United States
Environmental Protection
1=1 m m Agency
EPA/690/R-10/018F
Final
9-29-2010
Provisional Peer-Reviewed Toxicity Values for
Methyl Hydrazine
(CASRN 60-34-4)
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
Custodio V. Muianga, Ph.D., M.P.H.
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
CONTRIBUTORS
Jon Reid, Ph D, DABT
National Center for Environmental Assessment, Cincinnati, OH
Chris Cubbison, Ph.D.
National Center for Environmental Assessment, Cincinnati, OH
PRIMARY INTERNAL REVIEWERS
Q. Jay Zhao, Ph.D., M.P.H., DABT
National Center for Environmental Assessment, Cincinnati, OH
Geniece M. Lehmann, Ph.D.
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421
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
HISTORY 1
DISCLAIMERS 1
QUESTIONS REGARDING PPRTVS 2
INTRODUCTION 2
REVIEW 01 PERTINENT DATA 3
HUMAN STUDIES 3
ANIMAL STUDIES 4
Oral Exposure 4
Sub chronic-Duration Studies 5
Chronic-Duration Studies 6
Reproductive/devel opmental Studi es 9
Inhalation Exposure 10
Sub chronic-Duration Studies 10
Chronic-Duration Studies 12
OTHER STUDIES 18
Genotoxicity 18
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC ORAL RfD
VALUES I OR METHYL HYDRAZINE 21
SUBCHRONIC AND CHRONIC p-RfD 21
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC INHALATION
RfC VALUES I OR METHYL HYDRAZINE 22
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR METHYL HYDRAZINE 26
WEIGHT-OF -E VIDEN CE DESCRIPTOR 26
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK 26
Oral Exposure 26
Inhalation Exposure 27
REFERENCES 27
APPENDIX A. DERIVATION OF AN INHALATION SCREENING VALUE FOR
METHYL HYDRAZINE 32
APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING FOR
SUBCHRONIC AM) CHRONIC RfD 38
APPENDIX C. DETAILS OF BENCHMARK DOSE MODELING 43
I OR INHALATION UNIT RISK 43
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMD
benchmark dose
BMCL
benchmark concentration lower bound 95% confidence interval
BMDL
benchmark dose lower bound 95% confidence interval
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
POD
point of departure
RfC
reference concentration (inhalation)
RfD
reference dose (oral)
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFC
composite uncertainty factor
UFd
incomplete-to-complete database uncertainty factor
UFh
interhuman uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
METHYL HYDRAZINE (CASRN 60-34-4)
BACKGROUND
HISTORY
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1) EPA's Integrated Risk Information System (IRIS).
2) Provisional Peer-Reviewed Toxicity Values (PPRTVs) used in EPA's Superfund
Program.
3) Other (peer-reviewed) toxicity values, including
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values, and
~ EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's IRIS. PPRTVs are developed according to a Standard
Operating Procedure (SOP) and are derived after a review of the relevant scientific literature
using the same methods, sources of data, and Agency guidance for value derivation generally
used by the EPA IRIS Program. All provisional toxicity values receive internal review by a
panel of six EPA scientists and external peer review by three independently selected scientific
experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the multiprogram
consensus review provided for IRIS values. This is because IRIS values are generally intended
to be used in all EPA programs, while PPRTVs are developed specifically for the Superfund
Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a 5-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV documents conclude that
a PPRTV cannot be derived based on inadequate data.
DISCLAIMERS
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and Resource Conservation and Recovery Act (RCRA) program offices are advised to
carefully review the information provided in this document to ensure that the PPRTVs used are
appropriate for the types of exposures and circumstances at the Superfund site or RCRA facility
in question. PPRTVs are periodically updated; therefore, users should ensure that the values
contained in the PPRTV are current at the time of use.
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It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV document and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
QUESTIONS REGARDING PPRTVS
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
There are no RfD, RfC, or carcinogenicity assessments for methyl hydrazine (MH;
structure shown in Figure 1) on the IRIS database (U.S. EPA, 2009), or in the HEAST
(U.S. EPA, 1997), or on the Drinking Water and Health Advisories list (U.S. EPA, 2006). The
Chemical Assessments and Related Activities (CARA) database (U.S. EPA, 1991, 1994a) lists a
Health and Environmental Effects Profile (HEEP) for MH (U.S. EPA, 1984) that contains a
cancer assessment, in which a human oral slope factor (OSF) of 1.09 (mg/kg-day)"1 was derived
using liver tumor incidence data from hamsters in a chronic drinking water study (Toth and
Shimizu, 1973). Noncancer assessments were not developed in the HEEP.
H„C
3 \ /
N
H
NH_
Figure 1. Chemical Structure of Methyl Hydrazine
Occupational health guidelines and standards are available for MH. The American
Conference of Governmental Industrial Hygienists (ACGIH, 2001, 2008) recommends a
Threshold Limit Value-time-weighted average (TLV-TWA) of 0.01 ppm (0.019 mg/m3) with
skin irritancy and animal carcinogen (A3) notations. The National Institute for Occupational
Safety and Health (NIOSH, 2005) designated MH a potential human carcinogen and
"3
recommends a ceiling Recommended Exposure Limit (REL) (2 hours) of 0.04 ppm (0.08 mg/m )
and an Immediately Dangerous to Life or Health (IDLH) concentration of 20 ppm (38 mg/m3).
The Occupational Safety and Health Administration (OSHA, 2009) has promulgated a
Permissible Exposure Limit (PEL) of 0.2 ppm (0.38 mg/m3) for MH.
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ATSDR (1997) developed a toxicological profile for hydrazines, but this contains little or
no toxicological information about MH per se and no oral or inhalation Minimal Risk Levels
(MRLs) for MH. There is no World Health Organization (WHO, 2009) Environmental Health
Criteria Document for MH, and the carcinogenicity of MH has not been evaluated by the
National Toxicology Program (NTP, 2009, 2005) or the International Agency for Research on
Cancer (IARC, 2009). CalEPA (2009a,b,c) has not derived chronic oral or inhalation RELs or a
cancer potency factor for MH.
Literature searches were conducted for studies relevant to the derivation of provisional
toxicity values for MH. Databases searched included MEDLINE, TOXLINE (BIOSIS and
NTIS), TOXCENTER (Chemical Abstracts), CCRIS, DART/ETIC, DTIC, TSCATS/TSCATS 2,
GENETOX, HSDB, RTECS, and Current Contents. The time period covered by most of the
searches ranged from the 1960s through September 2010, although some searches covered the
early literature.
REVIEW OF PERTINENT DATA
HUMAN STUDIES
In an unpublished study conducted by the Aerospace Medical Research Laboratory
(AMRL), MacEwen et al. (1970) exposed volunteers to 90-ppm (170-mg/m3) MH for 10 minutes
(head-only exposure) and assessed subjective symptoms of irritation, clinical chemistry,
hematology, and respiratory parameters. The volunteers were all men whose average age was
31 years old (range up to 44 years old; minimum not available due to poor quality of the
available study); the group included nonsmokers, smokers, and former smokers. Preliminary
experiments with one volunteer each exposed to 50 or 70 ppm (94 or 130 mg/m3) for 10 minutes
and followed for 2 weeks were conducted prior to the experiment at 90 ppm. The authors
reported that there were no effects on these two volunteers, but they did not report the endpoints
that were examined. A group of five volunteers was then exposed to 90-ppm MH for
10 minutes, during which, each volunteer's subjective reports of nasal and eye irritation and odor
intensity were recorded. Each volunteer was subsequently exposed to 30- and 50-ppm ammonia
(in random order) for comparative information on irritation intensity. Blood samples for clinical
chemistry (electrolytes, calcium, inorganic phosphorus, cholesterol, total bilirubin, total protein,
albumin, glucose, creatinine, chloride, uric acid, blood urea nitrogen [BUN], lactate
dehydrogenase [LDH], alkaline phosphatase [ALP], and aspartate transaminase [AST, previously
Serum glutamic oxaloacetic transaminase, or SGOT]) and hematology (hematocrit [Hct];
hemoglobin [Hgb]; erythrocyte [red blood cell; RBC]; and leukocyte [white blood cell; WBC]
counts; reticulocytes; and Heinz bodies) were collected before exposure and 1, 7, and 14 days
after exposure; hematology was also assessed 60 days after exposure. Respiratory parameters
were evaluated before and after exposure as well as 60 days postexposure.
The authors reported increased moisture without tearing in most subjects, and slight
reddening of the eyes in some subjects exposed to 90-ppm MH for 10 minutes. The individual
reports of nasal and eye irritation intensity were not legible in the available study; however, the
authors reported that the 90-ppm MH concentration was "slightly more irritant than 30-ppm NH3
but considerably less than the 50-ppm NH3 atmosphere." Among hematology parameters, the
only finding was an increase in Heinz bodies (3-5% of erythrocytes by 7 days after exposure,
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dropping to the preexposure level of 0% by 60 days after exposure). Review of the data showed
that clinical chemistry and respiratory evaluations did not indicate an effect of exposure at any
time point, when compared with preexposure levels. The authors attributed individual changes
in respiratory parameters of two subjects to a respiratory infection in one subject and smoking in
another. In a transcript of a discussion following the study, the authors indicated that urine
samples were collected for analysis of glucose, albumin, and microscopic examination, and no
treatment-related effects were observed. This study was reviewed and approved by the Medical
Research Review Committee of the 6570th Aerospace Medical Research Laboratory at
Wright-Patterson Air Force Base (MacEwen et al, 1970).
In a review of data available for use in setting Spacecraft Maximum Acceptable
Concentrations (SMACs), Garcia et al. (1992) briefly reported the results of a study conducted
by the White Sands Facility of the NASA Johnson Space Center (Hoffman et al., 1976 cited by
Garcia et al., 1992). Efforts to obtain the original study were not successful. According to
information provided in Garcia et al. (1992), 42 volunteers inhaled 0.2-ppm (0.38 mg/m3) MH in
a single sniff (volume of 30 cm3). Garcia et al. (1992) reported that 75% of the subjects (32/42)
complained of an irritating odor, while 28% (12/42) exhibited evidence of significant nasal
pathology (not further characterized). No other details of the study population, exposure
conditions, toxicological evaluations conducted, or findings were available in the study by
Garcia et al. (1992).
ANIMAL STUDIES
Oral Exposure
The effects of oral exposure to MH in animals have been evaluated in one
subchronic-duration study in mice (Kelly et al., 1969) and four chronic-duration studies,
including a 40-week study in Swiss mice (Roe et al., 1967) and three lifetime exposure studies in
Swiss mice and Syrian golden hamsters (Toth, 1972; Toth and Shimizu, 1973; MacEwen et al.,
1970). There was also a developmental toxicity study in Sprague-Dawley rats (Slanina et al.,
1993). All these studies were published in peer-reviewed journals, except MacEwen et al.
(1970), which is a peer-reviewed technical report on cancer and noncancer effects. Tables 1 and
2 summarize dose-response information for the studies presented in this section.
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Table 1. Incidence of Tumors in Swiss Mice Exposed to Methyl Hydrazine or
Methyl Hydrazine Sulfate in Drinking Water for Life (Toth, 1972)
Tumor Type
Control3
0.01% MH
0.001% MH Sulfate
Male
Dose
0
17.8 mg/kg-day
2.8 mg/kg-day
Lung adenomas or adenocarcinomas
11/110
1 l/50b
23/50°
Malignant lymphoma
2/110
0/50
8/50°
Hepatoma
0/110
3/50b
0/50
Cholangioma
0/110
2/50
0/50
Cholangiocarcinoma
0/110
1/50
0/50
Angioma of liver and lymph node
0/110
1/50
1/50
Female
Dose
0
20.3 mg/kg-day
2.2 mg/kg-day
Lung adenomas or adenocarcinomas
14/110
12/50d
23/50°
Hepatoma
0/110
3/50b
0/50
Cholangioma
0/110
6/50°
0/50
Cholangiocarcinoma
0/110
1/50
0/50
Angioma of liver
0/110
4/50°
3/50b
"Untreated control data from a similarly designed study of hydrazine sulfate (Toth, 1969)
bSignificantly different from controls by Fisher's exact test conducted for this evaluation; p < 0.05
cp < 0.01
dp = 0.06
Table 2. Incidence of Tumors in Syrian Golden Hamsters Exposed to Methyl Hydrazine
in Drinking Water for Life (Toth and Shimizu, 1973)
Tumor Type
Control
0.01% MH
Male
Dose
0
8.2 mg/kg-day
Malignant histiocytoma of liver
0/97a
27/50b
Cecal tumor
1/97
7/5 0b
Female
Dose
0
9.0 mg/kg-day
Malignant histiocytoma of liver
0/99
16/49b
Cecal tumor
1/99
9/4 9b
aNumber affected/number exposed
bSignificantly different from controls by Fisher's exact test conducted for this evaluation; p < 0.01
Subchronic-Duration Studies—MH was evaluated in a subchronic-duration study of
carcinogenicity in mice (Kelly et al., 1969). Kelly et al. (1969) administered MH (purity not
specified) to 30 female CDFi mice (7-8 weeks old) by gavage in water at a total dose of
3.7 mg/mouse once a week for 8 weeks (0.53 mg/mouse-day) (Kelly et al., 1969). Using the
body weight of 27 g reported by the authors for treated mice at Study Week 12 (additional
body-weight data not reported), the estimated weekly dose per unit body weight was 20-mg/kg
MH. A control group of 10 females was given saline by gavage, and both treated and control
groups were observed for 20-25 weeks following the last dose. Body weight and mortality were
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recorded at Week 12 after study commencement. Upon sacrifice, gross observations for
pulmonary tumors and leukemia were undertaken and histologically verified by examinations of
lung, liver, thymus, spleen, kidney, lymph nodes, and other (unspecified) organs. Other
endpoints (e.g., nonneoplastic effects) were not investigated or not reported. Mortality was
increased in the treated mice (70% compared to 0% in controls); the authors did not discuss
timing or potential causes of death. There were no lung tumors or leukemias in the nine
surviving mice; data on other tumor types were not reported. This study identifies a Frank Effect
Level (FEL) of 20 mg/kg-week for mice based on high mortality.
Chronic-Duration Studies—MH sulfate (purity not specified) was administered to
25 female Swiss mice (age not reported) by gavage in water at a dose level of 0.5 mg/mouse for
5 days/week, for 40 weeks (Roe et al., 1967). A range-finding study using groups of five mice,
reported briefly by the study authors indicated that doses of 2, 8, or 32 mg/mouse, 5 days/week
were lethal to all mice within the first week of treatment, while all survived 0.5 mg/mouse. The
study authors chose this dose for the main study. Using the default reference average body
weight of 0.035 kg for chronic exposure in female mice (B6C3Fi strain used by default;
U.S. EPA, 1988), the estimated dose of MH sulfate per unit body weight was 14 mg/kg-day
(10 mg/kg-day adjusted for continuous exposure). A group of 85 untreated mice served as
controls. Evaluations were limited to gross and histological examinations for lung tumors in
10 treated and 37 control survivors at study Weeks 40-50 and in 9 treated and 42 control
survivors at Weeks 50-60. The incidence of lung tumors and the total number of lung tumors
were not increased by treatment with MH sulfate. Nonneoplastic effects were not reported, so
effect levels could not be determined.
Groups of 50 male and 50 female Swiss mice (6 weeks old) were exposed to drinking
water containing 0.01% MH or 0.001% MH sulfate (purities not reported) for life (Toth, 1972).
The experiments did not use concurrent control groups; rather, control data on 110 male and
110 female untreated mice from the same colony used in a similarly designed study of hydrazine
sulfate and reported by Toth (1969) were used for comparison. Reported average daily
consumption of MH was 0.66 mg/mouse for males and 0.71 mg/mouse for females; consumption
of MH sulfate was 0.102 mg/mouse for males and 0.078 mg/mouse for females. Using default
chronic reference average body weights of 0.037 and 0.035 kg for male and female mice,
respectively (B6C3Fi default; U.S. EPA, 1988), the estimated doses per unit body weight were
17.8 and 20.3 mg/kg-day for MH and 2.8 and 2.2 mg/kg-day for MH sulfate (males and females,
respectively). Treated drinking water was prepared three times per week. Survival, body
weight, comprehensive gross pathology, and limited histopathology (liver, kidneys, spleen,
lungs, and organs showing gross changes) were evaluated, but survival and tumor incidence and
latency data were the only results reported. Survival was reduced in the MH group, with no male
mice surviving past 60 weeks and no female mice surviving past 70 weeks, whereas MH sulfate
had no apparent effect on survival at Week 70 (83% in males and 84% in females compared to
67 and 37% in controls, respectively) or Week 110 (19% males and 10% females compared to
10 and 1% in controls). As survival was affected at the only dose of MH tested
(17.8-20.3 mg/kg-day), this dose is a FEL. Other effect levels could not be determined.
As evidenced in Table 1, incidences of lung tumors (adenomas and adenocarcinomas)
were higher in the mice exposed to 0.001% MH sulfate and 0.01% MH (Toth, 1972) in
comparison to the unexposed control incidences reported by Toth (1969). Although MH sulfate
was more potent than MH in inducing lung tumors, the average latency period was shorter in
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mice of both sexes exposed to 0.01% MH (51 vs. 74-95 weeks in controls and MH sulfate
treatment groups, respectively). The incidence of malignant lymphoma in male mice exposed to
MH sulfate was also significantly increased relative to the control data (see Table 1). The
authors noted that several other tumor types were observed in animals treated with MH (benign
and malignant liver and bile duct tumors in both sexes), but not in controls, and attributed the
tumors to treatment (see Table 1). Because the survival of mice treated with MH was markedly
reduced, the tumor incidences associated with this treatment may be underestimated relative to
longer-exposure durations. In addition, the lack of a concurrent control group increases the
uncertainty in the findings of this study.
Groups of 50 male and 50 female Syrian golden hamsters (6 weeks old) were exposed to
0.01% MH (purity not specified) in drinking water for life and compared with 100 male and
100 female untreated controls (Toth and Shimizu, 1973). Reported average daily intake of MH
for the males and females was 1.1 and 1.3 mg/hamster, respectively. Using default chronic
reference average body weights of 0.134 and 0.145 kg for male and female Syrian golden
hamsters (U.S. EPA, 1988), respectively, estimated doses per unit body weight for males and
females were 8.2 and 9.0 mg/kg-day, respectively. Treated drinking water was prepared three
times per week. Survival, body weight, comprehensive gross pathology, and limited
histopathology (liver, kidneys, spleen, bladder, thyroid, heart, pancreas, testes, brain, nasal
turbinate, lungs, and organs showing gross changes) were evaluated, but survival and tumor
incidence and latency data were the only results reported. Survival was reduced in treated male
and female hamsters during the second year of the study; survival to 90 weeks was 16 and 2% in
treated males and females, respectively, versus 32 and 20% in control males and females,
respectively. No treated animals remained alive after 100 weeks. Apart from survival, no
noncancer endpoints were reported; thus, nonfrank-effect levels cannot be identified for this
study. Incidences of liver and cecum tumors were statistically significantly increased in exposed
hamsters of both sexes (p < 0.01, Fisher's exact test conducted for this review; see Table 2). The
average latency period (animal age) for the liver tumors (malignant histiocytomas = Kupffer's
cell sarcomas) was 78 weeks in males and 70 weeks in females. The tumors were not seen in
any control animals. Tumors of the cecum (polyploid adenomas and adenocarcinomas) were
found with average latency periods of 77 and 64 weeks in males and females, respectively.
Cecal tumors were observed in only two control animals—one male after 84 weeks and one
female after 53 weeks. Other tumors occurred at low incidence and were not attributed to
treatment by the researchers.
An unpublished study designed to replicate the hamster carcinogenicity findings of
Toth and Shimizu (1973) was conducted by MacEwen and Vernot (1975). In the study, the
authors raised concerns regarding the stability of MH, postulating that the hamsters in the study
by Toth and Shimizu (1973) may not have been exposed to MH—but, rather, to its oxidation
products. Preliminary tests reported by MacEwen and Vernot (1975) showed that approximately
60% of the MH content could be lost over 24 hours from a 0.01% MH-tap water solution. The
authors also observed that the pH of the MH-water solution had an effect on decomposition, and
adjusting the pH to 3.5 with HC1 reduced the loss of the MH content to approximately 5%.
Additional experiments demonstrated that adjustment of the drinking water to pH 3.5 with HC1
did not affect body-weight gain or water consumption. Based on these observations, an
experiment was conducted using three groups of male Syrian golden hamsters: 30 were exposed
to 0.01%) MH as the free base in drinking water (unadjusted pH group), 30 were exposed to
0.01%) MH in drinking water adjusted to pH 3.5 with HC1 (acidic pH group), and 17 were
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exposed to drinking water alone adjusted to pH 3.5 with HC1 (control). Test solutions in the
main study were changed daily. No MH purity was reported. The hamsters (5 months old) were
exposed to the test solutions for life. Study endpoints included survival, body weight (monthly),
water consumption (daily), and limited hematology (RBC and Hct on five animals/group after 7,
11, and 15 months; bone marrow myeloid/erythroid (M/E) ratio in two acidic group animals and
one control animal at end of study [83 weeks]). The authors estimated the nominal (unadjusted
for loss) average daily doses of MH to be 7.3 mg/kg-day in the unadjusted pH group and
7.5 mg/kg-day in the acidic pH group. Complete necropsies with limited histopathology (liver,
kidney, spleen, heart, lung, trachea, esophagus, thyroid, urocyst, testes, and gross lesions) were
conducted on 13, 25, and 25 animals in the control, unadjusted, and acidic groups following
death or sacrifice at the end of the study.
Survival of hamsters through the 80th week of treatment was not affected by treatment in
either MH group (24, 17, and 17% in control, unadjusted MH, and acidic MH groups,
respectively) (MacEwen and Vernot, 1975). The authors reported that the hamsters exposed to
MH unadjusted for pH exhibited lower mean body weights beginning in the fourth month of
exposure (statistical analysis not reported; data presented graphically). Animals exposed to the
acidic MH solution had lower mean body weights after the 15th month of treatment. Based on
the graphical data, mean terminal body weights appeared to be about 20% lower than controls in
the unadjusted MH group and 10% lower than controls in the adjusted MH group. Hematology
analysis showed reduced RBC and Hct in both MH groups at all time points; however, statistical
analysis was not conducted, and the available data were inadequate for independent statistical
analysis (variability was not reported). Evaluation of the bone marrow M/E ratio showed
reduced values (0.5-0.7) for the two animals examined from the acidic MH group compared with
the ratio of 1.9 observed in the one control; however, the small number of animals evaluated
limits conclusions that can be drawn from this observation. Neither group exposed to MH
exhibited a statistically significant increase in any tumor type or in the total number of tumors
across sites. This study identifies a LOAEL of 7.3 mg/kg-day for body weight decrement of at
least 10%) and possible hematologic effects. A NOAEL was not determined.
There are several important differences in the two hamster studies that may have
contributed to the different results for carcinogenicity. First, the hamsters used in the
unpublished study were older at commencement of exposure (5 months) than the 6-week-old
hamsters in the study by Toth and Shimizu (1973); as a result, the exposure duration in the study
by MacEwen and Vernot (1975) was shorter (both studies featured lifetime exposure), and any
enhancement in carcinogenicity associated with exposure to younger animals would not have
been replicated. Second, MacEwen and Vernot (1975) used smaller groups (30/dose) of male
hamsters only, while Toth and Shimizu (1973) used 50 hamsters/sex/dose. Third, MacEwen and
Vernot (1975) took measures to ensure the stability of MH in water in their study, including
daily changing of test solutions (versus three times per week in the Toth and Shimizu, 1973
study) and inclusion of treatment groups with and without adjustment to pH 3.5, noting that at
this pH, the MH loss from 0.01%> solution over 24 hours was much lower (approximately 5%)
than at neutral pH (up to 60%>). Additionally, as already stated, MacEwen and Vernot (1975)
suggested that the hamsters in the study by Toth and Shimizu (1973) may have been exposed to
oxidation products of MH rather than the compound itself.
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Reproductive/developmental Studies—Slanina et al (1993) performed a embriotoxicity
and teratogencity study with rat intravenous (i.v.) infusion as pilot experiment (Experiment 1),
and a follow-up study with orally-administered MH (Experiment 2) as follow:
Experiment 1: Groups of three pregnant Sprague-Dawley rats received a constant i.v.
daily dose of 0 (physiological saline solution), 1.2, 3.0 (low dose range), 4.2, 6.0 (intermediate
dose range), 9.0, and 13.2 mg/kg-day (high dose range), respectively, at an infusion rate of
10 |iL/h from GDs 6 to 13 (Experiment 1). Throughout the experiment animals were inspected
daily for signs of maternal toxicity and weight gain. On Day 19 of gestation, the animals were
sacrificed with halothane and examined for embryotoxicity, external and skeletal malformations.
Signs of apparent maternal toxicity were seen after continuous i.v. infusion of MH in the
high dose range groups. Five out of 33 treated dams died before the end of the experiment in the
highest dose group (13.2 mg/kg-day). One or several occasions of convulsions were observed in
the high dose range groups (9.0-13.2 mg/kg-day). No apparent sings of maternal toxicity were
observed in the intermediate and low dose range groups (1.2-6.0 mg/kg-day). A significant
(p < 0.01, two-sample test of proportions) dose-related increase in the number of resorptions
starting with the second lowest dose group was observed. The number of dams with at least one
resorption was 70.6% and 100% in the 3.0- and 4.2-mg/kg-day dose groups, respectively, as
compared to 21.1% of controls. For the only dam which became pregnant in the 6.0-mg/kg-day
dose group, eight out of nine embryos were resorbed. A statistical difference (p < 0.01) for
pregnancy rate was observed in the dose groups 4.2 and 6.0 mg/kg-day (43% and 93% of the
animals, respectively, compared to 14% in control groups). The study authors indicated a
nonstatistically significant increase of some minor abnormalities (e.g., oedema and anemia,
extremely small fetuses), fetal body weights, or incidence of fetal malformations were observed
compared to the control group.
As follow-up study of the dramatically decreased pregnancy rate after continuous i.v.
infusion of MH pregnant rats observed in the experiment 1, Slanina et al. (1993) treated pregnant
(plug-positive) Sprague-Dawley rats with a single gavage dose of 0 (corresponding amount of
physiological saline), 1, or 5-mg/kg MH (with highest purity available) in distilled water on
Gestation Day (GD) 6 (Experiment 2). Determination of MH in serum was performed in 6 out of
16 animals in the low-dose group and in all animals in the high-dose group. There were 16 dams
in each exposure group and 24 controls. The dams were sacrificed on GD 19, at which time,
uteri were removed, and numbers of corpora lutea, implantations, resorptions, and live and dead
fetuses were recorded. Live fetuses were weighed and examined for external and skeletal
malformations; visceral malformations were not evaluated. Preimplantation loss (calculated as
percent of corpora lutea) was statistically significantly increased in the high-dose group. The
data was presented as mean ± SD preimplantation losses per litter as follow: control, low-, and
high-dose groups were 22.17 ± 0.50, 29.78 ± 0.40, and 40.83 ± 0.63, respectively; p < 0.05 for
the high-dose group. The incidence of females with resorptions or dead fetuses was higher in the
high-dose group (62.5%) than in controls (37.5%), but the difference was not statistically
significant. Fetal body weight and rates of external and skeletal malformations were not affected
by MH exposure. This study identifies a developmental LOAEL of 5 mg/kg based on increased
preimplantation losses; the NOAEL is 1 mg/kg. Maternal effect levels could not be determined
due to the lack of reported maternal evaluations. This effect is supported by the results of the
experiment 1, an intravenous (i.v.) experiment with six dose levels, which showed a significant,
dose-related increase in the incidence of females with resorptions/dead fetuses and a decrease in
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pregnancy rate with increasing doses of MH. Based on the results of the oral study, the
researchers attributed the decrease in pregnancy rate in the i.v. experiment to preimplantation
loss, which was not recorded directly in the i.v. experiment.
Inhalation Exposure
No published studies of inhalation exposure to MH in animals were identified in the
literature searches. The AMRL conducted a series of subchronic- and chronic-duration
inhalation studies (exposure durations from 3 months to 1 year) with a variety of species
(Kinkead et al., 1985; Darmer and MacEwen, 1973; MacEwen and Haun, 1971; Haun, 1970;
Kroe, 1971). None of these studies was published in peer-reviewed journals. Reports of these
studies (discussed below) were limited by poor descriptions of study design and findings, and
occasionally, legibility issues.
Subchronic-Duration Studies—Groups of 80 male Sprague-Dawley rats, 8 female
beagle dogs, and 4 female Rhesus monkeys were exposed to concentrations of 0-, 0.04-, or
0.1-ppm MH continuously (purity not specified) for 90 days (Darmer and MacEwen, 1973).
Body weights were measured before and after exposure, as well as at 2-week intervals during
exposure. Blood was collected from 30 rats/dose group after 45 and 90 days of exposure, and
from dogs and monkeys before exposure, biweekly during exposure, and at exposure
termination. Hematology parameters included Hct, Hgb, RBC, reticulocytes, WBC, and Heinz
bodies. In addition, serum levels of total inorganic phosphorus and ALP were analyzed.
Erythrocyte fragility tests were performed on blood samples collected from dogs at termination.
At the end of exposure, the 20 rats not sacrificed earlier for hematology analyses were sacrificed,
as were all dogs and monkeys, for gross pathology evaluations. Organ weights (heart, lung,
liver, spleen, and kidney) were measured in rats only. The authors reported that tissues were
collected for histopathology evaluation but did not present the findings of such evaluation.
Concentrations of MH were analyzed daily using a colorimetric method calibrated to a known
concentration of the compound; the authors reported average concentrations over the 90 days of
0.0462 and 0.100 ppm (0.087 and 0.19 mg/m ) for the low- and high-exposure groups,
respectively.
In rats, body weight was depressed in the high-exposure group through most of the
exposure period; the authors reported that the difference was statistically significantly different
from control at most measurements (Darmer and MacEwen, 1973). Based on data presented
graphically, the difference from control was much less than 10% throughout the study.
Statistically significant decreases in Hct (p <0.01), Hgb (p < 0.05), and RBC (p <0.01) were
observed in both exposure groups at the 45-day measurement; at the 90-day measurement, only
RBC (p < 0.01) in the high-exposure group was significantly different from controls. Table 3
shows the results. Total phosphorus was increased (8-13% higher than controls; p < 0.05) at
both exposure levels at the 90-day measurement; there was no effect on serum ALP. The
organ-weight data showed no effect of treatment on absolute or relative organ weights. The
authors reported that there were no gross necropsy findings. Histopathology findings were not
discussed. ALOAEL of 0.10 ppm (0.19 mg/m ) andNOAEL of 0.046 ppm (0.087 mg/m3) can
be identified in rats based on reduced RBC persisting to 90 days.
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Table 3. Hematology and Serum Chemistry Mean Values in Albino Sprague-Dawley Rats
Exposed to Methyl Hydrazine by Continuous Inhalation for 90 Days
(Darmer and MacEwen, 1973)
Control
0.046 ppm
0.10 ppm
Number of Animals Examined
30
30
30
45 days
Erythrocyte count (x 10)
8.2
7.6a
7.5a
Hemoglobin content (g %)
16.0
15.6b
15.0a
Hematocrit (vol %)
44
42a
41a
Total phosphorus (mg %)
7.1
8.1
7.3
Alkaline phosphatase (IU)
154
142
146
90 days
Erythrocyte count (x 10)
7.0
7.6
6.1b
Hemoglobin content (g %)
16.7
15.7
15.4
Hematocrit (vol %)
44
44
43
Total phosphorus (mg %)
6.2
6.7b
7.0b
Alkaline phosphatase (IU)
117
117
117
><0.01
bSignificantly different from control at p< 0.05
In dogs exposed to the high concentration, there was evidence of hemolysis (reduced Hct,
Hgb, and RBC, and increased osmotic fragility of erythrocytes) as well as increases in serum
phosphorus and ALP (Darmer and MacEwen, 1973). No effects on these parameters were
observed in the 0.046-ppm group. Table 4 provides the pertinent data. At necropsy, the livers of
the dogs in the high-exposure group exhibited a "nutmeg" appearance, which the authors
considered to be evidence of passive congestion; the incidence was not reported, and additional
"3
details were not provided. This study identified a LOAEL of 0.10 ppm (0.19 mg/m ) in dogs
based on hematology and serum chemistry changes and gross liver pathology; the NOAEL is
0.046 ppm (0.087 mg/m3).
A monkey in the 0.046-ppm group died after 10 days of treatment (Darmer and
MacEwen, 1973). Necropsy of the animal revealed amyloidosis, described as a preexisting
condition, and the authors did not consider the death to be related to MH exposure. The data
showed no effects of treatment on hematology parameters in monkeys (see Table 4). The
authors reported that no gross pathology related to treatment was observed. This study suggests
-3
a NOAEL of 0.10 ppm (0.19 mg/m ) in monkeys; although limited evaluations were performed
(as in the other species), and group sizes were small.
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Table 4. Hematology and Serum Chemistry Mean Values in Beagle Dogs and Rhesus
Monkeys Exposed to Methyl Hydrazine by Continuous Inhalation for 90 Days
(Results At Termination) (Darmer and MacEwen, 1973)
Control
0.046 ppm
0.10 ppm
Dogs
Number of Animals Examined
8
8
8
Hematology
Erythrocyte count (x 106)
6.26
5.41
4.73a
Hemoglobin content (g %)
18.1
17.0
is. r
Hematocrit (vol %)
49
47
44a
Clinical chemistry
Alkaline phosphatase
63
85
356a
Total phosphorus (mg %)
4.0
4.7
4.9b
Monkeys
Number of Animals Examined
4
4
4
Hematology
Erythrocyte count (x 106)
5.20
4.44
4.72
Hemoglobin content (g %)
13.8
12.7
13.8
Hematocrit (vol %)
40
38
38
><0.01
bSignificantly different from control at p< 0.05
Chronic-Duration Studies—Rats, mice, dogs, and monkeys were exposed to MH
(purity not specified) on an intermittent (6 hours/day, 5 days/week) or continuous basis for
approximately 6 months (MacEwen and Haun, 1971; Kroe, 1971; Haun, 1970). These
experiments are unpublished, and there are inconsistencies among the reports. Groups of 50
Wistar rats, 40 ICR mice, 8 beagle dogs, and 4 Rhesus monkeys were exposed to 0 or 0.2 ppm
3 3
(0 or 0.38 mg/m ) continuously; 0, 0.2, or 1 ppm (0, 0.38, or 1.9 mg/m ) intermittently; or 0, 2,
or 5 ppm (0, 3.8, or 9.4 mg/m3) intermittently. There were inconsistencies among the reports; for
example, MacEwen and Haun (1971) and Haun (1970) reported that female monkeys, dogs, and
mice were used in all of the 6-month studies, while Kroe (1971) reported that the mice exposed
to 2.0 and 5.0 ppm were male, and the dogs and monkeys exposed to 0.2 and 1.0 ppm were male.
The methods and evaluations conducted were poorly reported; however, based on the results
reported, the following parameters were evaluated: clinical signs and mortality in all animals;
biweekly body weight in rats; monthly electroencephalogram (EEG) measurements in monkeys;
biweekly hematology (8 indices) and clinical chemistry (15 indices) in dogs and monkeys; bone
marrow M/E ratio in dogs; gross pathology in all animals; organ weights (heart, lungs, liver,
kidneys, and spleen) in rats; and histopathology in 10 rats/group, 10 mice/group, and all dogs and
monkeys at the end of the exposure period. The histological examinations included the liver,
kidneys, spleen, heart, and lungs in all species, and the brain and unspecified endocrine glands in
dogs and monkeys. All result data were presented graphically, and incidences of histopathologic
findings were not reported.
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In rats, there were no deaths, and no clinical signs of toxicity were reported (MacEwen
and Haun, 1971; Kroe, 1971; Haun, 1970). Rats exhibited reduced body-weight gain with
continuous exposure at 0.2 ppm and intermittent exposure at >1 ppm; body weight was not
affected in the 0.2-ppm intermittent group. The authors did not indicate the methods of
statistical analysis; however, body-weight differences were reported to be statistically
significantly different from controls (p < 0.01) at Weeks 7, 9, and 13 for the 0.2-ppm continuous
exposure and at Weeks 1-9 for the 1-ppm intermittent exposure. Visual examination of the
growth curves indicate that the mean weights of the 0.2-ppm continuous and 1-ppm intermittent
exposure groups were no more than approximately 5% lower than the controls during the first
13 weeks; data after Week 13 were not considered reliable by the authors due to heat stress (from
equipment malfunction) in the control animals. At 2- and 5-ppm intermittent exposures, the
study authors not that rat growth was significantly decreased from Weeks 10 and 12,
respectively, until the end of the study; at Week 26, body weights in these groups were
approximately 10 and 20% less than controls, respectively (based on visual inspection of data
presented graphically). The study authors indicated that relative kidney and spleen weights were
significantly (p < 0.01) increased in rats exposed to 2 or 5 ppm intermittently, but data and
statistical methods were not given. It is not clear whether organ weight measurements were
made in the lower exposure groups. According to the study authors, histologic examination of
10 rats/group did not indicate treatment-related changes at any exposure level (data not shown).
A LOAEL of 2 ppm (3.8 mg/m3) for intermittent exposure (adjusted to 0.68 mg/m3 of continuous
exposure) is identified for rats based on body-weight reduction of about 10%. The NOAEL for
body-weight changes (organ weights not reported for this exposure group) was 1 ppm
3 3
(1.9 mg/m ) for intermittent (adjusted to 0.34 mg/m of continuous exposure). By continuous
exposure, 0.2 ppm (0.38 mg/m3) was a NOAEL in rats.
In mice, mortality was increased at >2 ppm; the authors reported 1/40, 6/40, and
9/40 deaths in the control, 2-, and 5-ppm intermittent exposure groups, respectively (MacEwen
and Haun, 1971; Haun, 1970; Kroe, 1971). Cause(s) and/or timing of deaths were not reported.
In addition to the deaths attributed to treatment, the study authors reported that seven other mice
from the 5-ppm exposure group died accidentally (no further information provided). Mice
exposed to 5 ppm reportedly showed clinical signs of rough yellowed coats and occasional
lethargy. Kroe (1971) reported pathology findings on 10 mice/group, but they did not report
incidences. Findings reported in mice exposed to 2- and 5-ppm MH included centrilobular or
periportal cholestasis, bile duct proliferation, centrilobular hepatic hemosiderosis, splenic
hemosiderosis, and renal tubular hemosiderosis. Hemosiderosis of the liver, spleen, and renal
tubules was also observed in the mice exposed to 0.2 ppm (intermittently or continuously) and
1 ppm; cholestasis and bile duct proliferation were not observed in these groups. The authors
reported that hemosiderosis was more severe in the 0.2-ppm continuous group, followed by the
1- and 0.2-ppm intermittent groups, and that the effects in the 0.2-ppm intermittent group was
"significantly more than nonexposed controls"; however, data and statistics were not reported.
These results suggest a LOAEL for hemosiderosis of the liver, spleen, and renal tubules in mice
3 3
given 0.2 ppm (0.38 mg/m ) by intermittent exposure (adjusted to 0.068 mg/m of continuous
exposure). The lack of incidence information for this effect, however, renders the LOAEL
uncertain.
No dogs died during the experiment (MacEwen and Haun, 1971; Kroe, 1971; Haun,
1970). Exposure to 5 ppm was associated with apparent conjunctivitis in dogs as indicated by
prominent nictitating membranes and photophobia. The effect on the nictitating membranes was
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observed as early as Week 2 and continued throughout the study, was minimal or absent
following weekends of no exposure, and increased in severity following several daily exposures.
No clinical signs were observed at <2 ppm. Hematological effects that were generally
concentration-related and suggestive of hemolytic anemia were observed at all exposure levels,
including both continuous and intermittent exposure to 0.2 ppm. These effects included
decreases in RBC, Hct, Hgb, and bone marrow M/E ratio and increases in methemoglobin, Heinz
body formation, and RBC fragility that were statistically significant at all exposure levels but
more severe at 2 and 5 ppm. Clinical chemistry findings in dogs included dose-related increases
in serum bilirubin, ALP, and total phosphorus levels in all exposure groups throughout the study,
with increases of 2-fold or greater in all but the 0.2-ppm intermittent exposure group.
Histopathology examinations in dogs revealed periportal intracanalicular cholestasis of the liver
at all concentrations and hepatic and renal hemosiderosis at 2 and 5 ppm. Moderate lymphoid
hyperplasia was reported in the lower exposure groups but not the higher exposure groups.
A LOAEL of 0.2 ppm (0.38 mg/m3), due to intermittent exposure (adjusted to 0.068 mg/m3 of
continuous exposure), is identified for dogs based on evidence of hemolytic anemia (hematology
and serum chemistry changes as well as histopathology) and hepatic cholestasis. A NOAEL was
not identified.
There were no deaths or clinical signs of toxicity among monkeys (MacEwen and Haun,
1971; Kroe, 1971; Haun, 1970). Body weights were not recorded. Hematology findings in
monkeys included decreased Hct, Hgb, and RBC, as well as increased reticulocytes. Based on
graphical presentation of data for the 2- and 5-ppm groups, RBC and reticulocyte counts were
affected at both concentrations, but Hct and Hgb levels were only clearly affected at 5-ppm MH.
No data were presented on these endpoints in monkeys exposed to 0.2 or 1 ppm. In addition to
the hematology changes noted above, the authors reported the presence of Heinz bodies (one to
five Heinz bodies in 100 RBCs) in all exposed groups of monkeys. No dose- or species-related
effects were evident in monkeys or dogs that were observed. There were no histopathology
findings in monkeys. Due to the lack of information on hematology results in the lower
exposure groups, it is not possible to determine effect levels for monkeys.
Kinkead et al. (1985) detailed the results of longer experiments by the same laboratory
(AMRL) in rats, mice, hamsters, and dogs exposed via inhalation (purity not reported) for
6 hours/day, 5 days/week, for 1 year. F344 rats (100/sex/group and 150/sex/controls, 10 weeks
of age at start) were exposed to concentrations of 0, 0.02, 0.2, 2.0, or 5.0 ppm (0, 0.038, 0.38,
3.8, or 9.4 mg/m ); C57BL/6J mice (400 females/group, 10 weeks of age at start) were exposed
to concentrations of 0, 0.02, 0.2, or 2.0 ppm (0, 0.038, 0.38, or 3.8 mg/m3); Syrian golden
hamsters (200 males/group, 12 weeks of age at start) were exposed to concentrations of 0, 0.2,
2.0, or 5.0 ppm (0, 0.38, 3.8, or 9.4 mg/m3); and beagle dogs (4/sex/group, 11-20 months of age
-3
at start) were exposed to 0, 0.2, or 2.0 ppm (0, 0.38, or 3.8 mg/m ). Body weights were recorded
biweekly for rats, hamsters, and dogs, and monthly for mice. Blood was collected biweekly
during the exposure period from dogs for hematology (Hct, Hgb, RBC, and WBC) and clinical
chemistry (ALP, alanine aminotransferase [ALT, previously serum glutamic pyruvic
transaminase, or SGPT], bilirubin, glucose, triglycerides, iron, and sedimentation rate)
evaluations. Methemoglobin levels were measured in dogs once every 3 months during
exposure. Liver function was assessed using the bromosulphophthalein (BSP) retention test in
dogs at the end of exposure. Rats, mice, and hamsters were observed untreated for 1 year after
the conclusion of the exposure period, while dogs were observed for 5 years. At the end of the
observation period, all animals were necropsied, and 33 tissues from each animal were examined
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microscopically. The prolonged postexposure observation period prior to sacrifice and
pathology evaluation (1 year in rodents and 5 years in dogs) is a limitation of the study for
assessment of noncancer effects, allowing ample time for recovery of reversible nonneoplastic
effects. No information on survival or clinical signs was reported for any species tested.
Statistical tests used in the study were not reported.
Growth curves for rats showed body-weight decrements for both sexes (Kinkead et al.,
1985). The authors reported that body weights were statistically significantly lower than controls
during the exposure period in all groups of treated males and in the 2- and 5-ppm groups of
females (data and p-walue not given). Examination of the graphs indicated that body weights at
the end of the exposure period were decreased by at least 10% in all exposed male rats and in the
5-ppm group of female rats. However, except for the 5-ppm groups in both sexes, there was no
evidence of a dose response. Body weights in the 0.02-, 0.2-, and 2-ppm groups were similar
throughout the exposure and postexposure periods in both sexes. It is, therefore, unclear whether
this represents a treatment-related effect at the lower exposure levels. Review of the data shown
indicated that there were no exposure-related increases in the incidences of any nonneoplastic or
neoplastic lesions upon histopathology examination 1 year after treatment ended; statistically
significant decreases in the incidences of some tumor types were associated with treatment. A
NOAEL of 2 ppm (3.8 mg/m3) and a LOAEL of 5 ppm (9.4 mg/m3) are identified for rats based
on decreased body weight clearly related to MH exposure.
Data on body weights of mice were not reported; histopathology findings were the only
results given (Kinkead et al., 1985). Tumors of the nasal mucosa, lung, and liver, as well as
hemangiomas (sites not specified), were increased in incidence in exposed mice when compared
with unexposed controls (Kinkead et al., 1985). Table 5 shows the tumor incidences. Although
the incidences of nasal tumors were not statistically distinguishable from controls, several
different tumor types (adenomas, adenomatous polyps, osteomas, and epithelial neoplasms) were
observed at the highest exposure, and no nasal tumors were observed in controls. The authors
indicated that nasal tumors are rare in untreated mice and considered these tumors to be
biologically significant. The incidence of hemangiomas at the highest exposure was not legible
in the available version of the report; however, the authors reported that the incidence was
"markedly increased." The incidences of several nonneoplastic lesions were also reported to be
increased in treated mice (see Table 5), although interpretation of these data is uncertain. Due to
the prolonged period of nonexposure and late sacrifice, reversible effects due to treatment would
not be observable, and age-related changes may confound treatment-related findings. Also in the
case of this study, tumors found in several organs may confound the results for nonneoplastic
lesions in the same organs. For example, it is uncertain whether the liver cysts that developed in
mice represent a distinct morphological entity from the adenomas and carcinomas that were
apparent in the same organ, or whether hepatocyte pleomorphism reported as a distinct
nonneoplastic lesion is related to the neoplastic effect in the liver. The low incidence and lack of
a clear step-wise dose-response for some lesions (e.g., nasal lesions) also argues against these
lesions being related to treatment. Due to the uncertain interpretation of the pathology data in
this study, a NOAEL and a LOAEL were not identified for the mouse.
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Table 5. Pathology Changes in Female C57BL/6J Mice Exposed to Methyl Hydrazine
by Inhalation for 1 Year (Kinkead et al., 1985)
Control
0.02 ppm
0.2 ppm
2.0 ppm
NeoplasticLesions
Nasal adenoma
0/367a
1/354
0/349
1/355
Nasal adenomatous polyp
0/367
0/354
0/349
4/355
Nasal osteoma
0/367
0/354
0/349
3/355
Nasal and respiratory epithelial neoplasms
0/367
2/354
1/349
4/355
Lung adenoma
13/364
16/354
23/347
56/360b
Lung carcinoma
0/364
1/354
2/347
3/360
Liver adenoma
6/373
2/357
5/357
20/363b
Liver carcinoma
2/373
4/357
4/357
14/363b
Hemangioma
5/387
9/371
5/368
2?/371b'°
Nonneoplastic Lesions
Nasal inflammation
10/367
35/354b
17/349
28/355b
Mandibular lymph node plasmacytosis
17/322
50/344b
46/3 30b
31/329
Mandibular lymph node hemorrhage
2/322
7/344
7/330
10/329d
Liver cysts
3/373
4/357
13/357d
39/363b
Bile duct hyperplasia
2/373
2/357
1/357
17/363b
Hepatocyte pleomorphism
11/373
6/357
11/357
33/363b
Gallbladder crystals
10/303
7/295
8/315
53/312b
Angiectasis
16/387
26/371
29/368d
59/37 lb
Kidney hydronephrosis
4/374
11/362
6/353
14/365d
aNumber affected/number examined
V<0.01
°? represents illegible digit in available report
dSignificantly different from control atp< 0.05
Treated male hamsters exhibited effects on growth (Kinkead et al., 1985). Body-weight
decrements were observed in all treated groups, but a clear dose-response relationship was not
evident. At the end of exposure, body weights of treated hamsters appeared to be between 5 and
10% lower than controls based on visual inspection of data presented graphically. Body weight
was decreased throughout exposure in the 5.0-ppm group, from about Week 16 on in the
2.0-ppm group, and from about Week 36 in the 0.2-ppm group. Neoplastic changes in the nares
and adrenals were observed in the hamsters (see Table 6). Nasal adenomas were increased in the
high-exposure group, and nasal adenomatous polyps were increased in both the mid- and
high-exposure groups. Benign adenomas of the adrenal cortex were also increased in incidence
in the high-exposure group. As was observed for mice, the incidence of a number of
nonneoplastic lesions were reported to be increased in treated hamsters (see Table 6), but, again,
the low incidence, lack of clear progressive dose-response, and/or potential confounding by
tumor- or age-related changes make interpretation of these data highly uncertain. Subsequently,
no NOAEL or LOAEL can be identified.
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Table 6. Pathology Changes in Male Syrian Golden Hamsters Exposed to Methyl
Hydrazine by Inhalation for 1 Year (Kinkead et al., 1985)
Control
0.2 ppm
2.0 ppm
5.0 ppm
Neoplastic Lesions
Nasal adenoma
1/1903
0/177
0/180
11X11°
Nasal adenomatous polyp
0/190
0/177
9/180°
11/177°
Adrenal cortical adenoma (benign)
16/191
16/173
10/172
23/176°
Nonneoplastic Lesions
Nasal submucosal cysts
35/190
52/177b
56/180°
46/177
Rhinitis
12/190
21/177b
25/180b
28/177°
Nasal hyperplasia
0/190
0/177
2/180
4/177
Pulmonary atelectasis
0/189
2/177
5/174b
7/174°
Hepatitis
20/194
15/175
24/177
31/174b
Biliary cysts
41/194
67/175°
73/177°
76/174°
Interstitial fibrosis of kidney
75/195
83/179
105/176°
96/177b
aNumber affected/number examined
bSignificantly different from control atp< 0.05
><0.01
Kinkead et al. (1985) reported hematology, clinical chemistry, and histopathology
findings for treated dogs. Apart from methemoglobin and serum ALT levels (shown in Table 7),
hematology and clinical chemistry data were reported graphically. A review of graphs of
hematology data indicated that both exposed groups of dogs exhibited dose-related decreases in
RBC, Hgb, and Hct throughout the exposure period. Methemoglobin levels were increased
relative to controls in the 2.0-ppm group throughout exposure and in the 0.2-ppm group at the
6-month time point (see Table 7). Serum ALT levels were markedly increased in the 2.0-ppm
group (between 4- and 6-fold higher than controls throughout the entire exposure period, based
on graphical data; levels at termination reported in Table 7) but were not different from controls
at 0.2 ppm. Liver function, as measured by BSP retention, was also affected at the high
exposure; retention was significantly (p < 0.01) increased in the 2.0-ppm group (see Table 7).
The authors reported that there were no treatment-related nonneoplastic or neoplastic lesions
observed upon histopathology examination of dogs 5 years after the end of exposure (data not
"3
shown). These data indicate a LOAEL of 0.2 ppm (0.38 mg/m ) for beagle dogs based on
hematology effects.
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Table 7. Selected Changes (Mean Values) in Dogs Exposed to Methyl Hydrazine
by Inhalation for 1 Year (Kinkead et al., 1985)
Control
0.2 ppm
2.0 ppm
Number of Animals Examined
4
4
4
Hematology
Methemoglobin(%Hgb): 3 months
0.791
0.753
1.0193
Methemoglobin (% Hgb): 6 months
0.806
1.084b
1.8333
Methemoglobin (% Hgb): 9 months
0.794
0.847
0.972b
Methemoglobin (% Hgb): 12 months
0.834
0.913
1.3363
Clinical chemistry
AST (IU/L): 12 months
50.0
75.0
228.3a
Other
BSP retention (% at 10 minutes): 12 months
14.9
20.3
34.5a
aSignificantly different from control atp< 0.05
V<0.01
OTHER STUDIES
Genotoxicity
Tables 8 and 9 summarize in vitro and in vivo genotoxicity data for MH, respectively.
Genotoxicity data for MH are mixed but suggest that this compound may be mutagenic under
some circumstances. Specifically, increased mutation frequencies have been observed in several
Salmonella typhimurium strains (TA100, TA1535, and TA1537) when tested in suspension
assays (Matsushita et al., 1993; Rogan et al., 1982; Brusick and Matheson, 1976), while negative
results were observed in these strains in plate incorporation assays (Mortelmans et al., 1986;
Brusick and Matheson, 1976). Poso et al. (1995) observed increased mutation frequencies when
S. typhimurium TA102 was tested in a plate incorporation assay; this strain also yielded positive
results in a suspension assay (Matsushita et al., 1993). Tests for mutations in DNA
repair-deficient strains of Escherichia coli have yielded positive results (Poso et al., 1995;
Von Wright et al., 1977). When MH was tested for forward mutation in Chinese hamster lung
fibroblast V79 cells, weakly positive results were reported (Kuszynski et al., 1981); this study
was reported only as an abstract. No increase in forward mutations was observed in mouse
lymphoma L5178Y cells (Rogers and Back, 1981; Brusick and Matheson, 1976). Tests for DNA
repair in ACI rat and C3H/HeN mouse hepatocytes were positive without metabolic activation
(Mori et al., 1988). MH did not induce unscheduled DNA synthesis in human fibroblast WI-38
cells when tested with or without metabolic activation (Brusick and Matheson, 1976). Ehrlich
ascites liver cells incubated with MH have shown evidence of single-strand DNA breaks
(Moroson and Furlan, 1969).
The few available in vivo studies of genotoxicity, including dominant lethal tests in mice
and rats exposed via intraperitoneal injection, as well as tests for unscheduled DNA synthesis in
the liver of rats exposed via gavage, have given negative results (Brusick and Matheson, 1976;
Beije and Olsson, 1990). Host-mediated assays using S. typhimurium in mice exposed orally to
MH gave equivocal results (Von Wright and Tikkanen, 1980b).
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Table 8. Results of In Vitro Genotoxicity Studies of Methyl Hydrazine
Test System
Endpoint
MH
Result"
Reference
Purity
Vehicle
Dose/Concentration
Tested
Metabolic activation
Cytotoxicity1"
+S9
-S9
S. typhimurium TA1535,
TA1537, TA1538, TA98,
TA100
Reverse mutation
(plate assay)
NR
DMSO
0.0001-5.0 |iL/platc
NR
Brusick and
Matheson, 1976
(unpublished)
S. typhimurium TA1535
Reverse mutation
(suspension
assay)
NR
DMSO
1-5 |iL/mL
NT
+
NR
Brusick and
Matheson, 1976
(unpublished)
S. typhimurium TA100
Reverse mutation
NR
NR
1-3 |imol
-
-
At >3 |imol
Von Wright and
Tikkanen, 1980b
S. typhimurium TA1535,
1537
Reverse mutation
(suspension
assay)
>98%
Distilled
water
100-1000 ng/plate
+ at cytotoxic
concentration
+ at cytotoxic
concentration
At 200-
500 ng/plate
Roganetal., 1982
S. typhimurium TA1535,
TA1537, TA97, TA98,
TA100
Reverse mutation
(plate assay)
NR
Distilled
water
1-100 ng/plate
NR
Mortelmans et al.,
1986
S. typhimurium TA102,
TA100
Reverse mutation
(suspension
assay)
NR
NR
Up to 2 nmol for TA100
and 10 junol for TA102
+
NR
Matsushita et al.,
1993
S. typhimurium TA102
Reverse mutation
(plate assay)
NR
Distilled
water
0.5- 2.0 |imo 1/plate
+
NT
NR
Poso et al., 1995
Saccharomyces cerevisae
D4
Gene recom-
bination
NR
DMSO
0.000 - 5.0 |iL/platc
NR
Brusick and
Matheson, 1976
(unpublished)
E. coli WPluvrA-
DNA repair
NR
DMSO
0.0001- 5.0 |iL/platc
NR
Brusick and
Matheson, 1976
(unpublished)
E. coli WP2 and CM871
DNA repair
NR
Distilled
water
NR
+ for repair-
deficient CM871
NT
NR
Poso et al., 1995
E. coli WP2 try,her
Reverse mutation
NR
Water
5-20 iig/mL
NT
+ at cytotoxic
concentration
Yes >5 iig/mL
Von Wright et al,
1977
E. coli pol Ai+, pol Af, WP2
try,her, B/r WP2/r>'
DNA repair
NR
NR
0.5-1.0 mg
NT
+
NR
Von Wright et al.,
1977
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Table 8. Results of In Vitro Genotoxicity Studies of Methyl Hydrazine
Test System
Endpoint
MH
Result"
Reference
Purity
Vehicle
Dose/Concentration
Tested
Metabolic activation
Cytotoxicity1"
+S9
-S9
E. coli WP2B/r trp; WP2B/r
uvrA, trp; CM871
uvrA, recA, lexA,trp
SOS induction
NR
NR
0.5-2.0 |imol (spot test);
0.5-1.0 |imol/mL (liquid
incubation test)
NT
+, greater response
in repair-deficient
strains
NR
Von Wright and
Tikkanen 1980a
Chinese hamster lung
fibroblasts V79 cells
Forward mutation
NR
NR
NR
Weakly +
Weakly +
NR
Kuszynski et al.,
1981 (abstract)
L5178Y mouse lymphoma
cells
Forward mutation
NR
DMSO
0.0005-0.1 nL/mL
NR
Brusick and
Matheson, 1976
(unpublished)
L5178Y mouse lymphoma
cells
Forward mutation
NR
DMSO
0.1-5 mM
-
-
At 5 mM
Rogers and Back,
1981
ACI rat hepatocytes
DNA repair
NR
NR
10"-Kr4 M MH sulfate
NT
+
At 10-3 M
Mori et al., 1988
C3H/HeN mouse
hepatocytes
DNA repair
NR
NR
10_5-10-3 M
MH sulfate
NT
+
None
Mori et al., 1988
Human fibroblast
WI-38 cells
Unscheduled
DNA synthesis
NR
DMSO
0.1-1.0 |iL/mL (without
activation)
0.1-0.5 |iL/mL (with
activation)
NR
Brusick and
Matheson, 1976
(unpublished)
a+ = active; - = inactive; NR = not reported; NT = not tested
bDefined for this review as survival < 50%
DMSO = dimethyl sulfoxide, DNA = deoxyribonucleic acid, NR = not reported, NT = not tested
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Table 9. Results of In Vivo Genotoxicity Studies of Methyl Hydrazine
Species/Test System
Endpoint
Dose and Route
Result"
Reference
Mouse
Dominant lethal
mutation
0.26-26 mg/kg via i.p.
injection
-
Brusick and Matheson,
1976 (unpublished)
Rat
Dominant lethal
mutation
0.22-22 mg/kg via i.p.
injection
-
Brusick and Matheson,
1976 (unpublished)
Rat liver
Unscheduled DNA
synthesis in liver
gavage, 30 mg/kg
-
Beije and Olsson, 1990
(abstract)
a - = inactive
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR METHYL HYDRAZINE
SUBCHRONIC AND CHRONIC p-RfD
There are no human studies of oral exposure to MH. The database of animal
toxicological studies of oral exposure includes one subchronic-duration study in mice
(Kelly et al., 1969), two chronic-duration studies each in mice and hamsters (MacEwen and
Vernot, 1975; Toth and Shimizu, 1973; Toth, 1972; Roe et al., 1967), and a developmental
toxicity study in rats (Slanina et al., 1993). The subchronic- and chronic-duration studies were
primarily aimed at assessing carcinogenicity of MH, and most did not report any noncancer
endpoints other than mortality. Of the available studies, only MacEwen and Vernot (1975) and
Slanina et al. (1993) provided enough information on noncancer endpoints to identify a LOAEL.
The LOAEL identified for the data in MacEwen and Vernot (1975) was 7.3 mg/kg-day,
the only dose tested, for reduced body weight (at least 10%) and possible hematologic effects in
hamsters. The use of a single dose level precluded modeling of these data. The developmental
toxicity study (Slanina et al., 1993) identified a LOAEL of 5 mg/kg (single dose) for increased
preimplantation losses; the NOAEL was 1 mg/kg. The data for percent preimplantation loss
were modeled using the EPA Benchmark Dose Software (BMDS v. 2.1), but adequate model fit
was not achieved. Appendix B presents details of the benchmark dose (BMD) modeling. As a
consequence, the NOAEL of 1 mg/kg-day associated with the developmental toxicity study
(Slanina et al., 1993) was selected as the point of departure (POD) for both subchronic and
chronic p-RfD derivation.
A subchronic and a chronic p-RfD were derived by dividing the NOAEL of
1.0 mg/kg-day by a UF of 1000, as shown below:
Subchronic and Chronic p-RfD = NOAEL UF
= 1.0 mg/kg-day 1000
= 0.001 mg/kg-day or 1 x 10 3 mg/kg-day
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The composite UF of 300 was composed of the following UFs:
• UFh: A UFh of 10 is applied for intraspecies differences to account for potentially
susceptible individuals in the absence of information on the variability of
response in humans
• UFa: A UFa of 10 is applied for animal-to-human extrapolation to account for
potential toxicokinetic and toxicodynamic differences between rats and humans.
There are no data to determine whether humans are more or less sensitive than
rats to the developmental toxicity of MH
• UFd: A UFd of 10 is selected because the database includes a single
developmental toxicity study in rats (Slanina et al., 1993) with only one day
dosing (GD 6) and limited developmental endpoints tested, no two-generation
reproduction studies, and there is a clear indication for hematological effects as
well as hemosiderosis of the liver, kidney, and spleen during inhalation exposure
(MacEwen and Haun, 1971; Kinkead et al., 1985)
• UFl: A UFl of 1 is applied because the POD was developed using a NOAEL.
• UFs: A UFs of 1 is applied because further adjustment for duration is not
warranted when developmental toxicity data are used to develop a POD.
Confidence in the key study (Slanina et al., 1993) is low. This study included three dose
groups with group sizes of 16-24 animals and identified clear NOAEL and LOAEL values.
However, the dams were exposed on a single day (GD 6), so effects on other developmental
stages could not be assessed. The toxicologic endpoints examined were uterine contents and
external and skeletal malformations; visceral malformations were not evaluated, and no
assessment of maternal toxicity was included. The oral experiment was supported by consistent
findings in an i.v. experiment with six dose levels reported in the same paper. Confidence in the
database for noncancer effects of oral MH is low. All oral subchronic- and chronic-duration
studies were designed as cancer bioassays, with little or no investigation of noncancer endpoints.
Developmental toxicity has been studied in only one species, and no studies of reproductive
toxicity are available. Low confidence in the subchronic and chronic p-RfD follows.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
INHALATION RfC VALUES FOR METHYL HYDRAZINE
There are no studies of subchronic- or chronic-duration human exposure to MH via
inhalation. All of the available animal studies of inhaled MH are unpublished. These include
90-day studies of continuous exposure to MH in rats, dogs, and monkeys (Darmer and
MacEwen, 1973); 6-month studies of intermittent (6 hours/day, 5 days/week) or continuous
exposure in rats, mice, dogs, and monkeys (MacEwen and Haun, 1971; Kroe, 1971; Haun,
1970); and 1-year studies of intermittent exposure in rats, mice, hamsters, and dogs
(Kinkead et al., 1985). Table 10 provides a summary of the effect levels identified from the
available studies. These studies provide consistent evidence of hemolytic effects in several
species, as well as hepatic changes in dogs and hamsters exposed for >90 days. Because all of
the relevant studies are unpublished, it is not appropriate to derive provisional values. However,
Appendix A of this document contains a screening value that may be useful in certain instances.
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Table 10. Summary of Inhalation Noncancer Dose-Response Information for Methyl Hydrazine
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Responses at the LOAEL
Comments
Reference
Sub chronic-Duration studies
Rats
80 M/group
0, 0.046, 0.1 ppm
(0,0.087,0.19
mg/m3), continuously
for 90 days
0.087
HEC:a
0.087
0.19
HEC:
0.19
Hematology changes (decreased
RBC)
Evaluations limited to body weight,
limited hematology and serum
chemistry, and gross pathology
Darmer and
MacEwen, 1973
Dogs
8 F/group
0, 0.046, 0.1 ppm
(0,0.087,0.19
mg/m3), continuously
for 90 days
0.087
HEC:
0.087
0.19
HEC:
0.19
Hematology (decreased RBC,
Hgb, Hct, increased osmotic
fragility) and serum chemistry
changes (increased ALP) and
gross liver pathology
Evaluations limited to body weight,
limited hematology and serum
chemistry, and gross pathology
Darmer and
MacEwen, 1973
Monkeys
4 F/group
0, 0.046, 0.1 ppm
0,0.087, 0.19 mg/m3),
continuously for
90 days
0.19
HEC:
0.19
NA
None
Evaluations limited to hematology
and gross pathology
Darmer and
MacEwen, 1973
Chronic-Duration studies
Wistar rats
50 M/group
0, 0.2, 1.0, 2.0,
5.0 ppm (0, 0.38, 1.9,
3.8, 9.4 mg/mg3)
6 hours/day,
5 days/week, for
about 6 months
1.9
HEC:
0.34
3.8
HEC:
0.68
Body weight decreased
approximately 10%
Hematology not examined in rats
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
Wistar rats
50 M/group
0, 0.2 ppm (0,
0.38 mg/m3)
continuously for
6 months
0.38
HEC:
0.38
NA
None
Hematology not examined in rats
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
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Table 10. Summary of Inhalation Noncancer Dose-Response Information for Methyl Hydrazine
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Responses at the LOAEL
Comments
Reference
F344 Rats
100/sex/treatment
group
150/sex controls
0, 0.02, 0.2, 2.0,
5.0 ppm (0, 0.038,
0.38, 3.8, 9.4 mg/m3)
6 hours/day,
5 days/week, for 1
year
3.8
HEC:
0.68
9.4
HEC:
1.68
Decreased body weight clearly
related to MH exposure
Body-weight decreases at lower
exposures not dose related
Kinkead et al.,
1985
ICR mice
40/group
0, 0.2, 1.0, 2.0,
5.0 ppm (0, 0.38, 1.9,
3.8, 9.4 mg/mg3)
6 hours/day,
5 days/week, for
about 6 months
NA
0.38
HEC:
0.068
Hemosiderosis of the liver,
spleen, and kidneys
Hematology not examined in mice.
Sex of treated mice reported
inconsistently by different authors
(see study summary)
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
ICR mice
40/group
0, 0.2 ppm (0,
0.38 mg/m3)
continuously for
6 months
NA
0.38
HEC:
0.38
Hemosiderosis of the liver,
spleen, and kidneys
Hematology not examined in mice.
Sex of treated mice reported
inconsistently by different authors
(see study summary)
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
Beagle dogs
8/group
0, 0.2, 1.0, 2.0,
5.0 ppm (0, 0.38, 1.9,
3.8, 9.4 mg/mg3)
6 hours/day,
5 days/week, for
about 6 months
NA
0.38
HEC:
0.068
Hematologic and histopathologic
evidence of hemolytic anemia;
liver cholestasis
Sex of treated dogs reported
inconsistently by different authors
(see study summary)
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
Beagle dogs
8/group
0, 0.2 ppm (0,
0.38 mg/m3)
continuously for 6
months
NA
0.38
HEC:
0.38
Hematologic and histopathologic
evidence of hemolytic anemia;
liver cholestasis
Sex of treated dogs reported
inconsistently by different authors
(see study summary)
MacEwen and
Haun, 1971;
Kroe, 1971;
Haun, 1970
Beagle dogs
4/sex/group
0, 0.2, 2.0 ppm (0,
0.38, 3.8 mg/m3)
6 hours/day,
5 days/week, for
1 year
NA
0.38
HEC:
0.068
Hematology changes (decreased
RBC, Hgb, Hct; increased
methemoglobin)
Kinkead et al.,
1985
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Table 10. Summary of Inhalation Noncancer Dose-Response Information for Methyl Hydrazine
Species and
Study Type
(«/sex/group)
Exposure
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Responses at the LOAEL
Comments
Reference
C57BL/6J mice
400 female/group
0, 0.2, 2.0 ppm (0,
0.38, 3.8 mg/m3)
6 hours/day,
5 days/week, for
1 year
NA
NA
NA
The low incidence and lack of a
clear step-wise-response for lesions
such as nasal lesions to be treatment
related
Kinkead et al.,
1985
Syrian golden
hamsters
200 males/group
0, 0.2, 1.0, 2.0,
5.0 ppm (0, 0.38, 1.9,
3.8, 9.4 mg/m3)
6 hours/day,
5 days/week, for
about 6 months
NA
NA
NA
Body weights of treated hamsters
was decreased throughout exposure
in the 5-ppm (9.4-mg/m3) group.
Nonstatistically significant increase
of nonneoplastic lesions
Kinkead et al.,
1985
aHEC calculated as follows: NOAELheC = NOAEL x exposure hours/24 hours x exposure days/7 days x dosimetric adjustment
For systemic effects, the dosimetric adjustment is the ratio of the animal:human blood:gas partition coefficients for MH (in the absence of experimental values,
a default value of 1 was used)
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PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR METHYL HYDRAZINE
WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), the
available evidence suggests that MH is "Likely to be Carcinogenic to Humans" based on positive
results in some (but not other) oral animal studies (discussed below), increased incidences of
lung and liver tumors and hemangiomas in female mice exposed via inhalation for 1 year
(Kinkead et al., 1985), and increased incidences of nasal and adrenal tumors in male hamsters
exposed via inhalation for 1 year (Kinkead et al., 1985). Genotoxicity data for MH are mixed
but suggest that this compound may be mutagenic under some circumstances.
The oral cancer data for MH include a study of increased incidences of lung and liver
tumors in male and female mice exposed via drinking water for life (Toth, 1972). However, this
study did not include a concurrent control group (the reference group was studied separately as
part of another, previous experiment), and animals may have been exposed to oxidation products
of MH, rather than the chemical itself (low stability of MH in water at neutral pH was
demonstrated by MacEwen and Vernot, 1975; solutions were changed only three times per week
in the Toth, 1972 study). Low survival in treated mice (no male mice surviving past 60 weeks
and no female mice surviving past 70 weeks) also complicates interpretation of these findings.
Also in this study, a 10-fold lower concentration of MH sulfate, which did not affect survival,
produced a much larger increase in lung tumor incidence. However, lung tumors were not
increased by MH sulfate in another study of mice that featured gavage (in water) exposure at a
higher dose (Roe et al., 1967) but was limited by small group size (n = 25) and relatively short
exposure duration (40 weeks).
The oral cancer data also include a study of increased incidences of malignant
histiocytomas of the liver and tumors of the cecum in male and female hamsters exposed via
drinking water for life (Toth and Shimizu, 1973). It has been suggested that exposure of treated
animals in this study was to oxidation products of MH, rather than the chemical itself (MacEwen
and Vernot, 1975). A second study in hamsters conducted at the same drinking water
concentration as the Toth and Shimizu (1973) study, but including daily changes of test solution
and adjustment of pH to 3.5 to reduce loss of MH from solution, found no increases in tumors
(MacEwen and Vernot, 1975). However, this study started with older animals (5 months versus
6 weeks), thereby reducing exposure duration (exposure was lifetime in both studies), and group
sizes were smaller (30 males in each of two treatment groups [adjusted pH and unadjusted] and
only 17 controls, versus 50/sex/treated group and 100/sex/ control group in Toth and Shimizu,
1973).
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
Oral Exposure
The database for oral carcinogenicity of MH is limited by (1) lack of a concurrent control
in the study of mice (Toth, 1972); (2) reduced survival in the study of mice (Toth, 1972); (3)
questions about the stability of the treatment compound in drinking water in both the Toth (1972)
mouse and Toth and Shimizu (1973) hamster studies; and (4) inconsistent findings in two studies
in hamsters (MacEwen and Vernot, 1975; Toth and Shimizu, 1973) that used the same exposure
concentration. Furthermore, available oral studies demonstrating treatment-related induction of
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tumors (Toth and Shimizu, 1973; Toth, 1972) each used a single concentration of MH in
drinking water, providing only limited dose-response information. As a consequence of the
uncertainties in the available database for oral carcinogenicity of MH, a provisional OSF was not
derived for this compound.
Inhalation Exposure
No human inhalation exposure data were located. The animal data available are from the
1-year bioassay conducted by Kinkead et al. (1985), an unpublished technical report for a
chronic-duration inhalation toxicity study. Kinkead et al. (1985) was the only study that
demonstrated increased incidences of tumors after inhalation exposure. In female B6C3Fi mice,
there were increased incidences of lung adenomas, liver adenomas, liver carcinomas, and
hemangiomas (Kinkead et al., 1985). In male hamsters, the incidences of nasal adenomas, nasal
adenomatous polyps, and adrenal cortical adenomas were significantly increased (Kinkead et al.,
1985). Because the incidence of hemangiomas in the high-dose group was illegible in the
available report, no modeling was performed on this data set. Because the principal study is
unpublished, it is not appropriate to derive provisional values. However, Appendix A presents a
screening provisional inhalation unit risk value (Screening p-IUR).
REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (2001) Documentation
of the threshold limit values for chemical substances. 7th Edition. Cincinnati, OH.
ACGIH (American Conference of Governmental Industrial Hygienists). (2008) Threshold limit
values for chemical substances and physical agents and biological exposure indices. Cincinnati,
OH.
ATSDR (Agency for Toxic Substances and Disease Registry). (1997) Toxicological profile for
hydrazines. U.S. Department of Health and Human Services, Public Health Service, Atlanta,
GA.
Beije, B. Olsson, U. (1990) Genotoxic activity of some hydrazines in the liver perfusion/cell
culture system and in the rat in vivo. Mutat Res 234:370-371.
Brusick, D; Matheson, D. (1976) Mutagenic evaluation of 1,1-dimethylhydrazine,
methylhydrazine and n-phenyl-alpha-naphthylamine, NTIS AD AMRL-TR-76-125.
CalEPA (California Environmental Protection Agency). (2009a) OEHHA/ARB approved
chronic reference exposure levels and target organs. Online http://www.arb.ca.eov/
toxics/healthval/chroni c.pdt'.
CalEPA (California Environmental Protection Agency). (2009b) Air chronic reference exposure
levels adopted by OEHHA as of February 2005. Online http://www.oehha.ca.gov/
air/chronic rels/AllChrels.html.
CalEPA (California Environmental Protection Agency). (2009c) Hot spots unit risk and cancer
potency values. Online http://www.oehha.ca.gov/air/hot spots/pdf/TSDlookup2002.pdf.
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Darmer, KI, Jr.; MacEwen, JD. (1973) Monomethylhydrazine. Chronic low level exposures and
24-hour emergency exposure limits. Aerospace Medical Research Laboratory, Aerospace
Medical Division, Air Force Systems Command, Wright-Patterson Air Force Base, Ohio, Report
No. AMRL-TR-73-125 (Proceedings of the Fourth Annual Conference on Environmental
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Garcia, HD; James, JT; Limero, T. (1992) Human exposure limits to hypergolic fuels. Govt
Reports Announcements & Index (GRA&I), Issue 16, 2092:7. In: 5th Annual Workshop on
Space Operations Applications and Research (Soar 1991), Volume 2 pp. 620-626.
Haun, CC. (1970) Chronic exposure to low concentrations of monomethylhydrazine.
Proceedings of the First Annual Conference on Environmental Toxicology, Paper No. 22, pp.
341-353. Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Ohio.
Report No. AMRL-TR-70-102. NTIS No. AD-727 526.
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Kelly MG; O'Gara, RW; Yancy, ST; et al. (1969) Comparative carcinogenicity of
N-\ sopropyl-a-(2-methyl hydrazine)-/>toluamide HC1 (procarbazine hydrochloride), its
degradation products, other hydrazines, and isonicotinic acid hydrazide. J Natl Cancer Inst
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Kinkead, ER; Haun, CC; Vernot, EH; et al. (1985) A chronic inhalation toxicity study on
monomethylhydrazine. Air Force Aerospace Medical Research Laboratory, Wright-Patterson
Air Force Base, Ohio. Report No. AFAMRL-TR-85-025. NTIS No. AD-A154 659.
Kroe, DJ. (1971) Animal pathology resulting from long-term exposure to low levels of
monomethylhydrazine. Proceedings of the Second Annual Conference on Environmental
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Wright-Patterson Air Force Base, Ohio. Report No. AMRL-TR-71-120. NTIS No. AD-751 441.
Kuszynski, C; Langenbach, R; Malick, L. (1981) Liver cell-mediated mutagenesis of v-79 cells
by hydrazine and related compounds. Environ Mutagen 3:323-324.
MacEwen, JD; Theodore, J; Vernot, EH. (1970) Human exposure to EEL concentrations of
monomethylhydrazine. Proceedings of the First Annual Conference on Environmental
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Report No. AMRL-TR-70-102 (Proceedings of the First Annual Conference on, 19701970
Environmental Toxicology, Fairborn, Ohio, September 9-11, 1970, pp. 355-363).
MacEwen, JD; Haun, CC. (1971) Chronic exposure studies with monomethylhydrazine.
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255-270. Report No. AMRL-TR-71-120. NTIS No. AD-751 440.
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MacEwen, JD; Vernot, EH. (1975) Studies on the effect of monomethylhydrazine in drinking
water on golden Syrian hamsters. In: Toxic Hazards Research Unit Annual Technical Report:
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Ohio. Report No. AMRL-TR-75-57. NTIS No. AD-A019 456.
Matsushita, H., Jr.; Endo, O; Matsushita, H. (1993) Mutagenicity of alkylhydrazine oxalates in
Salmonella typhimurium TA100 and TA102 demonstrated by modifying the growth conditions
of the bacteria. Mutat Res 301(4):213-222.
Mori, H., Sugie, S., Yoshime, N., Iwata, H., Nishikawa, A., Matsukubo, K., Shimizu, H. and
Hirono, I. (1988), Genotoxicity of a Variety of Hydrazine Derivatives in the Hepatocyte Primary
Culture/DNA Repair Test Using Rat and Mouse Hepatocytes. Cancer Science, 79: 204-211.
Moroson, H; Furlan, M. (1969). Single strand breaks in DNA of Ehrlich ascites tumor cells
produced by methyl hydrazine. Radiat Res 40:351-365.
Mortelmans, K; Haworth, S; Lawlor, T. (1986) Salmonella mutagenicity tests. 2.results from the
testing of 270 chemicals. Environ Mutagen 8(Suppl 7): 1-119.
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Rogers, AM; Back, KC. (1981) Comparative mutagenicity of hydrazine and 3 methylated
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Slanina, P; Cekan, E; Halen, B. (1993) Toxicological studies of the false morel (Gyromitra
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Toth, B. (1969) Lung tumor induction and inhibition of breast adenocarcinomas by hydrazine
sulfate in mice. J Nat. Cancer Inst 42:469-475.
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Swiss mice. Failure of ammonium hydroxide to interfere in the development of tumors. Int J
Cancer 9(1): 109-118.
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morphology of malignant histiocytomas. Cancer Res 33:2744-2753.
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monomethyl and dimethyl derivatives in bacterial test systems. Mutat Res 78(1): 17-23.
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APPENDIX A. DERIVATION OF AN INHALATION SCREENING VALUE
FOR METHYL HYDRAZINE
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for MH. However, information is available for this chemical which, although
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.
To provide a basis for comparing the studies, NOAEL and LOAEL values were adjusted
for continuous exposure and then converted to human equivalent concentrations (HECs). First,
exposure was adjusted to equivalent continuous exposure according to the equation below:
NOAELadj = NOAEL (mg/m3) x hours per day ^ 24 x days per week ^ 7
Then, treating MH as a Category 3 gas for effects on extrarespiratory endpoints, the dosimetric
adjustments were made using the ratio of animal :human blood:gas partition coefficients for MH
(U.S. EPA, 1994b). However, blood:gas partition coefficients for MH were not located for any
species. In the absence of blood:gas partition coefficients, the default ratio of 1.0 was used to
perform the dosimetric adjustment. For each study, the duration-adjusted effect level was
multiplied by the corresponding dosimetric adjustment to calculate the HEC:
NOAELhec = NOAELadj x Dosimetric Adjustment
Where:
Dosimetric Adjustment = ratio of animal :human blood:gas partition coefficients (default = 1).
Table 10 includes the HECs.
SCREENING SUBCHRONIC p-RfC
Table 10 summarizes inhalation noncancer dose-response data for MH and converted
HECs for both subchronic- and chronic-duration studies in rats, mice, hamsters, dogs, and
monkeys (Darmer and MacEwen, 1973; MacEwen and Haun, 1971; Kroe, 1971; Haun, 1970;
Kinkead et al., 1985). Darmer and MacEwen (1973) is the only available inhalation
subchronic-duration study and involved rats, dogs, and monkeys. Darmer and MacEwen (1973)
reported that hematologic effects of continuous exposure to 0.1 ppm (0.19 mg/m3) monomethyl
hydrazine (MMH) showed dose-response consistency with other previous studies. However,
continuous exposure at 0.046 ppm (0.08 mg/m3) did not significantly alter the hematology of the
treated and animals and had no effect on rat growth. On the basis of these changes in
hematologic parameters (RBC, Hgb, and Hct), as well as serum chemistry parameters (ALP and
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total phosphorus), in both rats and dogs (see Tables 3 and 4), NOAELhec and LOAELhec values
for rats and dogs were the same (0.087 and 0.19 mg/m3, respectively). The study in monkeys did
not identify any effects at the highest concentration tested (NOAELhec of 0.19 mg/m ). The data
were reported without any measures of variability (e.g., standard deviation), precluding
benchmark dose modeling. Consequently, the NOAELhec of 0.087 mg/m for both rats and dogs
was selected as the POD for screening subchronic p-RfC derivation.
The screening subchronic p-RfC for MH was calculated as the NOAELhec of
0.087 mg/m3 divided by an uncertainty factor (UF) of 300, as shown below:
Screening Subchronic p-RfC = NOAELhec ^ UF
= 0.087 mg/m3 - 300
= 0.00029 or 3 x 10"4 mg/m3
The composite UF of 300 was composed of the following UFs:
• UFa: A factor of 3 (10°5) is applied for animal-to-human extrapolation to account
for the toxicodynamic portion of the UFa because the toxicokinetic portion (10°5)
has been addressed in the dosimetric conversions.
• UFh: A factor of 10 is applied for intraspecies differences to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans
• UFd: A factor of 10 is selected because there are no acceptable two-generation
reproduction studies or developmental studies, and there is no indication of any
other studies that may be relevant to the database UF.
• UFl: A factor of 1 is applied because the POD was developed using a NOAEL.
• UFs: A factor of 1 is applied because the POD was developed using a
subchronic-duration study.
Confidence in the key study (Darmer and MacEwen, 1973) is low. This study included
three exposure groups with group sizes of 80 rats and eight dogs and identified clear NOAEL
and LOAEL values for effects (hematology changes) shown consistently in several studies and
species. However, the study is unpublished and, thus, has not been subjected to peer review.
Only a single sex (male rats and female dogs) was tested in each species. The toxicologic
endpoints examined were limited to body weight, hematology, serum chemistry, and gross
pathology; histopathology evaluations were not reported. Confidence in the database for
noncancer effects of subchronic-duration inhalation exposure to MH is low, as the database is
limited to one unpublished study in rats, dogs, and monkeys (Darmer and MacEwen, 1973).
There are no studies examining developmental or reproductive effects after inhalation of MH,
and developmental toxicity has been demonstrated in one oral study (Slanina et al., 1993). Low
confidence in the screening subchronic p-RfC follows.
SCREENING CHRONIC p-RfC
Table 10 shows the HECs for 6-month and 1-year studies in rats, mice, and dogs
(Kinkead et al., 1985; MacEwen and Haun, 1971; Kroe, 1971; Haun, 1970). As the table
indicates, the most sensitive endpoints were hematological and/or histopathological evidence of
hemolytic anemia in dogs and hemosiderosis of the liver, spleen, and kidneys in mice exposed to
"3
a HEC of 0.068 mg/m for 6 months or 1 year. Reported changes at this exposure level included
decreases in RBC, Hgb, and Hct; increases in RBC fragility, methemoglobin, and Heinz bodies;
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and hemosiderosis of the spleen, liver, and kidneys. Liver cholestasis, as indicated by
histopathology and serum chemistry (increased serum ALP and bilirubin), was also evident in
dogs exposed at this level in the 6-month study. Similar liver effects were seen in the 6-month
mouse study and the 1-year dog study but at higher concentrations. NOAEL values were not
established. No standard deviation was reported, which made the data in these studies
insufficient to perform benchmark dose modeling. Consequently, the LOAELhec of
0.068 mg/m for both mice and dogs was selected as the POD for screening chronic p-RfC
derivation.
The screening chronic p-RfC for MH was calculated as the LOAELhec of 0.068 mg/m
divided by a UF of 3000, as shown below:
Screening Chronic p-RfC = LOAELhec ^ UF
= 0.068 mg/m3 - 3000
= 0.00002 or 2 x 10"5 mg/m3
The composite UF of 3000 was composed of the following UFs:
• UFa: A factor of 3 (10°5) is applied for animal-to-human extrapolation to account
for the toxicodynamic portion of the UFa because the toxicokinetic portion (10°5)
has been addressed in the dosimetric conversions.
• UFh: A factor of 10 is applied for intraspecies differences to account for
potentially susceptible individuals in the absence of information on the variability
of response in humans
• UFd: A factor of 10 is selected because there are no acceptable two-generation
reproduction studies or developmental studies, and there is no indication of any
other studies that may be relevant to the database UF. All of the available studies
are unpublished, and none of them included lifetime exposure.
• UFl: A factor of 10 is applied for using a POD based on a LOAEL because a
NOAEL cannot be determined from the available data.
• UFs: A factor of 1 is applied because a chronic-duration study was utilized as the
critical study.
Confidence in the key mouse and dog studies (Kinkead et al., 1985; MacEwen and Haun,
1971; Kroe, 1971; Haun, 1970) is low. The studies included multiple exposure groups but did
not identify a NOAEL. Group sizes were large for mice but small for dogs. The studies are all
unpublished and, thus, have not been subjected to peer review; in addition, much of the data
were reported graphically and without information on the nature or results of statistical analysis.
The critical hematology endpoints were not measured directly in mice but were only indicated
indirectly by histopathology. The 1-year exposure duration, while chronic in duration, did not
represent a lifetime exposure in any of the species tested. Confidence in the database for
noncancer effects of chronic-duration inhalation exposure to MH is low. The database includes
6-month and 1-year studies in rats, mice, hamsters, dogs, and/or monkeys (Kinkead et al., 1985;
MacEwen and Haun, 1971; Kroe, 1971; Haun, 1970); however, few studies identified NOAEL
levels (see Table 10). All of the available studies are unpublished, and none of them included
lifetime exposure. There were reporting inconsistencies among reports of the same studies; for
example, MacEwen and Haun (1971) and Haun (1970) reported that female monkeys, dogs, and
mice were used in all of the 6-month studies, while Kroe (1971) reported that the mice exposed
to 2.0 and 5.0 ppm were male, and that the dogs and monkeys exposed to 0.2 and 1.0 ppm were
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male. The studies by Kinkead et al. (1985) employed prolonged postexposure observation
periods (1 year in rodents and 5 years in dogs) prior to pathology evaluations, allowing for
possible recovery from reversible noncancer effects and age- and tumor-related confounding of
noncancer lesions. There are no studies examining developmental or reproductive effects of
inhalation of MH; developmental toxicity has been demonstrated in one oral study (Slanina et al.,
1993). Low confidence in the screening chronic p-RfC follows.
SCREENING INHALATION UNIT RISK (IUR)
Inhalation data are sufficient to derive a quantitative estimate of cancer risk for MH; this
derivation is shown below. Data from the 1-year bioassay conducted by Kinkead et al. (1985)
were used as the basis for the quantitative cancer assessment, as this was the only study that
demonstrated increased incidences of tumors after inhalation exposure. In female B6C3Fi mice,
there were increased incidences of lung adenomas, liver adenomas, liver carcinomas, and
hemangiomas (Kinkead et al., 1985). In male hamsters, the incidences of nasal adenomas, nasal
adenomatous polyps, and adrenal cortical adenomas were significantly increased (Kinkead et al.,
1985). Because the incidence of hemangiomas in the high-dose group was illegible in the
available report, no modeling was performed on this data set. Table A-l shows the modeling
that was performed for the remaining data sets.
Table A-l. Dose-Response Data for Derivation of Inhalation Unit Risk
(Kinkead et al., 1985)
Species and Sex
Target Organ
Tumor Type
Exposure Concentration (mg/m3)
Control
0.038
0.38
3.8
Female mouse
Lung
Adenoma
13/3643
16/354
23/347
56/360b
Liver
Adenoma
6/373
2/357
5/357
20/363b
Liver
Carcinoma
2/373
4/357
4/357
14/363b
Control
0.38
3.8
9.4
Male hamster0
Nasal cavity
Adenoma
1/190
0/177
0/180
7/177d
Nasal cavity
Adenomatous polyp
0/190
0/177
9/180b
ll/177b
Adrenal cortex
Adenoma
16/191
16/173
10/172
23/176b
aNumber affected/number examined
V<0.01
Incidence of hemangioma data was not included because it was not quantified in the Kinkead et al. (1985) study
dSignificantly different from control atp< 0.05
Dose-response modeling of the data in Table A-l was performed to obtain a POD for a
quantitative assessment of cancer risk. The POD is an estimated concentration (expressed in
human-equivalent terms) near the lower end of the observed range that marks the starting point
for extrapolation to lower doses. Each tumor type was modeled individually. Combining tumors
at a single site (e.g., liver adenomas and carcinomas in mice, or nasal cavity adenomas and
adenomatous polyps in hamsters) was considered but was not possible because the summary data
were presented only for individual tumor types in the original study and the individual animal
data were not available.
Appendix C provides details of the modeling efforts and the selection of best-fitting
models. In accordance with EPA (2000) guidance, BMC and BMCL values associated with a
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BMR (benchmark response) of 10% extra risk were calculated. Table A-2 compares the BMCio
and BMCLio values estimated from the best-fitting models for the various tumor types. The
BMCLio values were adjusted to equivalent continuous exposure concentrations BMCLioadj,
and HECs were then calculated using the dosimetric adjustment appropriate to the observed
tumor type, as shown in the following equations:
BMCLioadj = BMCLio x exposure hours ^ 24 hours x exposure days ^ 7 days
= BMCLio x 6 hours ^ 24 hours x 5 days ^ 7 days
BMCLiohec = BMCLioadj x dosimetric adjustment
Table A-2. Comparison of BMCio and BMCLio Values for Lung, Nasal, and Adrenal
Tumors in Female Mice and Male Hamsters Exposed to Methyl Hydrazine3
Species
and Sex
Target Organ
Tumor Type
BMC10b
(mg/m3)
BMCL10b
(mg/m3)
BMCL10adj
(mg/m3)
Dosimetric
Adjustment11
BMCL10hec
(mg/m3)
Female
mouse
Lung
Adenoma
3.12
2.3
0.41
3.54 (RGDRpu)
1.4
Liver
Adenoma
8.82
5.8
1.04
1.0 (blood:gas)
1.0
Male
hamster
Nasal cavity
Adenoma
13.46
11.1
1.98
0.059 (RGDRet)
0.12
Nasal cavity
Adenomatous
polyp
12.36
8.7
1.55
0.059 (RGDRet)
0.092
Adrenal cortex
Adenoma
11.60
9.2
1.64
1.0 (blood:gas)
1.6
aSee Appendix C for details of modeling
bUnadjusted for continuous exposure and before conversion to human equivalent concentration
°Exposure concentration adjusted to equivalent continuous exposure concentration based on treatment 6 hours/day,
5 days/week, as follows: BMCLioadj = BMCL10 x 6 hours/24 hours x 5 days/7 days
dosimetric adjustment is calculated according to endpoint, as follows:
For systemic effects, it is the ratio of the animal:human blood:gas partition coefficients for MH (in the absence of
experimental values, a default value of 1 was used);
For respiratory effects, it is the RGDR for the affected portion of the respiratory tract (pulmonary for lung
adenomas in mice and extrathoracic for nasal tumors in hamsters), calculated as the ratio of the animal:human
minute volume/surface area ratios using default values from EPA (1994b)
eHuman equivalent concentration calculated as product of adjusted LOAEL and dosimetric adjustment, as follows:
BMCLiohec = BMCLioadj x Dosimetric adjustment
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For tumors in the respiratory tract, the dosimetric adjustment is the regional gas
deposition ratio (RGDR) for the affected portion of the respiratory tract (pulmonary for lung
adenomas in mice and extrathoracic for nasal tumors in hamsters). An RGDR(ET) was
calculated as the dosimetric adjustment for nasal (extrathoracic) tumors in male hamsters with
chronic-duration exposure, as follows (equation 4-18 and default values from U.S. EPA, 1994b):
Hamster RGDR(ET) = (MVa - Sa) - (MVh - Sh)
= (0.057 L/min/14 cm2) (13.8 L/min 200 cm2)
= 0.0041 L/min-cm2 0.069 L/min-cm2
= 0.059
Where:
RGDR(ET) = regional gas dose ratio for the extrathoracic area of the respiratory tract
MVa = animal minute volume (hamster = 0.057 L/min, based on default body weight of
0.134 kg for male hamster in a chronic-duration study; see U.S. EPA, 1994b)
MVh= human minute volume (13.8 L/min)
Sa= surface area of the extrathoracic region in the animal (hamster =14 cm2)
Sh = surface area of the extrathoracic region in the human (200 cm )
Similarly, an RGDR(PU) of 3.54 was calculated as the dosimetric adjustment for
pulmonary tumors in female mice with chronic exposure according to Equation 4-28 (pulmonary
RGDR for Category 1 gas) in EPA (1994b) using default values for body weight (0.0353 kg for
mice, 70 kg for humans), ventilation rate (0.0413 L/minute for mice, 13.8 L/minute for humans),
and pulmonary surface area (0.05 m2 for mice, 54 m2 for humans) in EPA (1994b).
The dosimetric adjustment for the remaining, systemic (extrarespiratory) tumors is the
ratio of animal :human blood:gas partition coefficients (U.S. EPA, 1994b). In the absence of
chemical-specific blood:gas partition coefficients for MH in humans or animals, the default
value of 1.0 was used.
Among the data sets modeled, the lowest BMCLiohec values calculated as described
above were 0.09 and 0.1 mg/m3 based on modeling of nasal adenomatous polyps and nasal
"3
adenomas in hamsters, respectively. The low value of 0.09 mg/m was selected as the POD for
derivation of the IUR.
The mode of action for tumors produced by MH has not been elucidated; thus, the default
linear methodology was applied. In order to linearly extrapolate cancer risks from the
BMCLiohec to the origin, a Screening p-IUR was calculated as the ratio BMR/BMCLiohec
(0.1/0.09 mg/m3), as follows:
Screening p-IUR = BMR BMCLiohec
= 0.1 ^ 0.09 mg/m3
= 1 (mg/m3) 1
The screening provisional IUR for MH should not be used with exposures exceeding the POD
(BMCLiohec = 0.09 mg/m3), because at exposures above this level, the fitted dose-response
model better characterizes what is known about the carcinogenicity of MH.
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APPENDIX B. DETAILS OF BENCHMARK DOSE MODELING
FOR SUBCHRONIC AND CHRONIC RfD
MODEL-FITTING PROCEDURE FOR CONTINUOUS DATA
The BMD modeling for continuous data was conducted with EPA's BMD software
(BMDS v. 2.1). For the continuous data, the original data were modeled with all the continuous
models available within the software with a default BMR of one standard deviation (SD). An
adequate fit was judged based on the goodness-of-fit p-value (p> 0.1), scaled residual at the
range of benchmark response (BMR), and visual inspection of the model fit. In addition to the
three criteria forjudging the adequate model fit, whether the variance needed to be modeled, and
if so, how it was modeled also determined the final use of the model results. If a homogenous
variance model was recommended based on statistics (Test 2) provided from the BMD modeling
results, the final BMD results would be estimated from a homogenous variance model. If the test
for homogenous variance (Test 2) was negative (p < 0.1), the model was run again while
applying the power model integrated into the BMDS to account for nonhomogenous variance
(known as the nonhomogenous variance model). If the nonhomogenous variance model did not
provide an adequate fit to the variance data (Test 3: p < 0.1), the data set would be considered
unsuitable for BMD modeling. Among all the models providing adequate data fit
(goodness-of-fit p-v alue > 0.1), the lowest BMDL will be selected if the BMDLs estimated from
different models vary over a wide range (not quantified); otherwise, the BMDL from the model
with the lowest Akaike Information Criterion (AIC) would be considered appropriate for the data
set.
MODEL-FITTING RESULTS FOR PERCENT PRE IMPLANTATION LOSS IN RATS
(SLANINA ET AL., 1993)
The data for percent preimplantation loss associated with MH administration (mean ± SD
\ri\ preimplantation losses per litter of 22.17 ± 0.50 [24], 29.78 ± 0.40 [16], and 40.83 ± 0.63%
[16], for 0-, 1-, and 5-mg/kg doses, respectively) were modeled using the EPA Benchmark Dose
Software (BMDS v. 2.1). The models were run with a BMR of 1 SD from the control mean, as
recommended by EPA (2000). With only three dose groups, the linear model was the only
continuous variable model available (the larger models have too many parameters for the number
of data points, leaving insufficient degrees of freedom to assess model fit). While the
homogenous variance model provided adequate fit to the variance data, the means did not fit the
linear model. Table B-l shows the modeling results. Thus, this data set was not suitable for
BMD modeling.
Table B-l. Model Predictions for Percent Preimplantation Loss in Rat
(Slanina et al., 1993)
Variance
Means
bmd1sd
BMDL1sd
Model
/7-Valuea
/7-Valuea
AIC
(mg/kg-day)
(mg/kg-day)
Linear, homogenous variance13
0.1975
<0.0001
126.81
0.50
0.43
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bCoefficients restricted to be positive
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Linear Model with 0.95 Confidence Level
40
35
CD
(f>
C
0
Q.
in
CD
iH
1 30
a)
25
Linear
BMDL
dose
11:13 08/09 2010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File: C:\USEPA\BMDS21\Data\MMH\lin_MMH-slamine_SDMHH-Slanine-
Linear.(d)
Gnuplot Plotting File: C:\USEPA\BMDS21\Data\MMH\lin_MMH-slamine_SDMHH-Slanine-
Linear.plt
Mon Aug 09 11:13:53 2010
BMDS Model Run
The form of the response function is:
Y[dose] = beta_0 + beta_l*dose + beta_2*doseA2 + ...
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
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Signs of the polynomial coefficients are not restricted
A constant variance model is fit
Total number of dose groups = 3
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 0.266104
rho = 0 Specified
beta_0 = 24.0167
beta 1 = 3.455
Asymptotic Correlation Matrix of Parameter Estimates
( * * * The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix)
alpha betaO betal
alpha 1 1.5e-009 2.8e-009
beta_0 1.5e-009 1 -0.63
beta 1 2.8e-009 -0.63 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
alpha 3.1815 0.601247 2.00308 4.35992
beta_0 23.5802 0.306592 22.9793 24.1811
beta 1 3.55573 0.112489 3.33525 3.7762
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
0 24 22.2 23.6 0.5 1.78 -3.87
1 16 29.8 27.1 0.4 1.78 5.93
5 16 40.8 41.4 0.63 1.78 -1.19
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Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Log(likelihood)
# Param's AIC
Al
10.610004
4
-13.220007
A2
12.232083
6
-12.464166
A3
10.610004
4
-13.220007
fitted
-60.405886
3
126.811772
R
-142.616844
2
289.233688
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (Al vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1
309.698
4
<0001
Test 2
3.24416
2
0.1975
Test 3
3.24416
2
0.1975
Test 4
142.032
1
<0001
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than . 1. A homogeneous variance
model appears to be appropriate here
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The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is less than . 1. You may want to try a different
model
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 0.501635
BMDL = 0.429715
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APPENDIX C. DETAILS OF BENCHMARK DOSE MODELING
FOR INHALATION UNIT RISK
MODEL-FITTING PROCEDURE FOR CANCER INCIDENCE DATA
The model-fitting procedure for dichotomous cancer incidence data is as follows. The
multistage cancer model in the EPA BMDS is fit to the incidence data using the extra risk option.
The multistage cancer model is run for all polynomial degrees up to n-1 (where n is the number
of dose groups including control). Adequate model fit is judged by three criteria: goodness-of-fit
p-walue (p > 0.1), visual inspection of the dose-response curve, and scaled residual at the data
point (except the control) closest to the predefined BMR. Among all of the models providing
adequate fit to the data, the lowest BMDL is selected as the POD when the difference between
the BMDLs estimated from these models is high (unquantified); otherwise, the BMDL from the
model with the lowest Akaike Information Criterion (AIC) is chosen. In accordance with EPA
(2000) guidance, BMDs and BMDL values associated with a BMR of 10% extra risk are
calculated.
MODEL-FITTING RESULTS FOR LUNG ADENOMA IN FEMALE MICE EXPOSED
TO METHYL HYDRAZINE BY INHALATION FOR 1 YEAR (KINKEAD ET AL., 1985)
Applying the procedure outlined above to the data for lung adenoma in female mice
exposed to MH, adequate fit was achieved with all models. Table C-l shows the modeling
results. The higher-degree polynomial models defaulted back to the 1-degree model, so that all
of the models gave the same result. Figure C-l shows the fit of the multistage cancer, 1-degree
model to the data. The benchmark concentration (BMCio) and associated 95% lower confidence
limit (BMCL10) for this data set were 3.12 and 2.34 mg/m3, respectively.
Table C-l. Model Predictions for Lung Adenoma in Female Mice Exposed to Methyl
Hydrazine by Inhalation for 1 Year (Kinkead et al., 1985)
Model
Degrees of
Freedom
x2
2
X
Goodness-of-Fit
/>-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
2
1.30
0.52
728.30
3.12
2.34
Multistage cancer, 2-degreeb
2
1.30
0.52
728.30
3.12
2.34
Multistage cancer, 3-degreeb
2
1.30
0.52
728.30
3.12
2.34
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
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Multistage Cancer Model wth 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.2
0.15
0.1
0.05
BMDL
BMD
0
0.5
1
1.5
2
2.5
3
3.5
14:37 04/132009
Figure C-l. Fit of Multistage Cancer, 1-Degree Model to Data on Lung
Adenoma in Female Mice Exposed to Methyl Hydrazine by Inhalation for
1 Year (Kinkead et al., 1985).
Note: BMC and BMCL indicated are associated with an extra risk of 10% and are in units of mg/m3.
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MODEL-FITTING RESULTS FOR LIVER ADENOMA IN FEMALE MICE EXPOSED
TO METHYL HYDRAZINE BY INHALATION FOR 1 YEAR (KINKEAD ET AL., 1985)
Applying the procedure outlined above to the data for liver adenoma in female mice
exposed to MH, adequate model fit was achieved with all models. Table C-2 shows the
modeling results. BMCLs from models providing adequate fit did not differ by more than
3-fold. In accordance with EPA (2000) guidance, the model with the lowest AIC, the 1-degree
model, was selected as the source of the POD. Figure C-2 shows the fit of the multistage cancer,
1-degree model to the data. For this data set, the resulting BMC10 and BMCL10 were 8.82 and
5.78 mg/m , respectively.
Table C-2. Model Predictions for Liver Adenoma in Female Mice Exposed
to Methyl Hydrazine by Inhalation for 1 Year (Kinkead et al. (1985)
Model
Degrees
of
Freedom
2
X
2
X
Goodness-of-Fit
/>-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
2
2.08
0.35
299.76
8.82
5.78
Multistage cancer, 2-degreeb
1
1.88
0.17
301.65
6.30
4.70
Multistage cancer, 3-degreeb
1
1.88
0.17
301.65
6.30
4.39
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
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Multistage Cancer Model with 0.95 Confidence Level
0.12
Multistage Cancer
Linear extrapolation
0.1
0.08
0.06
0.04
0.02
0
BMDL
BM
0
1
2
3
4
5
6
7
8
9
Dose
14:57 04/13 2009
Figure C-2. Fit of Multistage Cancer, 1-Degree Model to Data on Liver Adenoma in
Female Mice Exposed to Methyl Hydrazine by Inhalation for 1 Year (Kinkead et al.
1985).
Note: BMC and BMCL indicated are associated with an extra risk of 10% and are in units of mg/m3.
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MODEL-FITTING RESULTS FOR LIVER CARCINOMA IN FEMALE MICE
EXPOSED TO METHYL HYDRAZINE BY INHALATION FOR 1 YEAR
(KINKEAD ET AL., 1985)
Applying the procedure outlined above to the data for liver carcinoma in female mice
exposed to MH, all of the models achieved adequate fit, but the BMD calculations failed (see
Table C-3). Dropping the highest dose was considered but rejected because the only evidence
for an effect of treatment was at the high dose. This data set proved to be unsuitable for BMD
modeling.
Table C-3. Model Predictions for Liver Carcinoma in Female Mice
Exposed to Methyl Hydrazine by Inhalation for 1 Year
(Kinkead et al., 1985)
Model
Degrees of
Freedom
x2
2
X
Goodness-of-Fit
/j-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
2
0.69
0.71
235.98
Not applicable
Not applicable
Multistage cancer, 2-degreeb
2
0.69
0.71
235.98
Not applicable
Not applicable
Multistage cancer, 3-degreeb
2
0.69
0.71
235.98
Not applicable
Not applicable
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
MODEL-FITTING RESULTS FOR NASAL ADENOMA IN MALE HAMSTERS
EXPOSED TO METHYL HYDRAZINE BY INHALATION FOR 1 YEAR
(KINKEAD ET AL., 1985)
Applying the procedure outlined above to the data for nasal adenoma in male hamsters
exposed to MH, adequate model fit was achieved with multistage cancer, 2- and 3-degree
models. For the 1-degree model, model fit was marginal, and the BMD calculation failed.
Table C-4 shows the modeling results. BMCLs from models providing adequate fit did not
differ by more than 3-fold. In accordance with EPA (2000) guidance, the model with the lowest
AIC, the 3-degree model, was selected as the basis for the POD. Figure C-3 shows the fit of the
multistage cancer, 3-degree model to the data. For this data set, the resulting BMCio and
BMCL10 were 13.46 and 11.07 mg/m3, respectively.
Table C-4. Model Predictions for Nasal Adenoma in Male Hamsters Exposed to Methyl
Hydrazine by Inhalation for 1 Year (Kinkead et al. (1985)
Model
Degrees of
Freedom
x2
2
X
Goodness-of-Fit
/>-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
2
4.69
0.096
82.54
Not applicable
Not applicable
Multistage cancer, 2-degreeb
2
2.93
0.23
79.65
16.92
12.59
Multistage cancer, 3-degreeb
2
2.30
0.32
78.43
13.46
11.07
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.1
0.08
0.06
0.04
0.02
0
BMDL
BMD
0
2
4
6
8
10
12
14
14:18 04/14 2009
Figure C-3. Fit of Multistage Cancer, 3-Degree Model to Data on Nasal
Adenoma in Male Hamsters Exposed to Methyl Hydrazine by Inhalation for
1 Year (Kinkead et al. (1985).
Note: BMC and BMCL indicated are associated with an extra risk of 10% and are in units of mg/m3.
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MODEL-FITTING RESULTS FOR NASAL ADENOMATOUS POLYPS IN MALE
HAMSTERS EXPOSED TO METHYL HYDRAZINE BY INHALATION FOR 1 YEAR
(KINKKM) ET AL., 1985)
Applying the procedure outlined above to the data for nasal adenomatous polyps in male
hamsters exposed to MH, adequate model fit was achieved with all models. Table C-5 shows the
modeling results. The higher-degree polynomial models defaulted back to the 1-degree model,
so that all of the models gave the same result. Figure C-4 shows the fit of the multistage cancer,
1-degree model to the data. For this data set, the resulting BMC10 and BMCL10 were 12.36 and
8.74 mg/m , respectively.
Table C-5. Model Predictions for Nasal Adenomatous Polyps in Male Hamsters Exposed
to Methyl Hydrazine by Inhalation for 1 Year (Kinkead et al. (1985)
Model
Degrees of
Freedom
x2
2
X
Goodness-of-Fit
/j-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
3
3.04
0.39
159.26
12.36
8.74
Multistage cancer, 2-degreeb
3
3.04
0.39
159.26
12.36
8.74
Multistage cancer, 3-degreeb
3
3.04
0.39
159.26
12.36
8.74
aValues <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
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Multistage Cancer Model with 0.95 Confidence Level
0.12
Multistage Cancer
Linear extrapolation
0.1
'.08
'.06
'.04
'.02
0
BMDL
BMD
0
2
4
6
8
10
12
Dose
15:39 04/13 2009
Figure C-4. Fit of Multistage Cancer, 1-Degree Model to Data on Nasal
Adenomatous Polyps in Male Hamsters Exposed to Methyl Hydrazine by
Inhalation for 1 Year (Kinkead et al., 1985).
Note: BMC and BMCL indicated are associated with an extra risk of 10% and are in units of mg/m3.
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MODEL-FITTING RESULTS FOR ADRENAL CORTICAL ADENOMA IN
MALE HAMSTERS EXPOSED TO METHYL HYDRAZINE BY INHALATION
FOR 1 YEAR (KINKEAD ET AL., 1985)
Applying the procedure outlined above to the data for adrenal cortical adenoma in male
hamsters exposed to MH, adequate model fit was achieved with all models. Table C-6 shows the
modeling results. BMCLs from models providing adequate fit did not differ by more than
3-fold. In accordance with EPA (2000) guidance, the model with the lowest AIC, the 3-degree
model, was selected as the source of the POD. Figure C-5 shows the fit of the multistage cancer,
3-degree model to the data. For this data set, the resulting BMCi0 and BMCL10 were 11.60 and
9.19 mg/m3, respectively.
Table C-6. Model Predictions for Adrenal Cortical Adenoma in Male Hamsters Exposed
to Methyl Hydrazine by Inhalation for 1 Year (Kinkead et al., 1985)
Model
Degrees of
Freedom
x2
x2
Goodness-of-Fit
/>-Valuc
AIC
BMC10
(mg/m3)
BMCL10
(mg/m3)
Multistage cancer, l-degreeb
2
3.40
0.18
437.11
24.96
10.86
Multistage cancer, 2-degreeb
2
2.32
0.31
435.89
13.43
9.33
Multistage cancer, 3-degreeb
2
1.85
0.40
435.34
11.60
9.19
"Values <0.1 fail to meet conventional goodness-of-fit criteria
bBetas restricted to >0
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Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
BMO
BMDL
0
2
4
6
8
10
12
Dose
15:49 04/13 2009
Figure C-5. Fit of Multistage Cancer, 3-Degree Model to Data on Adrenal Cortical
Adenoma in Male Hamsters Exposed to Methyl Hydrazine by Inhalation for 1 Year
(Kinkead et al., 1985).
Note: BMC and BMCL indicated are associated with an extra risk of 10% and are in units of mg/m3.
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