank (HSDB) (NLM, 2008) report slightly different values in the rat
(989.6 mg/kg [males], 729.8 mg/kg [females]), and 1,105 mg/kg for male mice and 1,029 mg/kg
for female mice (gavage dosing). The acute inhalation toxicity (LC50) in the rat (4-hour
"3
duration) is also given as >0.853 mg/L (>0.853 mg/m ) (Brown et al., 1988). Dermal exposure
of rabbits produced only minor dermal irritation at 2 g/kg in the same report.
Guanidine chloride has roughly similar (within a factor of two) acute toxicities to
guanidine nitrate. Guanidine chloride is listed as having an oral LD50 of 475 mg/kg in the rat
(treatment conditions not specified) (NLM, 2008). Ecotoxicology data from Brown et al. (1988)
reports an EC50 (48-hour duration) for guanidine nitrate of 70.2 mg/L in Daphnia magna, a
cladoceran freshwater crustacean, with a lowest-observed-effect concentration (LOEC) of
4.2 mg/L and a no-observed-effect concentration (NOEC) of 2.9 mg/L based on immobilization
as the endpoint. In the less sensitive fathead minnow species (Pimephales promelas), the LOEC
was 424 mg/L and the NOEC was 181 mg/L (96-hour duration).
Available toxicity values for guanidine compounds are summarized in Table 2. There is
little information in the literature relative to the potential toxicity of guanidine or guanidine
compounds following subchronic- or chronic-duration exposure via the oral or inhalation routes.
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NH
HoN
NH
Figure 1. Guanidine Structure
Table 1. Chemical Properties of Guanidine Compounds
Property (unit)
Guanidine Nitrate
(CASRN 506-93-4)
Guanidine Chloride
(CASRN 50-01-1)
Molecular Weight (g/mol)
122.08a
95.53d-e
Boiling point (°C)
Decomposes13
Decomposes11
Melting point (°C)
213—215a
iso-iss46
Density (g/cm3)
1.44a
1.34d e-1.35f
Vapor pressure (Pa at 25°C)
Not available
Not available
pH (aqueous)
8-9.5°
7-8d
Solubility in water (g/100 mL at 20°C)
130 g/L3,0
2,000 g/Ld-2,280 g/Le'f
Relative vapor density (air = 1)
Not available
Not available
aSource: Chemical Book (no publication date [a], accessed 2-19-2014; guanidine nitrate).
bSource: NLM (2008).
°Source: CHEMICALLAND21 (no publication date, accessed 2-19-2014; guanidine nitrate).
dSource: CHEMICALLAND21 (no publication date, accessed 2-19-2014; guanidine hydrochloride).
eSource: Chemical Book (no publication date [b], accessed 2-19-2014; guanidine hydrochloride).
fSource: Chemical Book (no publication date [c], accessed 2-19-2014; guanidine hydrochloride).
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Table 2. Summary of Available Toxicity Values for Guanidine Nitrate
Source/
Parameter"
Value
(Applicability)
Notes
Reference
Date Accessed
Noncancer
ACGIH
NV
NA
ACGIH (2013)
NA
ATSDR
NV
NA
ATSDR (2013)
NA
Cal/EPA
NV
NA
Cal/EPA
(2013a,b)b
10-17-2013
NIOSH
NV
NA
NIOSH (2010)
NA
OSHA
NV
NA
OSHA (2006,
2011)
NA
IRIS
NV
NA
U.S. EPA
(2013)
10-17-2013
Drinking water
NV
NA
U.S. EPA
(2012)
NA
Drinking water
(Nitroguanidine)
RfD: 1 x KT1
mg/kg-d
Nitroguanidine is a different but structurally
related compound, which is also an explosive.
The RfD is based on reduced body weights of
rats (NOAEL of 316 mg/kg-d and a
composite uncertainty factor of 3,000).
U.S. EPA
(2012)
NA
HEAST
NV
NA
U.S. EPA
(2011)
NA
CARA HEEP
NV
NA
U.S. EPA
(1985, 1994)
NA
WHO
NV
NA
WHO
10-17-2013
U.S. Army
(Guanidine
nitrate)
RfD: 5 x KT1
mg/kg-d
This value is a Performance Standard for
guanidine nitrate with a surrogate RfD (based
on guanidine chloride) of 5 x 10 1 mg/kg-d,
intended as a guide in military cleanup
activities.
U.S. Army
(2012)
NA
NLM
NV
Mononitrate guanidine is included in the
Hazardous Substance Data Bank as cited by
ACGIH (2010). This citation mentions
human health effects (erythrocyte hemolysis
in vitro, and respiratory and dermal toxicity in
vivo), and nonhuman toxicity and ecotoxicity
values, but it does not include human
exposure limits.
NLM (2008)
NA
Cancer
IRIS
NV
NA
U.S. EPA
10-17-2013
HEAST
NV
NA
U.S. EPA
(2011)
10-17-2013
IARC
NV
NA
IARC (2013)
NA
NTP
NV
NA
NTP (2011)
NA
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Table 2. Summary of Available Toxicity Values for Guanidine Nitrate
Source/
Parameter"
Value
(Applicability)
Notes
Reference
Date Accessed
Cal/EPA
NV
NA
Cal/EPA (2009,
2013b)
NA
ACGIH
NV
NA
ACGIH (2013)
NA
"Sources: Integrated Risk Information System (IRIS) database; Health Effects Assessment Summary Tables
(HEAST); International Agency for Research on Cancer (IARC); National Toxicology Program (NTP); California
Environmental Protection Agency (Cal/EPA); American Conference of Governmental Industrial Hygienists
(ACGIH); Agency for Toxic Substances and Disease Registry (ATSDR); National Institute for Occupational Safety
and Health (NIOSH); National Library of Medicine (NLM); Occupational Safety and Health Administration
(OSHA); Chemical Assessments and Related Activities (CARA) list; Health and Environmental Effects Profile
(HEEP); World Health Organization (WHO).
bThe Cal/EPA Office of Environmental Health Hazard Assessment (OEHHA) Toxicity Criteria Database
(http://oehha.ca.gov/tcdb/index.asp) was also reviewed and found to contain no information on guanidine
compounds (CASRN 113-00-8 guanidine; CASRN 506-93-4 guanidine nitrate; CASRN 50-01-1 guanidine
chloride).
NA = not applicable; NV = not available; NOAEL = no-observed-adverse-effect level; RfD = reference dose.
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Literature searches were conducted on sources published from 1900 through
September 2013 for studies relevant to the derivation of provisional toxicity values for guanidine
(CASRN 113-00-8), guanidine nitrate (CASRN 506-93-4), and guanidine chloride
(CASRN 50-01-1). Searches were conducted using EPA's Health and Environmental Research
Online (HERO) database of scientific literature. HERO searches the following databases:
AGRICOLA; American Chemical Society; BioOne; Cochrane Library; DOE: Energy
Information Administration, Information Bridge, and Energy Citations Database; EBSCO:
Academic Search Complete; GeoRef Preview; GPO: Government Printing Office;
Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology; JSTOR: Mathematics
& Statistics and Life Sciences; NSCEP/NEPIS (EPA publications available through the National
Service Center for Environmental Publications [NSCEP] and National Environmental
Publications Internet Site [NEPIS] database); PubMed: MEDLINE and CANCERLIT databases;
SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network):
ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP,
GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search,
NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, and TRI; TSCATS; Virtual Health Library; Web
of Science (searches Current Content database among others); World Health Organization; and
Worldwide Science. The following databases outside of HERO were searched for relevant
health information: ACGM, AT SDR, Cal/EPA, EPA IRIS, EPA HEAST, EPA HEEP, EPA
OW, EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
The effects of oral or inhalation exposure to guanidine, guanidine nitrate, and guanidine
chloride have not been evaluated in any repeated-dose subchronic or chronic-duration,
developmental toxicity, reproductive toxicity, or carcinogenicity studies that are publically
available, in either humans or animals. However, in humans, guanidine chloride has been used
as an orally administered therapeutic in the management of Lambert-Eaton Myasthenic
Syndrome (LEMS), a rare autoimmune disease in which autoantibodies directed against
voltage-gated calcium channels at the presynaptic side of the neuromuscular junction result in
muscle weakness and autonomic dysfunction (see list of case reports in Table 3). Thus, the
occurrence of side effects induced by LEMS treatment with guanidine chloride is used as a basis
for the identification of toxicological effects for the derivation of provisional reference doses
(p-RfDs) in this PPRTV assessment. Examination of all citations listed within references
identified from the initial literature search revealed additional relevant case reports on LEMS
patients.
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Table 3. Summary of Potentially Relevant Data in LEMS Patients Treated Therapeutically With Guanidine Chloride
GC Dosimetry"
Effects Observed
NOAEL
LOAEL
Reference
Comments
Notesb
Human
1. Oral
20-30 mg/kg-d
Paresthesias (mouth)
NDr

Lambert (1966)
N= 1
Short-term-duration
PR; clear clinical and
electromyographic (EMG)
improvement.
14-42 mg/kg-d
Patient 1:
gastrointestinal (GI;
frequent bowel
movements), central
nervous system (CNS;
paresthesias, agitation,
insomnia, behavioral
disturbances
Patient 2: diarrhea and
nervous irritability
NDr

Kennedy and
Jimenez-Pabon (1968)
N= 2
Durations not
classifiable
PR; improvement in strength.
NA
None identified
NDr

Vroom and Engel
(1969)
N= 1
Duration not
classifiable
PR; no clinical or EMG improvement.
21 mg/kg-d
Atrial fibrillation and
hypotension
NDr

Nakano and Tyler
(1970)
N= 1
Short-term-duration
PR
12 mg/kg-d
None identified
12 mg/kg-d
(subchronic -
duration)

Oh (1972)
N= 1
Subchronic-duration
PR; "dramatic" clinical improvement.
12-58 mg/kg-d
Patient 1 = leukopenia
and mild
thrombocytopenia
Patient 2 = paresthesias
of tongue and fingers,
confusion, unsteady gait
Patient 3 = none
NDr
12 mg/kg-d
(subchronic-
duration)
Oh and Kim (1973)
N= 3
Patient 1 = short-term
duration;
Patient 2 =
subchronic-duration;
Patient 3 = duration
not provided.
PR; clinical improvement.
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Table 3. Summary of Potentially Relevant Data in LEMS Patients Treated Therapeutically With Guanidine Chloride
GC Dosimetry"
Effects Observed
NOAEL
LOAEL
Reference
Comments
Notesb
21-27 mg/kg-d
Nephrotoxicity (chronic -
duration interstitial
nephritis, and
leukopenia
NDr
24 mg/kg-d
(subchronic)
Cherington (1976)
N= 1
Subchronic-duration
PR
18-19 mg/kg-d
Patient 1 = renal,
hepatic, and pancreatic
damage, anemia
paresthesias
Patient 2 = paresthesias,
cold sensations and
pancytopenia
NDr
18 mg/kg-d
(subchronic-
duration)
Herriksson et al.
(1977)
N= 2
Patient 1 =
subchronic-duration;
Patient 2 =
short-term-duration.
PR
14 mg/kg-d
Fibrosing interstitial
nephritis
NDr
14 mg/kg-d
(chronic-duration)
Blumhardt et al.
(1977)
N= 1
Chronic-duration
PR; clinical improvement
30 mg/kg-d
Colicky pain,
postprandial fullness,
bloating, dark-colored
urine
NDr
NDr
Streib (1979)
N= 1
Short-term-duration
PR; also administered physostigmine
(dose not provided). EMG
improvement.
14-21 mg/kg-d
Leukopenia
NDr
NDr
Jenkynetal. (1980)
N= 1
Short-term-duration

12-45 mg/kg-d
Mild GI upset at
20 mg/kg-d; limb and
perioral paresthesias at
32 mg/kg-d; nausea,
tremors, confusion,
agitation at 45 mg/kg-d
12 mg/kg-d
(subchronic -
duration)
NDr
Streib and Rothner
(1981)
N= 3
Subchronic-durations
[Patient 2 was
previously recorded in
Streib (1979)];
Patient 3 received no
guanidine
PR; EMG and strength improvement.
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Table 3. Summary of Potentially Relevant Data in LEMS Patients Treated Therapeutically With Guanidine Chloride
GC Dosimetry"
Effects Observed
NOAEL
LOAEL
Reference
Comments
Notesb
16-29 mg/kg-d
Numbness, paresthesias,
coldness, emesis, renal
dysfunction
18 mg/kg-d
(subchronic -
duration)
16 mg/kg-d
(chronic-duration)
Dau and Denys (1982)
N= 5
Patients 1 and 3 =
subchronic-duration;
Patient 2 = short-
term-duration;
Patient 4 = chronic-
duration;
Patient 5 = duration
and dose not reported.
PR; other treatments concurrently
administered. Partial relief of
symptoms.
5 mg/kg-d
None reported
NDr
NDr
Jablecki (1984)
N= 1
Subchronic-duration
PR; slight improvement in strength.
10 mg/kg-d
None reported
NDr
NDr
Badyetal. (1987)
N= 1
Acute-duration
PR; EMG improvement.
5-20 mg/kg-d
Leukopenia
5 mg/kg-d
(subchronic -
duration)
12 mg/kg-d
(subchronic-
duration)
Silbert et al. (1990)
N= 1
Subchronic-duration
PR; reduction in muscular weakness.
5-36 mg/kg-d
Fainting spells, nausea,
vomiting, epigastric
pain, diarrhea,
paresthesias (fingers),
insomnia
5 mg/kg-d
(subchronic -
duration)
7 mg/kg-d
(chronic-
duration)
NDr
Ohetal. (1997)
N= 9
Patient 4 = acute-
duration;
Patients 1 and 5 =
short-term-duration;
Patients 2, 3,6, 7, and
9 = subchronic -
duration;
Patient 8 = chronic-
duration
PR; also administered physostigmine.
Clinical and EMG improvement.
6 mg/kg-d
None reported
NDr
NDr
Oh et al. (1998)
N= 1
Short-term-duration
PR; case report on one additional
patient from Oh et al. (1997).
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Table 3. Summary of Potentially Relevant Data in LEMS Patients Treated Therapeutically With Guanidine Chloride
GC Dosimetry"
Effects Observed
NOAEL
LOAEL
Reference
Comments
Notesb
Not Reported
None reported
NDr
NDr
Tim et al. (1998)
N= 51
Duration not
classifiable
PR; 7 of 57 patients received GC with
"moderate" improvement in 4 out of 7.
Not Reported
None reported
NDr
NDr
Tim et al. (2000)
N=13
Durations not
classifiable
PR; other treatments concurrently
administered. Nine of 73 patients
received GC with "marked"
improvement in 0%, "moderate"
improvement in 56%, "minimal"
improvement in 11%, and no
improvement in 33%.
aDoses are converted to mg/kg-d. If no patient weight was provided in the case reports, a default value of 70 kg was used in the conversion. If a range of doses was used
and details not specified, the mean of the range was used. Chronic-duration is defined as greater than 7 years; subchronic-duration is defined as greater than one year;
short-term-duration is defined as less than one year: acute duration refers to a single dose.
bPR = Peer reviewed, NDr = not determined.
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CASE REPORT SUMMARIES
As stated previously, the effects of oral or inhalation exposure to guanidine compounds
have not been evaluated in any repeated-dose sub chronic-duration, chronic-duration,
developmental toxicity, reproductive toxicity, or carcinogenicity publically available studies
conducted in either humans or animals. However, case reports on the treatment of LEMS in
humans by oral administration of guanidine chloride are reviewed below. Because LEMS results
from a lowering in acetylcholine release, some therapies involve cholinesterase inhibitors, such
as pyridostigmine, or potassium channel blockers (e.g., guanidine compounds, 4-aminopyridine,
3,4-diaminopyridine) which increase acetylcholine release. Guanidine inhibits potassium
channels by binding within the intracellular pore of the channel, whereby it interacts with its
hydrophobic subunit leading to the channel's closure (Kalia and Shwatz, 2011). Given that
LEMS is autoimmune in nature; immunosuppressive agents like the corticosteroid prednisone
have also been used therapeutically, sometimes in addition to guanidine.
The most important limitation in the use of guanidine compounds in the treatment of
LEMS is the occurrence of side effects, which include gastrointestinal effects (including nausea
and diarrhea), immunosuppression, and distal paresthesias. Throughout this section and in
Table 3 above, the word "symptom" is used to describe the severity of the muscular weakness or
other conditions characterized by LEMS, while "side effects" refer to negative effects of the
guanidine compound. The current treatment of choice for LEMS appears to be
3,4-diaminopyridine, but guanidine compounds are still occasionally used, in part due to the
unavailability of 3,4-diaminopyridine in some countries, including the United States. The
available data comes primarily from case reports of individual patients or small groups of LEMS
patients that received orally administered guanidine chloride. Only those case reports that
accurately report the dosimetry and side effects that might potentially identify points of departure
(PODs) or reveal additional side effects are summarized here. The purity of the guanidine
chloride used was not reported in any of the case reports, but current products have purities of
99% or greater. The durations that patients were treated with guanidine chloride range from
acute (i.e., single doses) to chronic durations. For the LEMS patients, treatment durations
greater than 7 years were considered to be chronic-duration, those less than 12 months were
considered short-term, and those between 12 months and 84 months were considered to be
subchronic durations.
Lambert (1966)
In an early case report, Lambert (1966) reported the treatment of a LEMS patient with
oral guanidine chloride in addition to several other potential treatments. Guanidine chloride was
used at doses of 20-30 mg/kg-day for at least 13 days, but the definitive treatment duration is
unknown. There was a reduction in the patient's neuromuscular weakness with treatment, and
the only side effect reported were paresthesias in the mouth that diminished with time. The
study author noted that the effect on the neuromuscular transmission peaked 45 to 60 minutes
after guanidine chloride administration, while the increase in strength lasted 3 to 4 days after the
discontinuation of the guanidine chloride. Neither a no-observed-adverse-effect level (NOAEL)
nor a lowest-observed-adverse-effect (LOAEL) is established for this paresthesia because the
duration of treatment was short-term.
Jimenez-Pabon (1968)
In a case report of two LEMS patients (both with lung cancer), Kennedy and
Jimenez-Pabon (1968) reported treatment with guanidine chloride. Patient 1 was treated with
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doses ranging from 14-42 mg/kg-day for approximately 5 months. At 32 mg/kg-day, there was
a resolution of the muscle weakness symptoms, but side effects occurred including paresthesias
of the mouth and tongue, momentary blurred vision, and an increase in the frequency of bowel
movements. Increasing the dose to 42 mg/kg-day caused agitation and more severe central
nervous system side effects including agitation, insomnia and behavioral effects. The behavioral
symptoms subsided when the dose was reduced to 25 mg/kg-day. Patient 2 was treated with
25 mg/kg-day guanidine chloride for 27 days but saw no resolution of symptoms, while side
effects including severe diarrhea and nervous irritability were noted. While these were
short-term treatments, an accurate determination of the treatment duration could not be made (the
initiation of therapy is given as "July"). Neither a NOAEL nor a LOAEL is established.
Vroom and Engel (1969)
In a case report from Vroom and Engel (1969), the study authors describe the treatment
of a 17-year-old female patient with LEMS with guanidine chloride (no dose reported), which
produced minimal resolution of the patient's muscle weakness. No duration of treatment or side
effects were reported. It was noted that prednisone produced a significant improvement at
10 mg/day. Because no dose was reported for the guanidine treatment, neither a NOAEL nor a
LOAEL is identified.
Nakano and Tyler (1970)
In a case report from Nakano and Tyler (1970) reported the treatment of a LEMS patient
with 21 mg/kg-day guanidine hydrochloride. Atrial fibrillation and hypotension ensued after
6 days, and the heart rhythm returned to normal when the treatment was discontinued. When
guanidine treatment was reinstituted 36 hours later, the patient again developed hypotension and
atrial fibrillation. Neither a NOAEL nor a LOAEL is established since the observed
cardiovascular effects could be considered frank effects, and the treatment duration (6 days) is
considered short-term.
Oh (1972)
A 50-year-old male with LEMS was treated with 12 mg/kg-day guanidine chloride for
2 years in a case report from Oh (1972). No side effects were noted, while the symptoms
resolved for the duration of the study. This dose (12 mg/kg-day) is considered a NOAEL, and
the treatment is considered of subchronic-duration.
Oh and Kim (1973)
Data from three LEMS patients were presented in a case report from Oh and Kim (1973).
Patient 1 was a 56-year-old male with a 2-month history of fatigability and muscle weakness.
The patient was treated with guanidine chloride with a gradual increase in dosage up to
58.3 mg/kg-day in 11 days. Leukopenia (2,200 white blood cells [WBCs]/m3) and mild
thrombocytopenia (113,000 thrombocytes/m ) were noted. The dosage was reduced to
29.2 mg/kg-day and blood cell counts returned to normal over the next 3 weeks. The patient was
continuously treated with guanidine chloride for approximately 2 months. Patient 2 was a
50-year-old male with a 3-month history of fatigability and muscle weakness, who was treated
with 12 mg/kg-day for approximately 2 years. Patient 2 exhibited both peripheral (paresthesias
of tongue and finger) and central (confusion and gait disturbances) nervous system side effects.
Patient 3 was a 64-year-old male who was treated with 32.8 mg/kg-day guanidine chloride
(duration not reported). No side effects were noted. Because the duration of treatment was
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short-term for Patient 1, and unknown for Patient 3, neither a NOAEL nor a LOAEL is
established from these patients. For Patient 2, a subchronic-duration LOAEL of 12 mg/kg-day is
established for peripheral nervous system effects (paresthesias).
Cherington (1976)
In a case report from Cherington (1976) a 38-year-old male with LEMS was treated with
21-27 (average = 24) mg/kg-day guanidine chloride for 4 years. At that point, guanidine
chloride was discontinued because of side effects that included leukopenia (depressed WBC
counts), nephrotoxicity (elevated blood urea nitrogen [BUN]), decreased serum calcium
(8.2 mg/100 mL) and creatine (17.4 mg/100 mL), and elevated serum phosphate
(5.7 mg/100 mL). According to the study author, blood chemistry results obtained prior to the
initiation of guanidine chloride treatment were within normal limits. Nephrotoxicity was
confirmed by biopsy. A subchronic-duration LOAEL of 24 mg/kg-day is established for
leukopenia and nephrotoxicity.
Henriksson et al. (1977)
A case report from Henriksson et al. (1977) described the treatment of two LEMS
patients with guanidine chloride. Patient 1 was treated with 18 mg/kg-day guanidine chloride for
12 months. At that time, renal (i.e., abnormal serum creatine levels), and pancreatic and hepatic
(abnormal alkaline phosphatase, gamma glutamate transferase, alanine and aspartate
aminotransferases, amylase) and anemia (hemoglobin = 79 g/L) side effects were noted.
Patient 2 was treated with 19 mg/kg-day for 7 months, and paresthesias and cold sensations were
noted as well as pancytopenia [hemoglobin =105 g/L, 60 x 109/L thrombocytes, 2 x 109/L
leukocytes (30% polymorphonuclear)]. Guanidine treatment was discontinued in both patients
because of these side effects. A LOAEL is established for serum chemistry changes indicative of
damage to multiple organs.
Blumhardt et al. (1977)
In a case report published by Blumhardt et al. (1977), a 61-year-old male patient with
LEMS was treated chronically with guanidine chloride at 14 mg/kg-day for 7 years. The most
notable side effect was interstitial fibrosis in both the cortex and medulla of the kidney. The
study authors thought the guanidine chloride was responsible for the nephrosis. A chronic-
duration LOAEL of 14 mg/kg-day is established.
Streib (1979)
In a case report published by Streib (1979), a 62-year-old male patient with LEMS was
treated with 30 mg/kg-day guanidine chloride for 10 days, with electromyographic (EMG)
improvement. The patient complained of gastrointestinal (GI) symptoms (colicky pain, nausea,
postprandial fullness, bloating) and dark-colored urine. Blood chemistry values for total
bilirubin, alkaline phosphatase, transaminases; serum glutamic oxaloacetic transaminase (SPOT)
and serum glutamic pyruvic transaminase (SGOT), lactate dehydrogenase, and porphobilinogen,
which had been within normal limits, became elevated by more than 10%. After discontinuation
of the guanidine chloride treatment, the blood chemistry values returned toward pretreatment
levels. These observed serum chemistry changes are consistent with liver injury, which has
rarely been mentioned in other case reports. No NOAEL or LOAEL is established because of
the short-term treatment duration.
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Jenkyn et al. (1980)
In a case report from Jenkyn et al. (1980), a 62-year-old male patient with LEMS was
treated with guanidine chloride initially at 14 mg/kg-day. The patient reported a significant
subjective increase in muscle strength. Two months later, the patient complained of increasing
weakness, and the dose was increased to 21 mg/kg-day. Two months following this dosage
increase, however, mild leukopenia was noted and the dose was reduced to 18 mg/kg-day. The
latter dosing regime was continued for another 5 months. No NOAEL or LOAEL is established
because of the short-term durations of exposure.
Strieb and Rothner (1981)
The treatment of three LEMS patients with guanidine chloride is described a series of
case reports from Strieb and Rothner (1981). Patient 1 was treated with several different doses
of guanidine chloride, with differing side-effects noted at each dose. The patient was initially
treated with 20 mg/kg-day, and mild gastrointestinal upset was noted. At 32 mg/kg-day,
peripheral paresthesias (limb and perioral) were noted. At 45 mg/kg-day, nausea was constant,
coarse tremors developed, and the patient became confused and agitated. The dose was
subsequently reduced to 12 mg/kg-day and was maintained for 3 years. Patient 2 was previously
described in Streib (1979), and a third patient was not treated with guanidine chloride. A
subchronic-duration NOAEL of 12 mg/kg-day is established for Patient 1.
Dau and Denys (1982)
In a series of case reports on five LEMS patients, Dau and Denys (1982) report the use of
plasmapheresis and plasmapheresis combined with immunosuppressive drugs in addition to
guanidine chloride. Patient 1 was treated with 18 mg/kg-day for 18 months without significant
side effects. Patient 2 was treated with 29 mg/kg-day for 6 months, but treatment was
discontinued after acral paresthesias developed. Patient 3 (also described in Cherington [1976])
was treated with 24 mg/kg-day but developed interstitial nephritis after 4 years of guanidine
chloride therapy. Patient 4 was treated with 16 mg/kg-day guanidine chloride for 12 years, and
side effects included mild nausea that was treatable with antacids. Patient 5 was treated with
guanidine chloride (dose not reported) for 1 month, with side effects that included nausea,
paresthesias, and cold extremities. A subchronic-duration NOAEL of 18 mg/kg-day is
established for Patient 1. The limited duration (acute) of treatment for Patient 2 precludes the
establishment of a NOAEL or LOAEL. A subchronic-duration LOAEL of 24 mg/kg-day was
previously characterized for Patient 3 in Cherington et al. (1976). A chronic-duration LOAEL of
16 mg/kg-day is established for Patient 4. No NOAEL or LOAEL is established for Patient 5
because of the incomplete reporting of dosimetry.
Jablecki (1984)
In a case report published by Jablecki (1984), a 50-year-old female LEMS patient was
treated with guanidine hydrochloride for 6 months. The initial dose was 5 mg/kg-day, and this
was slowly increased to an unreported dose. Slight clinical improvement in strength was
reported over the 6 month treatment; however, therapy was discontinued due to hemoptysis
(likely due to oat-cell carcinoma of lung). Because of the incomplete reporting of dosimetry, no
NOAEL or LOAEL is established.
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Bady et al. (1987)
In a case report published by Bady et al. (1987), a 4-year-old female with LEMS was
treated acutely (single dose) with 10 mg/kg guanidine chloride. Increased muscle tone was
reported with no side effects. Because of the acute treatment duration, no NOAEL or LOAEL is
established.
Silbert et al. (1990)
A 60-year old female patient with LEMS was treated chronically with guanidine chloride
for approximately 5 years (Silbert et al., 1990). This case is particularly interesting since several
doses were tried, and a dose response in the severity of side effects observed. In this case report,
the patient was treated initially with 10 mg/kg-day. Resolution of the muscle weakness ensued,
and the dose was increased to 20 mg/kg-day, with muscle power returning to baseline levels.
Five weeks later, however, blood tests revealed leukopenia (3.5 x 109/L, WBCs, 2.27 x 109/L
neutrophils, 0.91 x 109/L lymphocytes), and the dose was reduced to 12 mg/kg-day. The repeat
blood test again revealed leukopenia one week later (2.8 x 109/L WBCs, 1.14 x 109/L
neutrophils, 1.23 x 109/L lymphocytes). Therapy was discontinued, and a repeat blood test
conducted 4 days later showed normal WBC counts (6.6 x 109/L). A 10 mg/kg dose of
guanidine chloride was reinstituted every other day (5 mg/kg-day). Therapy was continued for
approximately 5 years without the return of side effects until the patient's death (unrelated to
therapy).
This particular case report was notable in that it revealed an apparently steep
dose-response curve within a single patient, allowing the determination of doses where
chronic-duration guanidine chloride treatment elicited no or negligible negative side effects, but
retention of the positive therapeutic effects and relief of LEMS symptoms. A
subchronic-duration NOAEL of 5 mg/kg-day (i.e., 10 mg/kg given on alternate days) is
established. A subchronic-duration LOAEL of 12 mg/kg-day is established for leukopenia.
Oh et al. (1997,1998)
A series of case reports is presented involving nine LEMS patients treated with guanidine
chloride was published by Oh et al. (1997). The duration of treatment ranged from 3 to
102 months. Patient 1 (a 46-year-old female) received 5 mg/kg-day for 20 months, with no
reported side effects. Patient 2 (a 26-year-old female) initially received 27 mg/kg-day for
10 months, but this dose was reduced because of a fainting spell. Patient 2 continued treatment
with 5 mg/kg-day for another 10 months, but this dose was discontinued because of nausea.
Patient 3 (a 53-year-old female) received 21 mg/kg-day, but the dose was reduced to
5 mg/kg-day due to nausea and vomiting. The duration of treatment for Patient 3 was
55 months. Patient 4 (a 58-year-old male) was initially treated with doses of 21, 27, and
36 mg/kg-day, but the dosage was reduced to below 15 mg/kg-day within 3 months because of
paresthesias of the fingers, insomnia, and diarrhea. Treatment of Patient 4 continued for
approximately 4 years. Patient 5 (a 70-year-old male) was treated with 5 mg/kg-day for
5 months, but treatment was discontinued because of nausea. Patient 6 (a 75-year-old male) was
treated with 7 mg/kg-day for 12 months and experienced some abdominal pain. Patient 7 (a
59-year-old female) was treated with 10 mg/kg-day for 57 months with no side effects. Patient 8
(a 36-year-old female) received the longest treatment duration with 7 mg/kg-day for 102 months,
with relief of LEMS symptoms and no reported side effects. Patient 9 (53-year-old male) was
treated for 36 months with 9 mg/kg-day, with resolution of muscular weakness but the
appearance of paresthesias. From these nine cases, a subchronic-duration NOAEL of
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5	mg/kg-day is established for Patient 1, a subchronic-duration NOAEL of 11 mg/kg-day is
established for Patient 7, and a chronic-duration NOAEL of 7 mg/kg-day is established for
Patient 8. A subsequent review by Oh et al. (1998) added one additional patient treated with
6	mg/kg-day guanidine chloride for 7 months with no side effects. However, the duration is
considered short-term for this additional patient, thus no NOAEL or LOAEL is established.
Tim et al. (1998, 2000)
The reviews by Tim et al. (1998, 2000) incompletely report the data, and neither
guanidine chloride treatment duration nor dose could be determined. As such, no NOAELs or
LOAELs are established.
GENOTOXICITY DATA
In the Ames test, guanidine nitrate has tested negative at 5,000 |ig/plate in Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with or without metabolic
activation. The chemical was also negative for chromosomal aberrations in hamster fibroblasts
(NLM, 2008). Guanidine chloride was also negative in the Hamster chromosome aberration test
at 5 mg/ml, and in the Ames test in Salmonella typhimurium strains TA98, TA100, TA1535,
TA1527, and TA1538 at up to 5,000 |j,g/plate with or without metabolic activation (NLM, 2008).
CATEGORICAL REGRESSION ANALYSIS
Since guanidine compounds act at the cholinergic neuromuscular junction when used
therapeutically, the methods used by Dourson et al. (1997) on categorical regression of aldicarb
(a carbamate insecticide that causes a similar elevation of acetylcholine) were followed as an
example since the constellations of side effects are similar. Dourson et al. (1997) used four
severity grades to characterize the observed effects, including severe effects, moderate, mild, and
none (no effects), with corresponding assigned values of 4, 3, 2, and 1, respectively. Using these
methods in the aldicarb IRIS assessment (U.S. EPA, 1993), the oral RfD was based on a POD for
a Level 2 effect (sweating), which was considered a NOAEL. For this PPRTV assessment, the
severity scores associated with the various side effects following guanidine chloride treatment of
LEMS patients are shown in Table 4.
Table 4. Description of Severity Scores Associated with Side Effects Following Guanidine
Chloride Treatment of LEMS Patients
Category
Side Effects
Potential
POD Groups
Category 1 (None)
No effects.
NOAELs
Category 2 (Mild)
Serum chemistry changes <10% or within one standard deviation.
NOAELs
Category 3 (Moderate)
Larger (>10%) serum chemistry changes, twitching, blurred vision, watery
eyes, excess salivation, hyperactivity, mild leukopenia, sweating,
clamminess, paresthesias, renal histopathology, or mild gastrointestinal
effects including irritation managed by antacids.
LOAELs
Category 4 (Severe)
Atrial fibrillation; hypotension; severe leukopenia; severe gastrointestinal
effects including unmanageable nausea, vomiting, or diarrhea; or central
nervous system effects including disorientation, confusion or seizures.
LOAELS or
frank effect
levels (FELs),
depending on
severity.
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Table 5 summarizes the oral doses given to LEMS patients in the case reports. The
duration of treatment in these cases ranges from acute to chronic-duration (single doses to
120 months). The most common side effects included gastrointestinal effects in 7 of 35 patients
and various paresthesias in 7 of 35 patients. Other less common side effects included renal
effects (in 3 of 35 patients) and various blood anemias and dyscrasias (most commonly
leukopenia), (in 5 of 35 patients). All of these observed side effects occurred at doses at or
above 10 mg/kg-day. A review of the available data reveals an apparent dose dependence of the
side effects following treatment, with no effects to mild effects observed between
5-10 mg/kg-day, various mild to moderate effects between 10-30 mg/kg-day, and more severe
effects >30 mg/kg-day. In particular, the results of Silbert et al. (1990) showing
immunosuppression and Streib and Rothner (1981) showing gastrointestinal and peripheral and
central nervous system effects indicate an increasing severity of these side effects with
increasing dose within individual patients.
Because much of the human data shown in Table 5 are from case reports of individual
patients with varying treatment durations, a conventional dose-response analysis of the data was
not possible. However, the overall approach used in categorical regression is useful in
characterizing and visualizing the data. In some of the clinical cases, LEMS patients were
started on a low dose of guanidine chloride and then titrated up in dose until side effects became
significant. Thus, there is sometimes a description of a dose-dependent decrease in LEMS
symptoms and a concomitant increase in side effects within the available data from a single
patient. In the cases where the study authors carefully describe what side effects occurred at
what dose, more than one severity score may be identified for an individual patient.
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Table 5. Severity Scoring of Guanidine Chloride Case Reports
Dosimetry"
Effects Observed
Severity Scoreb
Reference
10-30 mg/kg-d, 13 d
Paresthesias (mouth and tongue),
3 at 30 mg/kg-d
Lambert (1966
Patientl: 14-42 mg/kg-d, 5 mo
GI distress, paresthesias, agitation, insomnia
forgetfulness
3 at 32mg/kg-d, 4 at 42 mg/kg-d
Kennedy and
Jimenez-Pabon (1968)
Patient 2: 25 mg/kg-d, 27 d
Severe diarrhea, nervous irritability
4 at 25 mg/kg-d

NA
None identified
NA
Vroom and Engel (1969)
21 mg/kg-d, 1 wk
Atrial fibrillation and hypotension
4 at 21 mg/kg-d
Nakano and Tyler (1970)
12 mg/kg-d, 24 mo
None identified
1 at 12 mg/kg-d
Oh (1972)
12-58 mg/kg-d, 2-24 mo
Patient 1: Leukopenia, thrombocytopenia. High dose =
lid, low dose = 2 mo
Patient 2: Paresthesias at 2 yr, confusion, unsteady gait
P3: None
Patient 1: 4 at 58 mg/kg-d, 3 at 29 mg/kg-d
Patient 2: 4 at 12 mg/kg-d
Patient 3: 1 at 32.8 mg/kg-d
Oh and Kim (1973)
21-27 mg/kg-d 48 mo
Chronic interstitial nephritis and leukopenia
3 at 24 mg/kg-d
Cherington (1976)
Patient 1: 18 mg/kg-d, 12 mo
Renal, pancreatic, anemia, hepatic damage
Patient 1: 4 at 18 mg.kg-d
Henriksson et al. (1977)
Patient 2:19 mg/kg-d, 7 mo
Bone marrow damage (pancytopenia)
Patient 2: 4 at 19 mg/kg-d

14 mg/kg-d, 84 mo
Fibrosing interstitial nephritis
3 at 14 mg/kg-d
Blumhardt et al. (1977)
30 mg/kg-d, 10 d
Colicky pain, postprandial fullness, bloating,
dark-colored urine; elevated liver enzymes
3 at 30 mg/kg-d
Streib (1979)
14 mg/kg-d, 2 mo
18 mg/kg-d, 5 mo
21 mg/kg-d, 2 mo
Mild leukopenia at 21 mg/kg-d
1 at 14 mg/kg-d
1 at 18 mg/kg-d
3 at 21 mg/kg-d
Jenkynetal. (1980)
12-45 mg/kg-d, 36 mo
12 mg/kg-d, 36 mo
Mild GI upset at 20 mg/kg-d, limb and perioral
paresthesias at 32 mg/kg-d, nausea, tremors, confusion,
agitation at 45 mg/kg-d
1 at 12 mg/kg-d
3 at 20 mg/kg-d
3	at 32 mg/kg-d
4	at 45 mg/kg-d
Streib and Rothner (1981)
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Table 5. Severity Scoring of Guanidine Chloride Case Reports
Dosimetry"
Effects Observed
Severity Scoreb
Reference
18-29 mg/kg-d, 6-144 mo
Patient 1: No side effects reported at 18 mo
Patient 2: Numbness, paresthesia, coldness at 6 mo
Patient 3: Chronic interstitial nephritis and leukopenia at
48 mo
Patient 4: Emesis at 144 mo
Patient 5: Dose not reported at 1 mo
Patient 1: 1 at 18 mg/kg-d
Patient 2: 3 at 32 mg/kg-d
Patient 3: 3 at 24 mg/kg-d (same patient reported
in Cherington [1976])
Patient 4: 3 at 16 mg/kg-d
Patient 5: unable to score
Dau and Denys (1982)
5 mg/kg-d initially, 6 mo
None Reported
Unable to score
Jablecki (1984)
10 mg/kg-d, 2 hr
None Reported
lat 10 mg/kg-d
Bady etal. (1987)
5 mg/kg-d, 60 mo
10 mg/kg-d, 1 wk
12 mg/kg-d, 1 wk
20 mg/kg-d, 5 wk
Leukopenia
1 at 5 mg/kg-d
1 at 10 mg/kg-d
3 at 12 mg/kg-d
3 at 20 mg/kg-d
Silbert et al. (1990)
5-36 mg/kg-d, 3-102 mo
Patient 1: None at 20 mo
Patient 2: Fainting, nausea at 10 mo
Patient 3: GI at 55 mo
Patient 4: Paresthesias, GI at 48 mo
Patient 5: GI 5 at mo
Patient 6: GI 12 at mo
Patient 7: None at 57 mo
Patient 8: None at 102 mo
Patient 9: Paresthesia at 36 mo
Patient 1: 1 at 5 mg/kg-d, 1 at 3 mg/kg-d
Patient 2: 4 at 27 mg/kg-d, 4 at 5 mg/kg-d
Patient 3: 4 at 21 mg/kg-d, 3 at 5 mg/kg-d
Patient 4: 4 at 36 mg/kg-d, 3 at 11 mg/kg-d
Patient 5: 4 at 5 mg/kg-d
Patient 6: 4 at 5 mg/kg-d, 4 at 7 mg/kg-d
Patient 7: 1 at 7 mg/kg-d, 1 at 11 mg/kg-d
Patient 8: 1 at 7 mg/kg-d
Patient 9: 3 at 9 mg/kg-d
Oh etal. (1997)
6 mg/kg-d, 7 mo
None at 7 mo
1 at 6 mg/kg-d
Oh etal. (1998)
None reported
None reported
Unable to score
Tim etal. (1998)
None reported
None reported
Unable to score
Tim et al. (2000)
aDoses are converted to mg/kg-d. If no patient weight was provided in the case reports, a default value of 70 kg was used in the conversion. If a range of doses were
used, and details were not specified, the mean of the range was used.
Represents a severity score (as indicated in Table 4) for a given patient at a given dose in mg/kg-d.
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It is important to note that there is a negative correlation in the data between severity and
treatment duration, because in patients with severe side effects, the drug was discontinued or the
dose lowered, and the durations were therefore shorter. However, those patients with minimal
side effects continued to receive guanidine chloride for years. This affects the dose-response
modeling of the human data as will be discussed below. The observed side effects however,
appear relatively quickly (in days), compared to onset in months to years with many chemicals.
There did not appear to be a difference in the constellation of symptoms relative to the duration
of treatment.
While significant scatter is present in the dose-response data, the Cloglog model of the
categorical regression (CatReg) software (U.S. EPA, Version 3.0 [build 12/07/11] software
running on R Version 3.0.0, see Appendix C for additional details) does provide a statistically
significant fit to the data. All the severity scores and durations listed in Table 5 were used in the
categorical regression model. The CatReg software generated estimates for subchronic
(12-month) and chronic (84-month) durations separately. All the models provide effective
response concentration (ERCio) values very close to one another, and the choice of the best
fitting model is made by the lowest Akaike's Information Criterion (AIC), which in this case is
the Cloglog model with linear dose for both subchronic and chronic-duration guanidine chloride
treatment durations. For sub chronic-duration treatment, the Cloglog model provides an
estimated effective response concentration at a benchmark response (BMR) of 10% (ERCio; the
CatReg equivalent of a BMDio) of 4 mg/kg-day and a lower 95% confidence bound on this
ERC io (ER.CL10, the CatReg equivalent of a BlVIDLio) of 2 mg/kg-day. For chronic-duration
guanidine chloride treatment, the Cloglog model also provides an ERCio and ERCLio of
4 mg/kg-day and 2 mg/kg-day, respectively.
DERIVATION OF PROVISIONAL REFERENCE VALUES
Tables 6 and 7 present summaries of noncancer and cancer reference values for guanidine
chloride, respectively.
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Table 6. Summary of Reference Values for Guanidine Chloride
Toxicity Type (units)
Species/
Sex
Critical Effect
p-Reference
Value
POD
Method
POD
UFC
Principal Study
Subchronic p-RfD (mg/kg-d)
Human/Both
NA
2 x 1(T2
ERCL10
2 mg/kg-d
100
NA
Chronic p-RfD (mg/kg-d)
Human/Both
NA
2 x 1(T2
ERCL10
2 mg/kg-d
100
NA
Subchronic p-RfC (mg/m3)
NDr
Chronic p-RfC (mg/m3)
NDr
NDr = not determined, NA = not applicable.
Table 7. Summary of Cancer Values for Guanidine Compounds
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF (mg/kg-d)"1
NDr
p-IUR (mg/m3)
NDr
NDr = not determined.
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DERIVATION OF ORAL REFERENCE DOSES
Several case reports identify doses of guanidine chloride that are associated with negative
side effects including anemia and blood dyscrasias, peripheral and central nervous system
symptoms, gastrointestinal pain, and other frank effects. These same reports identify doses for
which no such side effects are noted, and these serve as potential PODs in the derivation of
p-RfD values for guanidine chloride. As stated above, limitations in the available data preclude
development of cancer and noncancer provisional toxicity values for any other guanidine
compounds. However, based on molecular weight adjustments and stoichiometric calculations,
p-RfDs derived for guanidine chloride can be used as the basis for the derivation of screening
toxicity values for other guanidine compounds when portal-of-entry effects are not expected.
Thus, summaries of screening subchronic and chronic p-RfDs for guanidine nitrate and
guanidine are presented in Tables A-l and A-2, respectively. For systemic effects, since
guanidine compounds are expected to ionize in the blood, the toxicities of the chemicals would
be due to guanidine (and not the particular salt), thus the toxicities of the guanidine compounds
would be expected to be similar on a molar basis. An exception may exist for guanidine nitrate,
because nitrate has intrinsic biological effects. However, a chronic RfD for nitrate itself
(U.S. EPA, 1991) is listed in IRIS as 1.6 mg/kg-day. The derivation for guanidine chloride
presented below results in subchronic and chronic p-RfDs that are approximately two orders of
magnitude lower, which would be protective of any potential health effects from nitrate itself.
Table 8. Potential PODs for Guanidine Chloride
Potential POD Type
Potential POD Value
Duration
Reference
ERCLjo
2 mg/kg-d:i
Subchronic
All available data used-12-mo duration
NO A F.I.
5 mg/kg-d
Subchronic
Oh et al. (1997)—Patient 1
NO A F.I.
5 mg/kg-d
Subchronic
Silbert et al. (1990)
NOAF.I.
6 mg/kg-d
Subchronic
Oh et al. (1997)—Patient 9
NO A F.I.
11 mg/kg-d
Subchronic
Oh and Kim (1973)—Patient 7
LOAEL
12 mg/kg-d
Subchronic
Silbert et al. (1990)
LOAEL
12 mg/kg-d
Subchronic
Oh and Kim (1973)—Patient 2
ERCL10
2 mg/kg-d
Chronic
All available data used-84-mo duration
NOAF.I.
7 mg/kg-d
Chronic
Oh et al. (1997)—Patient 8
LOAEL
14 mg/kg-d
Chronic
Blumhardt et al. (1977)
aERCL10 = lower 95% confidence bound on the ERCi0.
Derivation of Subchronic Provisional RfD (Subchronic p-RfD) for Guanidine Chloride
Although using case reports is not ideal because of the small sample size of LEMS
patients evaluated (N ranging from 1-9), the identification and reporting of side effects is
somewhat subjective, relatively few endpoints are evaluated (however, in some cases clinical
chemistry or pathologies are reported), and the durations of exposure are varied. However, the
case reports on treatment of LEMs patients with guanidine chloride provide the only data
available on guanidine compounds. The case reports identifying potential PODs are chosen
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because of relatively clear descriptions of the dosimetry, LEMS symptoms and side effects, and
the treatment durations. Following sub chronic-duration treatment with guanidine chloride,
potential PODs include NOAELs of 5 mg/kg-day established from both Oh et al. (1997) and
Silbert et al. (1990), aNOAEL of 6 mg/kg-day established from Oh et al. (1997), and the
categorical regression subchronic (12-month) ERCLio value of 2 mg/kg-day generated from a
meta-analysis of all the human data. LOAELs of 12 mg/kg-day are also potential PODs
established from both the Oh and Kim (1973) and the Silbert et al. (1990) data. The categorical
regression ERCLio value of 2 mg/kg-day is chosen as the POD because the categorical
regression uses all of the available data in the collective dose response, uses a dose-response
model (compared to a single point estimate from a NOAEL), provides a statistical basis for
estimating the confidence interval of the POD, and is the most sensitive potential POD.
The subchronic p-RfD for guanidine chloride, based on the subchronic ERCLio, is
derived as follows:
Subchronic p-RfD = ERCLio UFC
= 2 mg/kg-day -MOO
= 2 x 10" mg/kg-day
The composite uncertainty factor (UFq) for the subchronic p-RfD for guanidine chloride
is 100, as explained in Table 9 below.
Table 9. Uncertainty Factors for Subchronic p-RfD for Guanidine Chloride
UF
Value
Justification
UFa
1
A UFa of 1 has been applied for interspecies extrapolation to account for uncertainty in
extrapolating from laboratory animals to humans (i.e., interspecies variability) because human
data is used.
ufd
10
A UFd of 10 has been applied because there are no acceptable two-generation reproductive
toxicity or developmental toxicity studies via the oral route.
UFh
10
A UFh of 10 has been applied for inter-individual variability to account for human-to-human
variability in susceptibility in the absence of quantitative information to assess the toxicokinetics
and toxicodynamics of guanidine chloride in humans.
ufl
1
A UFl of lhas been applied for LOAEL-to-NOAEL extrapolation because the POD is an ERCL10.
UFS
1
A UFS of 1 has been applied because a subchronic-duration (12 mo) estimate generated by the
CatReg software was utilized.
UFC
100
Composite uncertainty factor (UFC = UFA x UFD x UFH x UFL x UFS)
The confidence in the subchronic p-RfD for guanidine chloride is low as explained in
Table 10 below.
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Table 10. Confidence Descriptors for Subchronic p-RfD for Guanidine Chloride
Confidence Categories
Designation"
Discussion
Confidence in studies
L
The case reports all evaluated small numbers of subjects.
Confidence in database
L
The database for repeat-dose oral exposures includes only
clinical studies, and no reproductive or developmental toxicity
studies are available.
Confidence in subchronic p-RfDb
L
The overall confidence descriptor is low.
aL= low; M= medium; H= high.
bThe overall confidence cannot be greater than the lowest entry in the table.
Derivation of Chronic Provisional RfD (chronic p-RfD) Guanidine Chloride
A chronic p-RfD is derived in an analogous manner to the subchronic p-RfD, except that
the potential PODs include a LOAEL of 14 mg/kg-day from Blumhardt et al. (1977), a NOAEL
of 7 mg/kg-day from Oh et al. (1997) (Patient 8), chronic (84-month) ERCLio value of
2 mg/kg-day. Ultimately, the categorical regression ERCLio value of 2 mg/kg-day is chosen as
the POD for the identical rationale applied to the derivation of the subchronic p-RfD as outlined
above.
The chronic p-RfD for guanidine chloride, based on the chronic ERCLio, is derived as
follows:
Chronic p-RfD = ERCLio UFc
= 2 mg/kg-day -MOO
= 2 x 10~2 mg/kg-day
The composite uncertainty factor (UFC) for the chronic p-RfD for guanidine chloride is
100, as explained in Table 11 below.
Table 11. Uncertainty Factors for Chronic p-RfD of Guanidine Chloride
UF
Value
Justification
ufa
1
A UFa of 1 has been applied for interspecies extrapolation to account for uncertainty in
extrapolating from laboratory animals to humans (i.e., interspecies variability) because human
data is used.
ufd
10
A UFd of 10 has been applied because there are no acceptable two-generation reproductive
toxicity or developmental toxicity studies via the oral route.
UFh
10
A UFh of 10 has been applied for inter-individual variability to account for human-to-human
variability in susceptibility in the absence of quantitative information to assess the toxicokinetics
and toxicodynamics of guanidine chloride in humans.
ufl
1
A UFl of lhas been applied for LOAEL-to-NOAEL extrapolation because the POD is an ERCLi0.
UFS
1
A UFS of 1 has been applied because a chronic-duration (84 mo) estimate generated by the
CatReg software was utilized.
UFC
100
Composite uncertainty factor (UFC = UFA x UFD x UFH x UFL x UFS)
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The confidence in the chronic p-RfD for guanidine chloride is low as explained in
Table 12 below.
Table 12. Confidence Descriptors for Chronic p-RfD of Guanidine Chloride
Confidence Categories
Designation"
Discussion
Confidence in studies
L
The case reports all evaluated small numbers of subjects.
Confidence in database
L
The database for repeat-dose oral exposures includes only
clinical studies, and no reproductive or developmental toxicity
studies are available.
Confidence in chronic p-RfDb
L
The overall confidence descriptor is low.
aL = low; M= medium; H= high.
bThe overall confidence cannot be greater than the lowest entry in the table.
As stated previously, screening toxicity values for guanidine nitrate and guanidine are
derived based on molecular weight adjustments and stoichiometric calculations (see
Appendix A). A computational approach to identifying additional potential surrogates for
guanidine nitrate was attempted, but no satisfactory surrogates were found (see Appendix B).
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
No studies have been identified to derive either subchronic or chronic p-RfCs for any
guanidine compounds.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
A paucity of data on the carcinogenicity of any guanidine compound precludes the
derivation of either provisional oral slope factors (p-OSFs) or inhalation unit risks (p-IUR).
CANCER WEIGHT OF EVIDENCE (WOE) DESCRIPTOR
Because no carcinogenicity data are available for any guanidine compounds, the Cancer
WOE descriptor for guanidine compounds is "Inadequate Information to Assess the
Carcinogenic Potential' for both oral and inhalation routes of exposure (see Table 13).
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Table 13. Cancer WOE Descriptor for Guanidine Compounds
Possible WOE Descriptor
Designation
Route of Entry
(Oral, Inhalation,
or Both)
Comments
"Carcinogenic to Humans"
NS
NA
NA
"Likely to Be Carcinogenic to
Humans"
NS
NA
NA
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
NA
"Inadequate Information to
Assess Carcinogenic
Potential"
Selected
Both
No studies on the carcinogenic potential of
guanidine compounds in animals or
humans via the oral or inhalation route
are available in the literature.
"Not Likely to Be
Carcinogenic to Humans"
NS
NA
NA
NS = not selected; NA = not applicable.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For the reasons noted in the main document, it is inappropriate to derive subchronic or
chronic provisional reference doses (p-RfDs) for guanidine nitrate due to the absence of specific
data. However, information is available for this chemical, which, while insufficient to support
derivation of a provisional toxicity value under current guidelines, may be of limited use to risk
assessors. In such cases, the Superfund Health Risk Technical Support Center summarizes
available information in an appendix and develops a "screening value." Appendices receive the
same level of internal and external scientific peer review as the PPRTV documents to ensure
their appropriateness within the limitations detailed in the document. Users of screening toxicity
values in an appendix to a PPRTV assessment should understand that there is considerably more
uncertainty associated with the derivation of an appendix screening toxicity value than for a
value presented in the body of the assessment. Questions or concerns about the appropriate use
of screening values should be directed to the Superfund Health Risk Technical Support Center.
CALCULATION OF SCREENING PROVISIONAL ORAL REFERENCES DOSES
The subchronic and chronic p-RfDs derived for guanidine chloride are used as the basis
for calculating the screening subchronic and chronic p-RfDs for additional guanidine
compounds. Because the toxicity of the various guanidine salts is expected to be due to
guanidine itself, the toxicity of such salts would be directly related to the fraction of the
molecular weight contributed from guanidine. Thus, based on molecular weight adjustments to
the subchronic and chronic p-RfDs derived for guanidine chloride (molecular weight =
95.53 g/mol) in this PPRTV assessment the resulting screening subchronic and chronic p-RfDs
are summarized in Table A-l and Table A-2, respectively, for guanidine nitrate and guanidine
(see calculations below).
Screening Subchronic Provisional RfD Calculations for Guanidine Compounds
Guanidine Nitrate:
Screening Subchronic p-RfD = Subchronic p-RfD for Guanidine Chloride x
for Guanidine Nitrate	(MW of Guanidine Nitrate ^ MW of
Guanidine Chloride)
= 2 x 10 2 mg/kg-day x (122.08 g/mol
95.53 g/mol)
= 3 x 10~2 mg/kg-day
Guanidine:
Screening Subchronic p-RfD
for Guanidine
Subchronic p-RfD for Guanidine Chloride x
(MW of Guanidine ^ MW of Guanidine Chloride)
2 x 10 2 mg/kg-day x (59.07 g/mol 95.53 g/mol)
1 x 10~2 mg/kg-day
MW = molecular weight
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Table A-l. Molecular Weights and Screening Subchronic p-RfDs for
Guanidine Compounds
Compound
Fraction as Guanidine (%)
Molecular Weight
(g/mol)
Subchronic Screening p-RfD
(mg/kg-day)
Guanidine Nitrate
48
122.08
3 x 10~2
Guanidine
100
59.07
1 x 10~2
Screening Chronic Provisional RfD Calculations for Guanidine Compounds
Guanidine Nitrate:
Screening Chronic p-RfD =
for Guanidine Nitrate
Guanidine:
Screening Chronic p-RfD =
for Guanidine
MW = molecular weight
Table A-2. Molecular Weights and Screening Chronic p-RfDs for
Guanidine Compounds
Compound
Fraction as Guanidine (%)
Molecular Weight (g/mol)
Chronic Screening p-RfD
(mg/kg-day)
Guanidine Nitrate
48
122.08
3x 10~2
Guanidine
100
59.07
1 x 10~2
Chronic p-RfD for Guanidine Chloride x
(MW of Guanidine Nitrate ^ MW of Guanidine
Chloride)
2	x 10 2 mg/kg-day x (122.08 g/mol 95.53 g/mol)
3	x 10~2 mg/kg-day
Chronic p-RfD for Guanidine Chloride x
(MW of Guanidine ^ MW of Guanidine Chloride)
2 x 10 2 mg/kg-day x (59.07 g/mol 95.53 g/mol)
1 x 10"2 mg/kg-day
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APPENDIX B. ALTERNATIVE METHODS
Several public databases (e.g., DSSTox, ChemlDplus) were screened for chemicals in
support of a computational approach to derive potential provisional reference values. The
standard approach involves the identification of a surrogate chemical with sufficient structural
and sufficient in vivo outcome similarities on which to base an extrapolation (Wang et al., 2012).
In this case, no chemicals were identified with >80% structural similarity to guanidine nitrate
that possessed repeated-dose toxicity information for comparison. Thus, a computational
toxicological surrogate-based approach was not feasible.
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APPENDIX C. CATREG OUTPUT FILES
The CatReg Version 3.0 (build 12/07/11) software running on R Version 3.0.0 was used
to model the guanidine chloride dose-response. The model form was Cumulative Odds, the
Extra Risk Option was used, the benchmark response (BMR) was set to 0.10 (10%), the
confidence interval was set to 95%, and linear dose and log time were used. Table C-l shows
the model fitting data. All of the models ran correctly when the time function was deleted from
the data. However, when the time functions were added, the CatReg software generated ERCio
and ERCLio values for the subchronic and chronic durations of interest with only the Cloglog
model. The Cloglog model with the lowest Akaike's Information Criterion (AIC) score is
deemed the best fit. The model outputs for 12 months are used for the subchronic p-RfD
derivation for guanidine chloride, while 84 months is used for the chronic p-RfD derivation.
Table C-l. CatReg Modeling Results
Model Name
AIC
ERC10 Value3
ERCL10 Valueb
/7-Value
CatReg Modeling Results (no time specified)
Logit Linear Dose
77.70715
2.87
1.68
<0.05
Probit Linear Dose
78.01757
3.12
1.95
<0.05
Cloglog Linear Dose
74.15734
3.65
1.92
<0.05
Logit Log Dose
78.77464
2.0924
0.6773
<0.05
Probit Log Dose
79.42696
2.0637
0.6521
<0.05
Cloglog Log Dose
75.40959
2.5387
0.9352
<0.05
CatReg Subchronic Modeling Results (12-mo)c
Logit Linear
No Fit
No Fit
No Fit
No Fit
Probit Linear Dose
No Fit
No Fit
No Fit
No Fit
Cloglog Linear Dose
72.84863
3.7305
2.2125
<0.05
Logit Log Dose
No Fit
No Fit
No Fit
No Fit
Probit Log Dose
No Fit
No Fit
No Fit
No Fit
Cloglog Log Dose
No Fit
No Fit
No Fit
No Fit
CatReg Chronic Modeling Results (84 mo)c
Logit Linear
No Fit
No Fit
No Fit
No Fit
Probit Linear Dose
No Fit
No Fit
No Fit
No Fit
Cloglog Linear Dose
72.84863
3.7233
1.7731
<0.05
Logit Log Dose
No Fit
No Fit
No Fit
No Fit
Probit Log Dose
No Fit
No Fit
No Fit
No Fit
Cloglog Log Dose
No Fit
No Fit
No Fit
No Fit
"ERC'i,, = estimated effective response concentration at a benchmark response (BMR) of 10%.
bERCLio = the lower 95% confidence bound on the ERCio °Model fits are reported at 12-month (subchronic) and
84-month (chronic) time periods.
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Duration (Hours) = 0 Stratum = SEV2
: When both concentration and time are not specified, Time=1(when logjTime)), Time=0(when Time) for Probability
CL
o
No effect
A
Severity 1
O
Severity 2
¦
Severity 3

Severity 4

Censored
5	10	20	50
Concentration (mg/m3)
Figure C-l. CatReg plot of Cloglog model run
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The dose-response curve is steep, the time-response models indicate a relatively small
change in ERCio values with time. Table C-2 shows the effects of time on the ERC values, with
time in months from subchronic to chronic durations.
Table C-2. ERCio at Various Times at Severity Score 2
Duration
ERC10
Lower Bound on ERCio
Upper Bound on ERCio
12 mo
3.7305
2.2125
5.2485
20 mo
3.7286
2.1642
5.2930
40 mo
3.7260
2.0152
5.4368
60 mo
3.7245
1.8917
5.5573
84 mo
3.7233
1.7731
5.6734
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