United States
Environmental Protection
1=1 m m Agency
EPA/690/R-10/008F
Final
9-29-2010
Provisional Peer-Reviewed Toxicity Values for
Cyclohexane
(CASRN 110-82-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Dan D. Petersen, Ph.D., DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
SRC, Inc.
7502 Round Pond Road
North Syracuse, NY 13212
PRIMARY INTERNAL REVIEWERS
Q. Jay Zhao, Ph.D., M.P.H., DABT
National Center for Environmental Assessment, Cincinnati, OH
Martin W. Gehlhaus, III, M.H.S.
National Center for Environmental Assessment, Washington, DC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
HISTORY	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVS	2
INTRODUCTION	2
REVIEW 01 PERTINENT DATA	3
Human Studies	3
Oral Exposure	3
Inhalation Exposure	3
Animal Studies	6
Oral Exposure	6
Inhalation Exposure	6
Sub chronic-duration Studies	6
Reproductive/developmental Studies	12
Other Studies	19
Neurotoxicity	19
Toxicokinetics	21
Genotoxicity	21
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL ORAL RfD
VALUES FOR CYCLOHEXANE	22
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL INHALATION
RfC VALUES FOR CYCLOHEXANE	22
Subchronic p-RfC	22
Chronic p-RfC	27
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR CYCLOHEXANE	27
Weight-of-Evidence Descriptor	27
Quantitative Estimates of Carcinogenic Risk	27
REFERENCES	28
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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
CYCLOHEXANE (CASRN 110-82-7)
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
Cyclohexane is a cycloalkane used as a nonpolar solvent in the chemical industry as well
as a feedstock for production of nylon. The IRIS (U.S. EPA, 2009a) database includes an RfC
"3
value of 6 mg/m for cyclohexane based on a two-generation reproduction study in rats
(i.e., Kreckmann et al., 2000) that identified a NOAEL of 6886 mg/m3 and a LOAEL of
"3
24,101 mg/m for developmental effects (reduced pup weight in F1 and F2 generation offspring).
IRIS includes discussions on oral toxicity and cancer, but, due to inadequate data, no RfD value
or quantitative cancer risk estimates were derived (U.S. EPA, 2009a). The source document for
the IRIS assessment is a Toxicological Review of Cyclohexane that was published in August
2003 (U.S. EPA, 2003). Cyclohexane is currently undergoing review as part of the IRIS Track
Report for Alkylates Assessment, which began in November 2008 (U.S. EPA, 2009b). See
Figure 1 for the chemical structure of cyclohexane.
Figure 1. Chemical Structure of Cyclohexane
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No RfD, RfC, or cancer assessment for cyclohexane is available on the HEAST
(U.S. EPA, 1997) or in the Drinking Water Standards and Health Advisories list (U.S. EPA,
2006). No relevant documents were located in the Chemical Assessments and Related Activities
(CARA) list (U.S. EPA, 1994, 1991). ATSDR (2009) has not published a Toxicological Profile
for cyclohexane, and no Environmental Health Criteria Document is available from the World
Health Organization (WHO, 2009). The carcinogenicity of cyclohexane has not been assessed
by the International Agency for Research on Cancer (IARC, 2009) or the National Toxicology
Program (NTP, 2009, 2005). The American Conference for Governmental Industrial Hygienists
(ACGIH, 2008) has adopted a threshold limit value-time-weighted average (TLV-TWA) of
100 ppm as protective against central nervous system (CNS) impairment. The National Institute
for Occupational Safety and Health (NIOSH, 2009) recommended exposure limit (REL) is
300 ppm based on irritation of eyes, skin, and respiratory system and CNS effects. The
Occupational Safety and Health Administration (OSHA, 2009) permissible exposure limit (PEL)
is 300 ppm.
Literature searches were initially conducted from the 1960s through July 2010 for studies
relevant to the derivation of provisional toxicity values for cyclohexane. Databases searched
included MEDLINE, TOXLINE (with NTIS), BIOSIS, TSCATS/TSCATS2, CCRIS, DART,
GENETOX, HSDB, RTECS, Chemical Abstracts, and Current Contents (February through July
2010).
REVIEW OF PERTINENT DATA
HUMAN STUDIES
Oral Exposure
No information was located regarding the subchronic or chronic oral toxicity of
cyclohexane in humans.
Inhalation Exposure
Two small studies have been conducted among workers primarily exposed to
cyclohexane (Yasugi et al., 1994; Yuasa et al., 1996). Both of these studies were reviewed by
EPA in the 2003 Toxicological Review of Cyclohexane (U.S. EPA, 2003) and are summarized
below.
Yasugi et al. (1994) surveyed women workers (20-60 years old) from a Japanese factory
where glue containing at least 75% cyclohexane was applied to surfaces by automated sprayers.
During the survey, each worker was equipped with a personal air monitor at the start of each
shift. Each worker was administered a questionnaire on subjective symptoms experienced within
the last 3 months both at home and at work. Health effects were compared between 38 exposed
(mean age 25.2) workers who were either directly involved in glue application or worked in the
vicinity of glue application for at least 1 year, and 9 nonexposed (mean age 25.5) clerical
workers employed at the same factory but located in a different building. Blood and urine
samples were collected from all women at the end of each shift on four separate occasions. In
addition, 14 exposed workers provided a preshift urine sample the morning following the study
day. Venous blood samples were submitted for hematology (white blood cell count [WBC], red
blood cell count [RBC], hemoglobin concentration [Hgb], and hematocrit [Hct]) and serum
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chemistry (total protein, blood urea nitrogen [BUN], creatinine, uric acid, total cholesterol,
HDL-cholesterol, triglyceride, aspartate aminotransferase [AST], alanine aminotransferase
[ALT], y-glutamyl transpeptidase [y-GTP], alkaline phosphatase [ALP], leucine aminopeptidase,
and lactate dehydrogenase [LDH]). The concentration of cyclohexane in blood was also
determined. Urine samples were analyzed to measure metabolite (cyclohexanone and
cyclohexanol) levels. In addition, blood samples from nine exposed and nine nonexposed
workers (five smokers and four nonsmokers from each group) were evaluated for the possible
effects of cyclohexane on the sister chromatid exchange (SCE) rates of peripheral lymphocytes.
There was a discrepancy in reporting of exposure levels in Yasugi et al. (1994); the
"3
geometric mean measured cyclohexane concentration was reported as 27 ppm (93 mg/m ) in the
text but as 18.2 ppm (63 mg/m3) in Table 1 of the report. The maximum air concentration of
"3
cyclohexane vapor was reported to be 274 ppm (943 mg/m ). Concentrations of cyclohexane in
blood and cyclohexanol in urine appeared to correlate with measured exposure levels. Based on
questionnaire responses, there was no difference in the prevalence of subjective symptoms
experienced at work between the exposed workers and controls. However, nonexposed subjects
complained of 57 symptoms while not at work, significantly more than exposed workers
(p < 0.01). There were no significant differences based on individual symptoms. Hematology
results revealed a difference in the prevalence of leukocytopenia between nonexposed (1/9) and
exposed (3/17 low exposure, 1/16 high exposure) workers that achieved marginal statistical
significance (0.05 
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Table 1. Summary of Responses to an Auditory Stimulus During Inhalation
Exposure to Cyclohexane Vapor for 90 Days
Response
Exposure Concentration (mg/m3)
0
1721
6886
24,101
90-Day rat study
Normal response
65/65a
61/65
0/65
0/65
Diminished response
0/65
4/65
16/65
1/65
No response
0/65
0/65
49/65
64/65
90-Day neurotoxicity rat study
Normal response
71/71
71/71
4/71
0/71
Diminished response
0/71
0/71
32/71
3/71
No response
0/71
0/71
35/71
68/71
90-Day mouse study
Normal response
67/67
67/67
7/67
3/67
Diminished response
0/67
0/67
47/67
17/67
No response
0/67
0/67
13/67
2/67
Hyperactivity
0/67
0/67
4/67
2/67
Abnormal behaviorb
0/67
0/67
0/67
61/67
aNumber of exposures when a given response was observed/total number of exposures. Animals in each exposure
group were observed together in the exposure chamber, and the response of the group as a whole to a standardized
auditory stimulus (prior to exposure, after 2, 4, and 6 hours of exposure, and 30 minutes after exposure ended) was
subjectively characterized as normal, diminished, absent, or hyperresponsive; observers were not blind to exposure
status of the animals.
bClinical signs of abnormal behavior in mice at 24,101 mg/m3 included hyperactivity, jumping, hopping, circling,
flipping, rear leg kicking, standing on front legs, and excessive grooming, These behaviors frequently prevented
determination of response to a sound stimulus in this group.
Source: Malley et al. (2000).
Urinary cyclohexanol measurements ranged from 0.12 to 8.23 mg/L (geometric mean of
0.55 mg/L) and were highly correlated to ambient cyclohexane levels in the workplace
(Yuasa et al., 1996). Responses from the health effects questionnaire included complaints of
fatigue in 9/18 exposed workers and 4/15 controls, headaches in 10/18 exposed workers and
7/15 controls, and dizziness in 7/18 exposed workers and 4/15 controls. In the
neurophysiological examination, no significant differences in nerve conduction velocities
(NCVs) were observed between exposed workers and controls, but the ulnar and peroneal MDLs
were significantly shorter in exposed workers than in controls (p < 0.05). During the follow-up
study 1 year later, significant improvements in NCV and MDL of exposed workers were
observed. These results suggest that past //-hexane exposures may have impacted the initial
results greater than in the follow-up study. Based on limitations of this study, including past
exposures to //-hexane, small group sizes, and poorly matched controls, the effects of
cyclohexane exposure in these workers cannot be adequately assessed.
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Lammers et al. (2009) conducted a physiologically based pharmacokinetic (PBPK)
modeling study that involved human volunteers. The neurobehavioral effects of inhaled
cyclohexane in rats and humans were investigated to define relationships between internal doses
and acute CNS effects. Cyclohexane concentrations in blood were measured to assess internal
"3
exposure. Human volunteers were exposed for 4 hours to 86 or 860 mg/m in two test sessions.
Neurobehavioral effects were measured using a computerized neurobehavioral test battery. In
rats, slight reductions in psychomotor speed in the high-exposure group—but minimal CNS
effects—are evident. In humans, there are no significant treatment-related effects at the levels
tested. While a NOAEL of 86 mg/m for neurobehavioral outcomes may be suggested from these
data, the small number of volunteers and the number of replicates make this determination
uncertain. Additionally, because these are acute studies (4 hours), this value would not
necessarily be protective of additional outcomes from longer exposures.
EPA (2003) considered these studies, as well as additional studies of occupational
exposure to a mixture of solvents where the primary exposures were to solvents other than
cyclohexane, to be inadequate for dose-response assessment because of limitations in study
design and potential confounding by coexposures to other compounds.
ANIMAL STUDIES
Oral Exposure
No data were located on the subchronic and/or chronic oral effects of cyclohexane in
animals.
Inhalation Exposure
Subchronic-duration Studies—There are four sub chronic-duration studies in the
literature; one in rats, one in mice, and two in rabbits. All of the studies of subchronic-duration
inhalation exposure of cyclohexane in animals identified in the literature search were reviewed
by EPA (2003), except for an older, 40-day French study in rabbits (Fabre et al., 1952). The
subchronic-duration inhalation studies for cyclohexane are summarized below.
Two unpublished, 90-day inhalation toxicity studies conducted with cyclohexane in
Crl:CD BR rats and CD-I mice (Haskell Laboratory, 1996a,b) were later summarized and
published as parts of Malley et al. (2000). In these studies, groups of rats and mice
(20/gender/species/concentration for control and high-concentration groups and
10/gender/species/concentration for low- and intermediate-concentration groups) were exposed
to cyclohexane (99.9% purity) vapor at 0, 500, 2000, or 7000 ppm (0, 1721, 6886, or
"3
24,101 mg/m ) 6 hours/day, 5 days/week, for 13-14 weeks, for a total of at least 65 exposures.
Exposure concentrations were selected based on the results of 2-week range-finding studies in
rats and mice (Haskell Laboratories, 1995) and knowledge of the explosive properties of
cyclohexane. All rats and mice were monitored during and immediately after the daily exposure
periods for clinical signs of distress and for their response to an auditory-alerting stimulus, and
weekly for changes in body weight and food consumption. Blood samples were collected from
10 rats/sex/concentration at 45 and 90 days for hematology (RBC, platelet count, Hgb, Hct, mean
corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular
hemoglobin concentration [MCHC], and total and differential WBC) and clinical chemistry
(ALP, ALT, AST, sorbitol dehydrogenase [SDH], y-GTP, creatine phosphokinase, LDH, BUN,
glucose, calcium, phosphate, bilirubin, cholesterol, creatinine, triglycerides, total protein,
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albumin, globulin, sodium, potassium, and chloride). Blood samples from mice were evaluated
for the same hematology parameters as evaluated in the rat and for plasma protein. Urine
samples were also collected from rats for urinalysis (volume, osmolality, urobilinogen, pH, Hgb,
glucose, protein, bilirubin, and ketone). Both rats and mice were subjected to ophthalmological
examination both prior to the start of each study and just prior to the end of the 90-day
exposures. At the end of 90 days, 10 rats and 10 mice/species/gender/concentration were
sacrificed and necropsied. After a 1-month recovery period, 10 remaining rats and
mice/species/sex from the control and high-dose groups were sacrificed and necropsied. The
lungs, brain, heart, liver, spleen, kidneys, ovaries, adrenal glands, and testes were weighed, and a
complete histological examination was conducted.
There were no treatment-related deaths, and no significant effects were observed based
on body weight or food consumption in cyclohexane-treated rats (Malley et al., 2000). Rats
exposed at 6886 or 24,101 mg/m3 demonstrated a diminished or absent alerting response each
day in the exposure chamber, as detailed in Table 1. The researchers considered the effect on
alerting response to represent a compound-related sedative effect. The effect was transient, as no
clinical signs of compromised neurological function were evident when the rats were observed
individually upon removal from the exposure chamber 30 minutes after the end of the daily
exposure. The only observations in rats at that time were transient signs of stained and/or wet
fur, which the researchers attributed to salivation in response to the taste of residual cyclohexane
experienced by the rats while grooming themselves upon removal from the inhalation chambers.
Although there were a small number of instances of diminished alerting response at the low
"3
exposure level of 1721 mg/m , the researchers did not consider them to be treatment related due
to the low frequency, the lack of time-dependent pattern, and the possibility of misclassification
due to the subjective nature of the observations (see Table 1).
No significant treatment-related effects were observed in rats based on ophthalmology or
hematology (Malley et al., 2000). Decreases in the activity of some serum enzymes related to
hepatic function (AST, SDH, LDH, and creatine phosphokinase) were statistically significant
(p < 0.05), (Cochran-Armitage trend test) but these parameters generally did not exhibit a
dose-response relationship. Although increases in such enzyme activities can indicate tissue
damage, the biological significance of decreases in these enzyme activities is not known. Males
"3
exposed to 24,101 mg/m demonstrated a significant increase in mean relative liver weights
(relative to body and brain weights) both at 90 days and after the 1-month recovery period, as
shown in Table 2. Histology revealed centrilobular hepatocellular hypertrophy in these rats at
the end of the 90-day exposure period (see Table 2) at the high dose. Although livers of female
rats were not significantly enlarged compared to controls following cyclohexane treatment, 50%
of female rats exposed to 24,101 mg/m3 exhibited similar pathological changes as reported in the
male rats. Similar liver changes were not observed microscopically in rats of either sex at the
end of the 1-month recovery period. Malley et al. (2000) considered the liver changes observed
in treated rats to represent an adaptive response rather than an adverse effect. No other
significant changes in organ weights or histology were reported in rats.
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Table 2. Summary of Liver Changes in Rats Exposed to Cyclohexane Vapor
for 90 Days
Parameter
Test Day
Exposure Concentration (mg/m3)
0
1721
6886
24,101
Males
Mean absolute liver weight
(g)
90
18.81 ± 1.97a
17.64 ±3.40
16.86 ±2.11
19.91 ±2.53
123
20.23 ± 2.49
NDb
ND
22.37 ±3.58
Mean relative liver weight
(% of body weight)
90
3.65 ±0.21
3.56 ±0.30
3.52 ±0.24
4.00 ± 0.27°
123
3.78 ±0.24
ND
ND
4.01 ±0.31
Mean relative liver weight
(% of brain weight)
90
8.70 ±0.71
8.30 ±0.95
8.03 ±0.87
9.39 ± 1.09
123
9.22 ± 1.00
ND
ND
10.37 ± 1.31°
Incidence of hepatomegaly
90
0/10
0/10
0/10
10/10
123
0/10
0/10
0/10
4/10
Incidence of hypertrophy
90
0/10
0/10
0/10
9/10
123
0/10
ND
ND
0/10
Females
Incidence of hepatomegaly
90
0/10
0/10
0/10
0/10
123
0/10
0/10
0/10
0/10
Incidence of hypertrophy
90
0/10
0/10
0/10
5/10
123
0/10
ND
ND
0/10
aMean ± standard deviation.
bNot determined at this time point.
Significantly different from controls (p < 0.05, Dunnett's t-test).
Source: Malley et al. (2000).
In the corresponding mouse study, there were no treatment-related deaths, and no
significant effects were observed based on body weight or food consumption (Malley et al.,
2000; Haskell Laboratories, 1996b). Similar to rats, mice exposed to 6886 or 24,101 mg/m3
generally demonstrated a diminished or absent alerting response while in the exposure chamber,
although hyperactivity in response to alerting stimulus was observed near the end of the
experiment (Exposures 64-67) in these groups (see Table 1). Furthermore, group observations
during exposure showed that mice exposed to 24,101 mg/m3 had marked CNS stimulation
characterized by circling, jumping/hopping, excessive grooming, kicking of the rear legs,
standing on the front legs, and an occasional flipping behavior that persisted for a short period
after the end of each daily exposure. These clinical observations were apparent by the fourth
exposure and persisted throughout the remaining exposures. The abnormal behavior frequently
prevented assessment of auditory stimulus response in this group. In the individual observations
for clinical signs performed 30 minutes after the end of exposure, mice in the 24,101-mg/m3
group showed increases in abnormal gait/mobility, excessive grooming, hyperactivity,
hyperreactivity, leg spasms, and ruffled fur (see Table 3). These clinical signs were not observed
during the recovery period.
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Table 3. Incidence of Clinical Signs in Mice Exposed to Cyclohexane Vapor
for 90 Days
Clinical Observation
Exposure Concentration (mg/m3)
0
1721
6886
24,101
Males
Abnormal gait or mobility
0/20a
0/10
0/10
2/20b
Excessive grooming
0/20
0/10
0/10
2/20b
Hyperactive
0/20
0/10
0/10
3/20b
Hyperreactive
1/20
1/10
0/10
8/20b
Spasms rear leg(s)
0/20
0/10
0/10
2/20b
Ruffled fur
9/20
6/10
1/10
4/20
Females
Abnormal gait or mobility
0/20
0/10
0/10
1/20
Excessive grooming
0/20
0/10
0/10
0/20
Hyperactive
0/20
0/10
1/10
1/20
Hyperreactive
1/20
0/10
2/10
6/20b
Spasms rear leg(s)
0/20
0/10
0/10
1/20
Ruffled fur
1/20
1/10
0/10
5/20b
aNumber of responders/number in group. Based on individual animal observations performed each exposure day
upon removal from the exposure chamber 30 minutes after the end of the exposure period.
Significantly different from controls (p < 0.05, Cochran-Armitage test for trend).
Source: Malley et al. (2000).
Hematology revealed statistically significant increases in RBC and Hct among all
exposed male groups at 90 days and among females of the highest exposure group at 90 days and
after the 1-month recovery period, as shown in Table 4 (Malley et al., 2000). Hgb concentrations
were also elevated among both sexes at the highest exposure level. In addition, plasma protein
was significantly elevated in males from the highest exposure group. The pattern of changes
suggests a possible hemoconcentration effect, perhaps secondary to dehydration. However, the
changes were small in magnitude, not clearly related to dose, and within the range of biological
variation for control animals, and so not considered biologically relevant by the researchers. As
shown in Table 5, increased mean liver weights were observed in male mice from the
24,101-mg/m3 exposure group. However, no corresponding histological changes were observed.
Malley et al., 2000 considered the liver-weight changes observed in treated mice to represent an
adaptive response rather than an adverse effect. No other gross or histological changes were
observed in mice.
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Table 4. Summary of Changes in Hematology and Clinical Pathology
Parameters in Mice Exposed to Cyclohexane Vapor for 90 Days
Parameter
Test Day
Exposure concentration (mg/m3)
0
1721
6886
24,101
Males
RBC(xl06/ul)
45
9.29 ± 0.54a
9.78 ±0.83
9.58 ±0.70
9.96 ±0.86
90
9.28 ±0.68
10.14 ±0.71b
10.35 ±0.86b
10.16 ±0.74b
123
8.99 ±0.73
ND°
ND
9.54 ±0.61
Hct (%)
45
44 ±2
46 ±3
45 ±2
48 ±4
90
44 ±3
48 ± 4b
48 ± 3b
49 ± 3b
123
42 ±2
ND
ND
45 ± 3b
Hgb (g/dl)
45
16.0 ±0.7
16.7 ±0.9
16.6 ±0.9
17.9 ± 1.4b
90
16.5 ±0.7
17.5 ± 1.1
17.6 ± 1.5
17.6 ± 1.5
123
15.1 ±0.4
ND
ND
15.9 ± 0.9b
Plasma protein
45
6.2 ±0.3
6.2 ±0.3
6.3 ±0.5
6.8 ± 0.5b
90
6.1 ±0.4
6.7 ±0.3
6.5 ±0.5
6.5 ±0.4
123
6.4 ±0.4
ND
ND
6.5 ±0.4
Females
RBC(x106/uL)
45
9.39 ±0.52
9.53 ±0.74
9.26 ±0.94
10.26 ± 1.57
90
9.14 ±0.84
9.14 ±0.80
9.11 ±0.61
9.98 ± 0.73b
123
8.99 ±0.86
ND
ND
9.79 ± 0.73b
Hct (%)
45
45 ±3
46 ±3
45 ±4
50 ± 7b
90
43 ±3
44 ±3
44 ±3
49 ± 3b
123
43 ±4
ND
ND
45 ±4
Hgb (g/dL)
45
16.1 ±0.8
16.6 ± 1.3
16.1 ± 1.4
18.2 ± 2.8b
90
16.1 ±0.9
16.1 ±0.7
15.8 ±0.9
17.4 ± 1.2b
123
15.8 ± 1.3
ND
ND
16.6 ± 1.1
Plasma protein
45
5.9 ±0.3
6.2 ±0.2
5.9 ±0.3
6.2 ±0.6
90
5.8 ±0.3
6.1 ±0.3
6.1 ±0.3
6.1 ±0.3
123
6.0 ±0.3
ND
ND
6.1 ±0.4
aMean ± standard deviation.
bSignificantly different from controls (p < 0.05, Dunnett's t-test).
°Not determined at this time point.
Source: Malley et al. (2000).
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Table 5. Summary of Liver Changes in Mice Exposed to Cyclohexane
Vapor for 90 Days
Parameter
Test Day
Exposure concentration (mg/m3)
0
1721
6886
24,101
Males
Mean absolute liver weight (g)
90
1.28 ± 0.16a
1.46 ±0.21
1.42 ±0.22
1.50 ± 0.10b
123
1.52 ±0.18
ND°
ND
1.50 ±0.14
Mean relative liver weight
(% of body weight)
90
4.15 ±0.41
4.65 ± 0.50b
4.55 ±0.53
4.82 ± 0.3 lb
123
4.52 ±0.43
ND
ND
4.43 ±0.27
Mean relative liver weight
(% of brain weight)
90
2.58 ±0.39
2.90 ±0.39
2.87 ±0.46
3.06 ± 0.21b
123
3.18 ±0.37
ND
ND
3.12 ±0.34
Females
Mean absolute liver weight (g)
90
1.03 ±0.18
1.06 ±0.15
1.04 ±0.17
1.12 ±0.09
123
1.22 ±0.19
ND
ND
1.29 ±0.19
Mean relative liver weight
(% of body weight)
90
4.27 ±0.39
4.39 ±0.30
4.53 ±0.40
4.73 ± 0.3 lb
123
4.61 ±0.47
ND
ND
4.87 ±0.60
Mean relative liver weight
(% of brain weight)
90
2.14 ±0.37
2.16 ±0.28
2.23 ±0.32
2.31 ±0.18
123
2.5 ±0.30
ND
ND
2.69 ±0.38
aMean ± standard deviation.
bSignificantly different from controls (p < 0.05, Dunnett's t-test).
°Not determined at this time point.
Source: Malley et al. (2000).
-3
For the purposes of this review, NOAEL and LOAEL values of 1721 mg/m (500 ppm)
and 6886 mg/m3 (2000 ppm), respectively, are identified for cyclohexane in these studies in rats
and mice based on neurobehavioral effects (diminished response to a sound stimulus, and in
mice at 24,101 mg/m3, hyperreactivity and other behavioral changes). Mild liver changes
(increased relative liver weight, and in rats, centrilobular hypertrophy) were found at
24,101 mg/m3 in both species.
In the third sub chronic-duration study, Treon et al. (1943) exposed groups of four white
rabbits (gender/strain not specified) whole-body to cyclohexane (purity not reported) vapor at 0,
434, 435, 786, 3330, 7444, 9220, 12,574, 18,565, or 56,572 ppm (0, 1494, 1498, 2706, 11,465,
25,629, 31,744, 43,292, 63,918, or 194,820 mg/m3) 6 hours/day, 5 days/week, for 2, 5, 10, or
26 weeks. In addition to the rabbits, one Rhesus monkey was exposed to cyclohexane vapor at
"3
1243 ppm (4,730 mg/m ) by the same schedule for 10 weeks. Evaluations during exposure and
up to 2 months after exposure termination included clinical signs, survival, body weight, and
hematology (RBC, Hgb, WBC). Gross pathology and histopathology (tissues not specified)
were conducted following the 2-month postexposure observation period.
"3
All rabbits exposed at the highest concentration of 91,486 mg/m died during the 1-hour
exposure period (Treon et al., 1943). These rabbits exhibited frank effects including severe,
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rapid, extensive rhythmic movements of feet, tremors, rapid narcosis, and opisthotonos prior to
death. Treon et al. (1943) observed that mortality appeared to be related more to concentration
"3
than exposure duration since prolonged exposures to low concentrations (<11,465 mg/m ) did
not result in similar effects on survival. Additional fatalities were observed among rabbits at
"3
>25,629 mg/m . Rabbits at these concentrations showed a concentration-related increase in the
presence and severity of clinical signs ranging from lethargy, light narcosis, increased
"3
respiration, and diarrhea among rabbits exposed to 25,629 mg/m for a total of 60 hours to
rhythmic movement of feet, tremors, spasmodic jerking, narcosis, temporary paresis of legs,
salivation, conjunctival congestion, and labored respiration among rabbits exposed to
63,918 mg/m3 for a total of 60 hours. No noteworthy clinical signs were observed among rabbits
3	3
exposed to <11,465 mg/m . Rabbits exposed to cyclohexane at >31,744 mg/m generally
showed decreased body weights (ranging from -188 to -311 g/animal). Treon et al. (1943)
reported that the corresponding control group behaved similarly, but these data are not shown.
Treon et al. (1943) reported that the monkey exposed to 4730 mg/m3 survived the duration of the
study without demonstrating any noteworthy clinical signs of intoxication other than a reduction
in body weight (-333 g/animal). No significant hematological changes were observed at any
concentration. Treon et al. (1943) does not specifically report the incidence of pathological
changes among treated rabbits. However, the study authors did indicate that gross and
microscopic tissue changes produced by inhalation of cyclohexane were not specific for the
group as a whole or for individuals within it. This study is limited by small group sizes and
insufficient detail in reporting of results. For the purposes of this review, a NOAEL of
3330 ppm (11,465 mg/m3) and a LOAEL of 7444 ppm (25,629 mg/m3) are identified based on
clinical signs of toxicity.
For the fourth subchronic-duration study, (a French study with an English abstract)
groups of white rabbits (strain/gender unspecified) were exposed to cyclohexane vapor (purity
"3
unspecified) at concentrations ranging from 2.7 to 22 mg/L (2700-22,000 mg/m ) 8 hours/day,
6 days/week, for 40 days (Fabre et al., 1952). Rabbits in this study were observed for changes in
growth, clinical signs, hematology, and pathological changes in the liver, kidneys, spleen, heart,
lungs, adrenal glands, intestines, and brain. No significant effects were observed on growth,
clinical signs, hematology, or pathology. Additionally, rabbits received daily cutaneous
applications of 10 mL of cyclohexane for an unspecified number of days and subcutaneous
injection of 2 mL of cyclohexane daily for 20 days. Rabbits observed for hematological changes
following these applications did not exhibit any significant changes in RBCs or WBCs, but the
study authors reported observing an increase in the percentage of monocytes among treated
rabbits and a slight increase in the coagulation time of the blood. Based on the lack of additional
details for this study, these data cannot be used to inform toxicity value derivation.
Reproductive/developmental Studies—there are two developmental studies in the
literature: one in rats and one in rabbits. In addition, there is a multigenerational reproductive
study in rats. All of the reproductive and developmental toxicity studies of cyclohexane that
were identified in the literature search were previously reviewed by EPA (2009a, 2003).
Unpublished reports of a two-generation reproduction toxicity study in rats and prenatal
developmental toxicity studies in rats and rabbits exposed to cyclohexane by inhalation were
submitted by industry (Haskell Laboratories, 1997a,b,c) and later summarized and published by
Kreckmann et al. (2000). These studies are summarized below.
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As part of a two-generation reproduction study in rats, groups of 30 Crl:CD BR rats/sex/
concentration were exposed whole-body to cyclohexane (99.9% purity) vapor at 0, 500, 2000, or
7000 ppm (0, 1721, 6886, or 24,101 mg/m3) 6 hours/day, 5 days/week, for 10 weeks prior to
mating, during mating, and during Gestation Days (GDs) 0-20 (Haskell Laboratories, 1997a;
Kreckmann et al., 2000). Exposure concentrations were selected based on a range-finding
developmental study in rats (Haskell Laboratories, 1997a). Females were not exposed during the
period between GD 21 and Lactation Day (LD) 4. Exposure resumed on LD 5 and continued
through weaning. Males continued to be exposed 5 days/week, for up to 106 days, from the start
of the exposure period. At least 11 weeks after weaning, 30 F1 rats/sex/concentration were bred
with their respective treatment groups to produce the F2 litters. F1 animals were exposed to
cyclohexane as described above for the parental animals. Rats in both generations were
monitored weekly for changes in body weights and clinical observations, including response to
an auditory stimulus. F1 and F2 pup body weights and clinical observations were recorded on
Postnatal Days (PNDs) 0, 4, 7, 14, 21, and 25. Following litter production, all parental rats and
20 of the F1 and F2 weanlings/sex/concentration were sacrificed and subjected to gross
examination. Reproductive organs and pituitary glands from adult rats in the control and
high-exposure groups were collected and examined microscopically.
No significant treatment-related effects on the survival of parental rats were observed
(Kreckmann et al., 2000; Haskell Laboratories, 1997a). Clinical observations during the
exposure period showed a diminished or absent alerting response to a sound stimulus beginning
at Days 16 and 15 in animals exposed to 6886 and 24,101 mg/m3, respectively. The study
authors characterized this observed sedation as transient because the effect was no longer
apparent shortly after the rats were removed from the exposure chamber. Adult male rats of both
generations from the two highest exposure groups and females of both generations from the
high-exposure group also demonstrated clinical signs of toxicity possibly related to sedation
including increased salivation, stained perioral area, and wet chin. As described above in the
subchronic-duration rat study, such clinical signs are considered to be associated with the
propensity of treated rats to groom themselves following removal from the exposure chambers.
Kreckmann et al. (2000) did not consider these clinical signs to be toxicologically important.
Table 6 summarizes the effects on mean body weight and body-weight gain in adult PI
and F1 rats. (Kreckmann et al., 2000; Haskell Laboratories, 1997a). As shown, there were no
significant reductions in mean body weight or mean body-weight gain during the exposure
period among PI males at any concentration. However, for most data points, mean body weight,
and mean body-weight gain were significantly reduced throughout the course of the study among
"3
F1 males exposed to 24,101 mg/m compared to controls (6%). Females of both generations
exposed to 24,101 mg/m3 demonstrated significant reductions in both mean body weights at the
end of the premating periods (6-8%) and overall mean body-weight gains for the premating
period (8—13%). At this concentration, mean female body weights were also significantly
reduced during gestation and lactation (7—8%) (LD 25 was not significantly reduced). However,
no significant reductions in mean body-weight gains during these periods were observed at any
concentration, and in fact, cyclohexane-exposed PI rats gained significantly more weight than
controls during the lactation period. Therefore, the reductions in mean gestation and lactation
body weights were most likely due to the preexisting body-weight deficits established during the
premating period. No significant changes in food consumption or food efficiency were observed
among males of either generation. Food consumption was comparable between treated and
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control rats during the premating and lactation periods, but food consumption among the
24,101 mg/m3 PI females was significantly less than the controls during GDs 0-7 by
approximately 14% (p < 0.05). Food consumption among these rats through the remainder of
gestation was comparable to controls, and no significant effects on overall food efficiency were
observed throughout the gestation period. Food efficiency was significantly reduced in females
exposed to 24,101 mg/m3 from both generations during the premating period (10 and 5% in PI
and F1 females, respectively,/* < 0.05). In general, effects on food efficiency correlated with the
observations in body-weight gains. Overall, although significant body-weight reductions were
observed among adult PI females and F1 males and females, the magnitude of the changes
compared to controls was generally <10%, and appeared reversible in some cases, as
body-weight gains were increased over controls among treated females in later stages of the
study.
Table 6. Summary of Changes in Body Weight and Body-Weight Gain in
Rats Exposed to Cyclohexane Vapor as Part of a Two-Generation
Reproduction Study
Parameter
Test Day
Exposure concentration (mg/m3)
0
1721
6886
24,101
PI Males
Mean body weight (g)
1
266.5 ± 18.2a
266.5 ± 19.7
266.6 ± 19.5
264.6 ± 17.3
71b
488.3 ±53.5
489.4 ±49.8
494.0 ±48.2
468.0 ±53.9
106
519.0 ±58.5
515.6 ±55.6
530.8 ±60.3
509.2 ±58.7
Mean body-weight gain (g)
1-71
221.8 ±42.5
222.9 ±34.0
227.3 ±42.3
203.4 ±43.4
71-106
30.7 ± 15.1
26.2 ± 17.0
38.4 ±9.6
41.2 ±20.3
1-106
252.5 ±48.6
249.1 ±40.7
264.1 ±54.9
244.6 ± 49.2
F1 Males
Mean body weight (g)
1
69.9 ±7.9
70.5 ±6.3
70.0 ±6.7
63.4 ± 5.5°
78b
480.6 ±47.8
484.9 ±45.6
487.4 ±39.9
452.6 ± 45.9C
106
524.1 ±49.0
524.9 ±48.6
531.5 ± 41.3
497.5 ± 46.6
120
547.9 ±51.4
547.5 ± 52.7
548.0 ±42.5
513.2 ±48.2C
Mean body-weight gain (g)
1-78
410.8 ±44.6
414.4 ±42.2
417.4 ±37.7
389.1 ±42.5
78-120
67.2 ± 16.2
62.9 ± 15.5
59.0 ±32.1
61.9 ± 10.7
1-120
478.0 ±48.7
477.0 ±48.5
478.2 ±39.7
449.7 ±44.5C
PI Females
Mean body weight (g)
1
190.4 ± 11.3
191.1 ± 12.3
191.8 ± 12.0
186.6 ± 15.4
71b
282.0 ±26.3
283.5 ± 19.1
280.9 ±22.3
265.8 ±21.4C
GD0
288.4 ±27.4
288.2 ±24.3
286.3 ±23.0
268.6 ± 24.2°
GD 21
418.4 ±42.7
421.6 ±25.9
415.8 ±28.8
391.1 ± 31.2°
LD0
320.9 ±34.6
319.1 ±25.2
320.0 ±26.2
296.2 ± 26.2°
LD 25
315.3 ±28.3
321.9 ± 18.7
327.6 ±26.9
305.9 ±22.9
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Table 6. Summary of Changes in Body Weight and Body-Weight Gain in
Rats Exposed to Cyclohexane Vapor as Part of a Two-Generation
Reproduction Study


Exposure concentration (mg/m3)
Parameter
Test Day
0
1721
6886
24,101
Mean body-weight gain (g)
1-71
91.5 ± 18.6
92.4 ± 14.7
89.1 ± 15.9
79.2 ± 13.4°

GD 0-21
130.0 ±23.9
133.4 ± 14.3
129.5 ±21.6
122.5 ± 13.9

LD 0-25
-5.6 ± 15.3
2.8 ± 13.8
7.6 ± 19.8°
9.6 ± 14.2°
F1 Females
Mean body weight (g)
1
64.8 ±8.3
64.5 ±5.3
64.8 ±7.4
60.1 ±5.8C

78b
290.1 ±34.1
285.2 ±24.6
292.4 ±28.5
267.6 ±23.4C

GD 0
301.5 ±28.4
300.5 ±30.3
310.0 ±33.3
277.7 ± 27.6°

GD 21
445.2 ±34.7
439.6 ±35.7
460.5 ±42.1
415.6 ±32.8C

LD 0
339.3 ±23.1
333.5 ±29.6
346.9 ±32.7
309.3 ±25.2C

LD 25
349.7 ± 16.3
336.9 ±29.8
349.6 ±23.8
324.5 ±23.6C
Mean body-weight gain (g)
1-78
225.2 ±32.0
220.9 ±21.3
227.6 ± 26.0
207.5 ± 19.1°

GD 1-21
143.7 ± 19.8
139.1 ±24.4
150.5 ±21.0
137.9 ± 17.3

LD 0-25
10.4 ± 16.9
3.4 ± 14.2
2.6 ± 19.7
15.2 ± 13.5
aMean ± standard deviation.
bEnd of premating period.
Significantly different from controls (p < 0.05, One-Way ANOVA and Dunnett's test).
GD = gestation day; LD = lactation day.
Source: Haskell Laboratories (1997a); Kreckmann et al. (2000)
There were no significant differences in mating, fertility, or gestation indices,
implantation efficiency, or gestation length in either the PI or the F1 generations
(Kreckmann et al., 2000; Haskell Laboratories, 1997a). No significant effects were observed on
the mean number of implantation sites or mean number of pups/litter for both F1 and F2 litters.
Among F1 litters exposed to 24,101 mg/m3, the mean percent of pups born alive was
significantly lower than controls (see Table 7). However, this observation was not repeated
among the F2 litters, and the observed percentage of 98.1% was within the range of historical
control data (97.5%; range of 92.5-100%). Mean pup weight was significantly reduced from
Postnatal Day 7 through the remainder of the lactation period for both F1 and F2 litters of the
high-exposure group (7—15%) (see Table 8). No treatment-related effects on organ weights,
gross observations, or microscopic findings were observed. EPA (2003) identified a NOAEL for
developmental effects in this reproductive toxicity study of 6886 mg/m3 (2000 ppm) based on
reduced rat pup weights during lactation in the two generations tested. The corresponding
LOAEL is 24,101 mg/m3 (7000 ppm). EPA (2003) identified the diminished alerting response at
"3
>6886 mg/m as the most sensitive effect in parental rats in this study. Body weights were
slightly reduced in parental rats at 24,101 mg/m3.
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Table 7. Mean Pup Numbers and Survival Among F1 and F2 Litters from Rats
Exposed to Cyclohexane Vapor During a Two-Generation Reproduction Study
Parameter
Exposure concentration (mg/m3)
0
1721
6886
24,101
F1 Generation
Number of litters/group
28
27
27
28
Number of implantation sites
13.1
12.5
13.0
13.5
Mean number of pups born/litter
12.7
12.7
12.7
12.4
Mean number of pups born alive/litter
12.7
12.3
12.6
12.2
Sex ratio (males)
0.5
0.55
0.46
0.5
Implantation efficiency (%)
90.2
90.2
94.2
92.1
Gestation Index (%)
100
96.3
100
96.4
Mean % born alive
100
96.3
99
98.la
F2 Generation
# litters/group
22
25
22
24
# implantation sites
13.6
12.2
14.5
14.3
Mean # of pups born/litter
12.9
13.0
15.0
13.5
Mean # of pups born alive/litter
12.7
12.5
14.7
13.5
Sex ratio (males)
0.54
0.48
0.56
0.51
Implantation efficiency
93.2
96.3
96.1
91.2
Gestation Index
100
96
100
100
Mean % born alive
97.9
95.0
98.7
100
Significantly different from controls (p < 0.05, Jonekheere's test).
Source: Kreckmann et al. (2000).
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Table 8. Mean Pup Weights Among F1 and F2 Litters from Rats Exposed to
Cyclohexane Vapor During a Two-Generation Reproduction Study
Exposure day
Exposure concentration (mg/m3)
0
1721
6886
24,101
F1 Generation
Number of rats
28
26
27
27
Day 0
6.7 ± 0.6a
6.7 ±0.6
6.7 ±0.6
6.6 ±0.5
Day 4 Preculling
11.0± 1.6
11.0 ± 1.3
11.2 ± 1.6
10.6 ± 1.1
Day 4 Postculling
11.0± 1.6
11.0 ± 1.3
11.3 ± 1.6
10.6 ± 1.1
Day 7
16.2 ±2.0
16.2 ± 1.8
16.3 ±2.0
15.1 ± 1.4b
Day 14
30.0 ±3.1
29.9 ±2.6
29.7 ±2.9
26.5 ± 2.0b
Day 21
48.5 ±5.0
48.5 ±3.9
48.3 ±4.8
43.1 ±3.9b
Day 25
67.5 ±7.3
67.8 ±4.6
68.3 ±5.9
62.2 ± 4.7b
F2 Generation
Number of rats
21-22
21-24
22
24
Day 0
6.4 ±0.9
6.6 ±0.5
6.3 ±0.5
6.3 ±0.6
Day 4 Preculling
10.8 ± 1.7
10.8 ± 1.3
10.1 ± 1.3
10.2 ± 1.7
Day 4 Postculling
10.9 ± 1.7
10.8 ± 1.4
10.1 ± 1.2
10.1 ± 1.7
Day 7
16.3 ±2.4
16.0 ± 1.8
15.3 ± 1.8
14.3 ±2.1b
Day 14
31.0 ± 3.2
30.2 ±3.1
28.9 ±2.6
26.2 ± 3.4b
Day 21
50.0 ±5.4
48.3 ±5.5
46.4 ±5.9
42.8 ± 6.6b
Day 25
69.3 ±6.9
67.1 ±6.4
65.6 ±6.9
61.3 ±7.8b
aValues reported in grams.
bSignificantly different from controls (p < 0.05, Analysis of Covariance with litter size and sex ratio as covariates).
Sources: Kreckmann et al. (2000); Haskell Laboratories (1997a).
In the developmental study in rats, groups of 25 assumed-pregnant Crl:CD BR
rats/concentration were exposed whole-body to cyclohexane (99.9% purity) vapor at
concentrations of 0, 500, 2000, or 7000 ppm (0, 1721, 6886, or 24,101 mg/m3), for 6 hours/day,
on GDs 6-15 (Kreckmann et al., 2000; Haskell Laboratories, 1997b). In addition to the standard
control group, a pair-fed control group was included; this group received an amount of food
equal to the cumulative average amount of food consumed by the high-concentration group on
the corresponding gestation day. Maternal body weights and food consumption were recorded
daily during the exposure period, and clinical signs were recorded before and after exposure.
Dams were sacrificed on GD 21, and the organs of the thoracic and abdominal cavities were
examined grossly. The types of implants observed in the uterus were counted, and their relative
positions were recorded. Fetuses were weighed, sexed, and examined for external and skeletal
abnormalities; one-half of the fetuses were examined for visceral and head abnormalities.
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Mean maternal body-weight gain was significantly reduced during GDs 7-17 (GDs 6-16
as reported in Kreckmann et al., 2000) by approximately 11 and 31% for the 6886 and
"3
24,101 mg/m exposure groups, respectively (see Table 9) (Haskell Laboratories, 1997b;
Kreckmann et al., 2000). Kreckmann et al. (2000) attributed the reduction in maternal
"3
body-weight gain at 6886 mg/m to biological variation because the mean value (57.5 g) fell
above the mean value for historical controls (53.5 g, range of 40.7-65.5 g). Adjusted mean
maternal body-weight gain (adjusted based on final body weight minus gravid uterine weight)
was also significantly reduced among 24,101 mg/m3 rats compared to controls (25%).
"3
Reductions in food consumption in the 24,101-mg/m exposure group (11%) corresponded with
observations of body-weight deficiencies in this group. Mean body-weight gain was also
significantly reduced in the pair-fed control group. This suggests that the reduction in
body-weight gain in the high-exposure groups is most likely a consequence of diminished food
consumption. Similar to the reproductive study, a sedative effect was observed among dams at
6886 and 24,101 mg/m3 and was characterized by transient, diminished-alerting responses. In
"3
addition, dams exposed to 24,101 mg/m exhibited a significant increase in the incidence of fur
stains and wetness, likely related to salivation and grooming activity following removal from the
exposure chambers as described in the other rat studies. As described in the reproductive
toxicity study, although these clinical signs were considered by the study authors to be treatment
related, they were not considered to be toxicologically relevant.
Table 9. Mean Maternal Body-Weight Gain Among Rats Exposed to
Cyclohexane Vapor During GDs 6-15
GDs
Exposure concentration (mg/m3)
0
0 (pair-fed)
1721
6886
24,101
Number of rats evaluated3
21
25
22
23
23
1-7
18.7 ±6.71
22.4 ±8.23
22.3 ± 8.09
24.6 ±7.80
22.1 ± 10.19
7-17
64.2 ± 10.64
32.1 ± 8.03b
60.1 ± 11.28
57.2 ± 8.68°
44.2 ± 9.58°
17-22
80.5 ± 10.21
68.8 ± 12.16b
72.1 ± 12.42
75.8 ± 16.50
81.4 ± 11.13
7-22d
49.6 ±7.97
12.5 ± 14.88b
43.0 ± 11.94°
43.0 ± 9.53°
37.1 ± 10.01c
aData from females that were not pregnant were excluded.
bSignificantly different from controls (p < 0.05, ANOVA, and Dunnett's test).
Significantly different from controls (p < 0.05, linear contrast of means from ANOVA).
dWeight changes calculated using the final body weight minus weight of the intact uterus.
Source: Haskell Laboratories (1997b).
There were no significant effects of cyclohexane exposure on the pregnancy rates,
delivery rates, abortion rates, resorption rates, mean number of live fetuses per litter, or sex ratio
(Kreckmann et al., 2000; Haskell Laboratories, 1997b). A significant reduction in the mean
"3
number of implantations for female rats in the 24,101 mg/m group was observed. However,
implantation occurred prior to cyclohexane exposure, so this effect is attributed to normal
biological variation. Necropsy revealed no gross lesions. No significant effects were observed
on mean fetal weights or fetal development. The total incidence of fetal malformations was four
3	3
fetuses from four litters in the 24,101-mg/m group, one fetus in one litter in the 6886-mg/m
group, none in the 1721 -mg/m3 group, two fetuses in two litters from pair-fed controls, and none
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in the ad libitum-fed control group. EPA (2003) noted that although finding one defective fetus
in all four litters is of greater concern than an observation of four such fetuses in one litter, the
"3
malformations in the four fetuses from the 24,101 mg/m were of different types, malformations
were observed in fetuses from two litters from the pair-fed controls, and no other signs of
developmental toxicity were noted by the study authors. EPA (2003) identified a NOAEL of
24,101 mg/m3 (7000 ppm) for developmental toxicity in rats for this study. EPA (2003)
identified NOAEL and LOAEL values of 1721 (500 ppm) and 6886 mg/m3 (2000 ppm),
respectively, for maternal toxicity on the basis of the transient sedative effect.
In a companion developmental study in rabbits, groups of 20 pregnant New Zealand
white rabbits/concentration were exposed whole-body to cyclohexane (99.9% purity) vapor at
concentrations of 0, 500, 2000, or 7000 ppm (0, 1721, 6886, or 24,101 mg/m3), for 6 hours/day,
on GDs 6-18 (Kreckmann et al., 2000; Haskell Laboratories, 1997c). Rabbits were observed for
the same endpoints as described above in the rat developmental toxicity study. No significant
effects on survival, mean body weight or mean body-weight gain, or maternal food consumption
were observed. No significant differences in the incidences of clinical observations were noted
in exposed rabbits as compared to controls. There were no significant effects of cyclohexane
exposure on the pregnancy rates, delivery rates, abortion rates, resorption rates, or mean number
of live fetuses per litter. A significant reduction in the mean number of corpora lutea was
observed among does in the 6886- and 24,101-mg/m3 groups (8.9 and 8.8, respectively)
(p < 0.05). However, these values were within the range of historical controls (7.0-10.9), and
this effect occurred prior to cyclohexane treatment. Therefore, these differences are attributed to
normal biological variation. There was a significant trend in sex ratio (number of males/total
number of pups), with the ratios being higher for the 6886- and 24,101-mg/m3 groups (0.59 and
0.54, respectively, compared with the control ratio of 0.48). However, Kreckmann et al. (2000)
did not consider the changes in sex ratio to be related to cyclohexane treatment based on the
3	3
disparity between the ratios for the 1721-mg/m (0.42) and the 6886-mg/m (0.59) groups, the
absence of a true dose response, and the observation that the reported values generally fell within
the historical control range (0.40-0.56). Necropsy revealed no gross lesions. No significant
effects were observed on mean fetal weights or fetal development. Based on the absence of
significant treatment-related effects on rabbits in this study, EPA (2003) identified a NOAEL of
24,101 mg/m3 (7000 ppm) for both maternal and developmental toxicity in rabbits.
OTHER STUDIES
Neurotoxicity
Concern about neurotoxic effects exists because of the similarity of cyclohexane to
//-hexane, a widely recognized neurotoxicant. The neurotoxicity of cyclohexane has been
evaluated in rats in an acute operant behavior study (Christoph et al., 2000; Haskell Laboratories,
1996c), a 90-day inhalation neurotoxicity study (Malley et al., 2000; Haskell Laboratories,
1996d), a 30-week inhalation neurotoxicity study (Frontali et al., 1981), and a
subchronic-duration Russian study (Khanin, 1969). These studies are summarized below.
The acute operant behavior study and the 90-day inhalation neurotoxicity study have
been reviewed by EPA (2009a, 2003). In the acute operant behavior study, groups of 10 male
Crl:CD:BR rats were exposed whole-body to cyclohexane (99.97% purity) vapor at 0, 500, 2000,
or 7000 ppm (0, 1721, 6886, or 24,101 mg/m3) for 6 hours (Christoph et al., 2000; Haskell
Laboratories, 1996c). During the exposure period, all animals exhibited a normal startle
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response, and there were no obvious signs of intoxication. These rats had undergone a 6-week
training of food presentation through the pressing of a lever during a fixed feeding schedule. At
"3
24,101 mg/m , a transient decrease in the mean fixed-ratio rate of responding by approximately
11% was apparent on the treatment day relative to the pretreatment period. Fixed-ratio pause
duration showed a slight, nonsignificant increase after exposure, which is difficult to interpret
since the mean fixed-ratio pause duration for the two high-dose groups was considerably lower
prior to exposure than for the other groups.
In the 90-day inhalation neurotoxicity study, groups of 12 Crl:CD BR rats/sex/group
were exposed whole-body to cyclohexane vapor at 0, 500, 2000, or 7000 ppm (0, 1721, 6886, or
"3
24,101 mg/m ) 6 hours/day, 5 days/week, for approximately 90 days (Malley et al., 2000;
Haskell Laboratories, 1996d). Rats were subjected to a functional observation battery (FOB) and
motor activity (MA) assessments prior to exposure and during Weeks 4, 8, and 13 on
nonexposure days. Rats were monitored weekly for changes in body weights and food
consumption, they were monitored daily for clinical signs and for their response to an
auditory-alerting stimulus. After at least 65 days of exposure to cyclohexane, six rats/sex/group
were sacrificed and grossly examined. Sections of the brain, spinal cord, muscle, sciatic and
tibial nerves, gasserian ganglia, dorsal root fibers and ganglia, and ventral root fibers were
"3
collected from rats exposed at 0 or 24,101 mg/m and examined histologically. No
treatment-related effects were found on food consumption or body weights at any exposure
"3
concentration. Rats exposed to 6886 and 24,101 mg/m demonstrated diminished and/or absent
responses to an alerting stimulus. All rats from these exposure groups exhibited an increased
incidence in stained and/or wet chins. The study authors characterized these clinical signs as
transient because they were only observed immediately following removal from the exposure
chambers. No treatment-related effects were observed based on the 34 parameters evaluated
during the FOB assessment. Similarly, no treatment-related effects were observed on forelimb
or hindlimb grip strength, or hindlimb foot splay. Motor activity was significantly decreased
from controls at 6886 mg/m3 in males at Week 13. Motor activity was also decreased compared
"3
to controls at 24,101 mg/m among males at Week 13, but the difference was not statistically
significant and was increased over the 6886-mg/m3 group. Microscopic evaluation revealed no
morphological differences from control rats. In summary, no statistically significant, treatment-
related effects on FOB, MA, or neuropathology measures were found in rats following exposure
"3
to cyclohexane vapor up to 24,101 mg/m for 90 days. However, NOAEL and LOAEL values of
1721 mg/m3 (500 ppm) and 6886 mg/m3 (2000 ppm), respectively, are identified based on
diminished response to a sound stimulus during exposure.
Frontali et al. (1981) exposed groups of 6-9 male Sprague-Dawley rats to cyclohexane
(99.5% purity) at 1500 ppm (5160 mg/m3), 9 hours/day, 5 days/week, for 7, 14, and 30 weeks,
"3
and 2500 ppm (8610 mg/m ), 10 hours/day, 6 days/week, for 30 weeks. Controls were exposed
to room air. Rats were monitored weekly for changes in body weight. Neuropathological
response was evaluated based on a "hindlimb spread" test and central-peripheral distal
axonopathy, for which sections of the tibial nerve supplying the calf muscles were examined
histopathologically at termination. Frontali et al. (1981) compared the results of these
evaluations to a control group, although no description of this group is provided. No significant
effects on body weight or hindlimb spread were observed. No significant neuropathological
alterations were observed among cyclohexane-treated rats. Therefore, for the purposes of this
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"3
review, the highest concentration tested of 8610 mg/m is considered a NOAEL for neuropathy
in rats in this study.
In addition, a Russian study for which an English abstract is available, apparently
observed signs of toxicodystrophic encephalopathy of the CNS and inflammation of internal
organs in rats (gender/strain not specified) following inhalation exposure to toxic substances
"3
including cyclohexane (purity unspecified) at concentrations of 0.06-0.1 mg/m for 70-82 days
(Khanin, 1969). It is unclear from the study abstract if cyclohexane was administered by itself or
as a mixture with gasoline and other hydrocarbons. Based on the absence of additional
information, these data cannot inform toxicity value derivation.
Toxicokinetics
Hissink et al. (2009) describes a PBPK model for cyclohexane and its use in comparing
internal doses in rats and volunteers following inhalation exposures. Parameters describing
saturable metabolism of cyclohexane were measured in rats and used along with experimentally
determined partition coefficients. The model was evaluated by comparing predicted blood and
brain concentrations to data from studies in rats and then allometrically scaled the results to
humans. Levels of cyclohexane in blood and exhaled air were measured in human volunteers
and compared with model values. The model predicted that exposure of volunteers to
cyclohexane at levels of 4100 mg/m3 (approximately 1200 ppm) would result in brain levels
"3
similar to those in rats exposed to 8000 mg/m (the no-effect level for acute CNS effects). There
were no acute CNS effects in humans exposed to 860 mg/m3, consistent with model predictions
that current occupational exposure levels for cyclohexane protect against acute CNS effects.
Genotoxicity
The results of standard genotoxicity tests have been mixed. Most of the studies regarding
the genotoxicity of cyclohexane identified in the literature search have been reviewed by EPA
(2003). Genotoxicity data for cyclohexane are summarized below.
In several independent reverse-mutation assays, cyclohexane tested negative for
mutagenicity in bacterial tests using Salmonella typhimurium strains TA1535, TA1537, TA98,
TA97, and TA100 (Mortelmans et al., 1986; Haskell Laboratories, 1982a; Maron et al., 1981;
McCann et al., 1975) in both the presence and absence of metabolic activation.
Salmeen et al. (1989) observed a weak positive response in S. typhimurium strain TA98 in the
absence of metabolic activation but without a clear dose-response relationship. The study
authors classified Cyclohexane was as only very weakly mutagenic in this assay. Cyclohexane
did induce forward mutations in cultured L51578Y mouse lymphoma cells in the presence of
metabolic activation (Haskell Laboratories, 1982b). However, a second study did not observe an
increase in forward mutations in mouse lymphoma cells either in the presence or absence of
metabolic activation (i.e., Litton Bionetics, 1982). Cyclohexane did not induce sister chromatid
exchange (SCE) in cultured Chinese hamster ovary (CHO) cells at exposure levels up to that
which inhibited cell growth in the presence or absence of metabolic activation (Haskell
Laboratories, 1982c). Perocco et al. (1983) did not observe any effects on DNA synthesis in
cultured human lymphocytes as measured by [H] thymidine uptake in the presence or absence of
metabolic activation. Kubinski et al. (1981) reported equivocal results in Escherichia coli in a
DNA cell-binding assay. In vivo, no significant increase in chromosome structural aberration
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frequency was observed in bone marrow cells of male or female rats exposed by inhalation for
5 consecutive days to levels of cyclohexane up to 1000 ppm (Litton Bionetics, Inc., 1981).
Cyclohexane was found to be a weak inducer of autosomal recessive lethal mutations and
sex-linked recessive lethal mutations in Drosophila melanogaster (Shetty and Rangaswamy,
1984).
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL
ORAL RFD VALUES FOR CYCLOHEXANE
No data are available on the effects of cyclohexane in humans or animals exposed orally.
The absence of data precludes the derivation of provisional RfD values for cyclohexane.
DERIVATION OF SUBCHRONIC AND CHRONIC PROVISIONAL
INHALATION RFC VALUES FOR CYCLOHEXANE
SUBCHRONIC P-RFC
As described above, EPA (2003) determined that the available human data, which are
limited to occupational exposures to mixed solvents, or are limited by small numbers of subjects
are not useful for the derivation of the p-RfC values. Two 90-day studies in rats and mice
(Malley et al., 2000; Haskell Laboratories, 1996a,b), an 10-week study in rabbits
(Treon et al., 1943), a two-generation reproduction study in rats (Kreckmann et al., 2000;
Haskell Laboratories, 1997a), developmental toxicity studies in rats and rabbits
(Kreckmann et al., 2000; Haskell Laboratories, 1997b,c), and neurotoxicity studies in rats
(Christoph et al., 2000; Malley et al., 2000; Haskell Laboratories, 1996c,d; Frontali et al., 1981)
are, however, available for use in deriving a subchronic p-RfC for cyclohexane. These data are
summarized in Table 10, which also includes calculation and presentation of human equivalent
concentrations (HECs) for the identified NOAELs and LOAELs.
EPA (2009a, 2003) considered all but one of these same studies in deriving the
chronic-RfC for cyclohexane. Frontali et al. (1981) was not reviewed as part of the 2003
Toxicological Review (U.S. EPA, 2003), but this study did not observe any significant
"3
neuropathological changes in rats exposed to cyclohexane concentrations up to 8610 mg/m for
30 weeks and supports the findings from the acute (Christoph et al., 2000; Haskell Laboratories,
1996c) and 90-day neurotoxicity (Malley et al., 2000; Haskell Laboratories, 1996d) studies that
were reviewed by EPA.
After careful consideration of the available inhalation studies and benchmark
concentration (BMC) modeling of a number of endpoints from several different studies, EPA
(2009a, 2003) selected the BMCLisdhec value of 1822 mg/m3 based on reduced F2 pup weight
gain in the two-generation reproduction toxicity study in rats (Kreckmann et al., 2000; Haskell
Laboratories, 1997a) as the point of departure (POD) for the subchronic p-RfC. A duration
adjustment was first made to adjust for the 6-hour dosing regimen, by multiplying by 6/24. The
ainblood partition coefficients (1.39 for rat, 1.41 for human) were not significantly different and
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a value of 1 was used in the calculation of a human equivalent concentration (HEC). The HEC
values were modeled and a one-standard deviation benchmark concentration of 1822 mg/m3 was
calculated.
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Table 10. Summary of Inhalation Noncancer Dose-Response Information for Cyclohexane
Species and study
type («/sex/group)
Exposure
NO A EL'
(mg/m3)
LOAEL'
(mg/m3)
Responses at the LOAEL
Comments
Reference
Rat
(10-20/sex/group)
0, 1721, 6886, or
24,101 mg/m3
6 hours/day,
5 days/week for
90 days.
1721
HEC: 307
6886
HEC: 1230
Transient sedative effect
Increased liver wt and centrilobular
hepatic hypertrophy at 24,101 mg/m3
Malley et al., 2000;
Haskell
Laboratories, 1996a
Mouse
(10-20/sex/group)
0, 1721, 6886, or
24,101 mg/m3
6 hours/day,
5 days/week for
90 days.
1721
HEC: 307
6886
HEC: 1230
Transient sedative effect
Clinical signs of CNS stimulation and
increased liver weight at
24,101 mg/m3.
Malley et al., 2000;
Haskell
Laboratories, 1996b
Rabbit (4/group)
0, 1494, 1498,
2706, 11,465,
25,629, 31,744,
43,292, 63,918,
or 91,486 mg/m3
6 hours/day,
5 days/week for
up to 10 weeks.
11,465
HEC: 2047
25,629
HEC: 4576
Clinical signs such as
lethargy, light narcosis,
increased respiration,
diarrhea
More severe clinical signs including
rhythmic movement of the feet,
tremors, spasmodic jerking, narcosis,
salivation, temporary paresis of legs,
and labored respiration were
observed at higher concentrations.
Treonetal., 1943
Rat (30/sex/group)
0, 1721, 6886, or
24,101 mg/m3 6
hours/day,
5 days/week prior
to mating, during
mating, and
through lactation.
Maternal:
1721
HEC: 307
Developmental:
6886
HEC: 1230
Maternal:
6886
HEC: 1230
Developmental:
24,101
HEC: 4304
Transient sedative effect
in dams. Reduced body
weight in F1 and F2 pups
during lactation
Depressed body-weight gain among
PI females during the premating
period at 24,101 mg/m3. Significant
decreases were also observed in mean
body weights during gestation and
lactation among female rats, but since
overall mean body-weight gain was
not significantly affected, these
changes were most likely due to
preexisting body weight deficits
established during the premating
period.
Kreckmann et al.,
2000; Haskell
Laboratories, 1997a
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Table 10. Summary of Inhalation Noncancer Dose-Response Information for Cyclohexane
Species and study
type («/sex/group)
Exposure
NOAEL3
(mg/m3)
LOAEL'
(mg/m3)
Responses at the LOAEL
Comments
Reference
Rat (25/group)
0, 1721, 6886, or
24,101 mg/m3
6 hours/day on
GDs 6-15.
Maternal:
1721
HEC: 430
Developmental:
24,101
HEC: 6025
Maternal:
6886
HEC: 1722
Developmental:
NA
Transient sedative effect
in dams
No developmental effects at any
concentration.
Kreckmann et al.,
2000; Haskell
Laboratories, 1997b
Rabbit (20/group)
0, 1721, 6886, or
24,101 mg/m3
6 hours/day on
GDs 6-18.
Maternal:
24,101
HEC: 6025
Developmental:
24,101
HEC: 6025
Maternal:
NA
Developmental:
NA
NA
No maternal or developmental effects
at any concentration.
Kreckmann et al.,
2000; Haskell
Laboratories, 1997c
Rat (12/sex/group)
0, 1721, 6886, or
24,101 mg/m3
6 hours/day,
5 days/week for
90 days.
1721
HEC: 307
6886
HEC: 1230
Transient sedative effect
No significant effects on FOB, motor
activity or neuropathology.
Malley et al., 2000;
Haskell
Laboratories, 1996c
Rat (6-
9 males/group)
0, 5160
9	hours/day, 5
days week or
8610 mg/m3
10	hours/day,
6 days/week for
up to 30 weeks.
8610
HEC:
3,075
NA
NA
No significant neuropathological
changes were observed.
Frontali et al., 1981
aHEC calculated as follows: NOAELhec = NOAEL x exposure hours ^ 24 hours x exposure days ^ 7 days x dosimetric adjustment. For nonrespiratory effects (no
respiratory effects were reported for cyclohexane), the chemical is treated as a Category 3 gas (per U.S. EPA, 1994b) and the dosimetric adjustment is the ratio of the
animal:humanblood:gas partition coefficients for cyclohexane (as per U.S. EPA, 1994b). A default value of 1 was used because the ratio of measured animal:human
blood:gas partition coefficients for cyclohexane is not statistically different than 1[U.S. EPA, 2009a, 2003]).
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While the hepatic endpoint described by Malley et al. (2000) is a potential critical effect,
it would be less sensitive than the reproductive endpoint. For this review, Kreckmann et al.
(2000) data were selected for use as the POD in deriving the subchronic p-RfC for cyclohexane.1
EPA (2003) stated the following bout the transient sedative effect noted during exposure:
The clinical observation of diminished response to a sound stimulus while in the exposure
chamber, noted in many of the studies as the most sensitive endpoint, added information
to the qualitative assessment of the toxicity of cyclohexane but does not provide data of
the quality necessary for the quantitative estimation of an RfC (US.EPA, 1994b). These
are subjective observations (the observers know which treatment group they are
observing), and are made on a per group basis rather than an individual test animal
basis (only a few animals in the exposure chamber are visible when the chamber is hit
with the rod to produce an alerting stimulus) (Malley et al., 2000).
Further detail on the selection of the critical study, the modeling efforts, and the POD is
available in Section 5 and Appendix B of the 2003 Toxicological Review of Cyclohexane
(U.S. EPA, 2003).
"3
For derivation of the subchronic p-RfC, the BMCLisdhec of 1822 mg/m for reduced
body weight of F2 rat pups calculated by EPA (2003) was divided by an UF of 100 to yield the
subchronic p-RfC for cyclohexane, as shown here:
Subchronic p-RfC = BMCLisdhec ^ UF
= 1822 mg/m3- 100
= 18 mg/m3
The composite UF of 100 is composed of the following:
•	UFh: A factor of 10 is applied for extrapolation to a potentially susceptible human
subpopulation because data for evaluating a susceptible human response are
insufficient.
•	UFa: A partial UF of 3 (10°5) is applied for interspecies extrapolation to account
for potential pharmacodynamic differences between rats and humans. Two lines
of evidence support reducing this UF: First, converting the rat data to human
equivalent concentrations by the dosimetric equations accounts for
pharmacokinetic differences between rats and humans; thus, an UF of 3 is applied
for interspecies extrapolation.
•	UFd: A partial UF of 3 (10°5) is applied for database inadequacies, primarily
reflecting the lack of a developmental neurotoxicity study; the database includes
subchronic-duration studies in rats and mice, a two-generation reproduction study
in rats, and developmental studies in rats and rabbits.
•	UFl: A factor of 1 is applied for extrapolation from a LOAEL to a NOAEL
because BMD modeling was used.
1 No attempt was made to duplicate the modeling efforts conducted for the 2003 Toxicological Review of
Cyclohexane (U.S. EPA, 2003).
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EPA (2003) describes confidence in the principal study (Kreckmann et al., 2000; Haskell
Laboratories, 1997a) as high based on adequate numbers of study animals and exposure levels to
evaluate an adequate set of endpoints. The database includes subchronic-duration toxicity
studies in rats, mice, and rabbits, a two-generation reproduction study in rats, developmental
toxicity studies in rats and rabbits, and neurotoxicity studies in rats. EPA (2003) describes
confidence in the database for the purposes of deriving a chronic p-RfC value as
low-to-moderate. This assessment is based on the lack of data for long-term or lifetime
exposures or for developmental neurotoxicity. However, for the purposes of deriving a
subchronic p-RfC, confidence in the database is moderate- reflecting primarily the lack of
developmental neurotoxicity testing. Moderate confidence in the subchronic p-RfC value
follows.
CHRONIC P-RFC
A chronic p-RfC of 6 mg/m3 is available on IRIS (U.S. EPA, 2009a) based on the
two-generation reproduction study in rats (i.e., Kreckmann et al., 2000; Haskell Laboratories,
1997a). The RfC was calculated from a BMCLisdhec of 1822 mg/m3 for decreased pup body
weight and a composite UF of 300 (3 [10°5] for interspecies extrapolation, 10 for intraspecies
variability among the human population, and 10 for database deficiencies, in particular, the lack
of a chronic-duration inhalation study.
PROVISIONAL CARCINOGENICITY ASSESSMENT
FOR CYCLOHEXANE
WEIGHT-OF-EVIDENCE DESCRIPTOR
Under the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005), there is
"Inadequate Information to Assess [the] Carcinogenic Potential" of Cyclohexane. No
information was located on the carcinogenicity of cyclohexane in humans or animals.
Genotoxicity data provide little evidence to suggest that cyclohexane is mutagenic. In vitro data
suggest that cyclohexane was not mutagenic in bacterial mutation assays with S. typhimurium
(Haskell Laboratories, 1982a; McCann et al., 1975; Mortelmans et al., 1986, Maron et al., 1981).
Cyclohexane tested positive for induction of forward mutations in cultured mouse lymphoma
cells in the presence of metabolic activation in one study (Haskell Laboratories, 1982b), but was
negative in another study (Litton Bionetics, 1982). Cyclohexane tested negative for induction of
SCE in CHO cells (Haskell Laboratories, 1982c) and for effects on DNA synthesis in human
lymphocytes (Perocco et al., 1983). Equivocal results were reported for a DNA cell-binding
assay in E. coli (Kubinski et al., 1981). Results were weakly positive in Drosophila (Shetty and
Rangaswamy, 1984). No signs of genotoxicity were observed in vivo in rats (Litton Bionetics,
Inc., 1981).
QUANTITATIVE ESTIMATES OF CARCINOGENIC RISK
The paucity of suitable data precludes the derivation of quantitative estimates of cancer
risk for cyclohexane.
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Values for Chemical Substances and Physical Agents and Biological Exposure Indices. ACGIH,
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ATSDR (Agency for Toxic Substances and Disease Registry). (2009) Toxicological Profile
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Christophe, GR; Kelly, DP; Krivanek, N. (2000) Acute inhalation exposure to cyclohexane and
schedule-controlled operant performance in rats: comparison to D-amphetamine and
chlorpromazine. Drug Chem Toxicol 23(4):539-553.
Fabre, R; Truhaut, R; Peron, M. (1952) Toxicological studies of solvents replacing benzene. I.
Cyclohexane. Arch Maladies Profess Med Travail et Securite Sociale 13:437-448.
Frontali, N; Amantini, MC; Spagnolo, A; et al. (1981) Experimental neurotoxicity and urinary
metabolites of the c5-c7 aliphatic hydrocarbons used as glue solvents in shoe manufacture. Clin
Toxicol 18(12): 1357-1367.
Haskell Laboratories. (1982a) Salmonella typhimurium mammalian microsome plate
incorporation assay: cyclohexane. Final report. Submitted by Phillips Petroleum Co. to
U.S. EPA under TSCA Section 4; EPA Document No. 40-8623065; Fiche No. OTS0527456.
Haskell Laboratories. (1982b) Mouse lymphoma forward mutation assay: cyclohexane. Final
report. Submitted under TSCA Section 4; EPA Document No. 40-8623065; NTIS No.
OTS0527456.
Haskell Laboratories. (1982c) In vitro sister chromatid exchange in Chinese hamster ovary
cells. Final report. Submitted under TSCA Section 4; EPA Document No. 40-8623065; NTIS
No. OTS0527456.
Haskell Laboratories. (1995) Two week inhalation range-finding studies with cyclohexane in
rats and mice. Submitted under TSCA Section 8E; EPA Document No. 40-8623065; NTIS
No. OTS0572154-1
Haskell Laboratories. (1996a) 90-day inhalation toxicity study with cyclohexane in rats, with
cover letter dated 11/18/96. Submitted under TSCA Section 4; EPA Document No. 44634; NTIS
No. OTS0558873.
Haskell Laboratories. (1996b) 90-day inhalation toxicity study with cyclohexane in mice, with
cover letter dated 8/16/96. Submitted under TSCA Section 4; EPA Document No. 44631; NTIS
No. OTS0558870.
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Haskell Laboratories. (1996c) Final report, acute operant behavior study of cyclohexane
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