United States
Environmental Protection
1=1 m m Agency
EPA/690/R-12/01 OF
Final
12-04-2012
Provisional Peer-Reviewed Toxicity Values for
Cyclohexene
(CASRN 110-83-8)
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
Chris Cubbison, PhD
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Ghazi Dannan, PhD
National Center for Environmental Assessment, Washington, DC
Q. Jay Zhao, PhD, MPH, DABT
National Center for Environmental Assessment, Cincinnati, OH
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 at 513-569-7300 or via e-mail to Superfund_STSC@epa.gov.
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVs	1
INTRODUCTION	2
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	3
HUMAN STUDIES	7
Oral Exposures	7
Inhalation Exposures	7
ANIMAL STUDIES	7
Oral Exposures	7
Sub chronic-Duration Studies	7
Chronic-Duration Studies	8
Reproductive/Developmental Studies	8
Carcinogenicity Studies	9
Inhalation Exposures	9
Subchronic Studies	9
Chronic Studies	9
Developmental Studies	11
Reproductive Studies	11
Carcinogenicity Studies	11
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	12
Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity	19
Other Toxicity Studies (Exposures Other than Oral or Inhalation)	19
Short-Term Studies	19
Metabolism/Toxicokinetic Studies	19
Mode of Action/Mechanistic Studies	19
Immunotoxicity	19
Neurotoxicity	19
DERIVATION 01 PROVISIONAL VALUES	20
DERIVATION OF ORAL REFERENCE DOSES	21
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	21
Derivation of Chronic Provisional RfD (Chronic p-RfD)	25
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	27
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)	27
Derivation of Chronic Provisional RfC (Chronic p-RfC)	27
Justification	27
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR	28
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	28
Derivation of Provisional Oral Slope Factor (p-OSF)	28
Derivation of Provisional Inhalation Unit Risk (p-IUR)	28
APPENDIX A. PROVISIONAL SCREENING VALUES	30
APPENDIX B. DATA TABLES	32
APPENDIX C. BMD OUTPUTS	35
APPENDIX D. REFERENCES	39
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower bound 95% confidence interval
BMD
benchmark dose
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
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional reference concentration (inhalation)
p-RfD
provisional reference dose (oral)
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
CYCLOHEXENE (CASRN 110-83-8)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (http://www.epa.eov/iris). the respective PPRTVs are
removed from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
Cyclohexene (see Figure 1), CAS No. 110-83-8, is used as an industrial solvent as well as
an intermediate in a variety of industrial processes. Cyclohexene has a high vapor pressure
(119 hPa at 25°C), suggesting it has high volatility; however, it is moderately soluble in water
(0.250 g/L at 25°C) (OECD SIDS, 2002). A table of physicochemical properties is provided
below (see Table 1).
Figure 1. Cyclohexene Structure
Table 1. Physicochemical Properties for Cyclohexene (CASRN 110-83-8)a
Property (unit)
Value
Boiling point (°C)
83
Melting point (°C)
-103.5
Density (g/cm3 at 20°C)
0.810
Vapor pressure (hPa at 25°C)
119
Solubility in water (g/L at 25°C)
0.250
Relative vapor density (air =1)
No data
Molecular weight (g/mol)
82.15
aOECD SIDS (2002).
No Reference Dose (RfD), Reference Concentration (RfC), or cancer assessment for
cyclohexene is included in the United States Environmental Protection Agency (U.S. EPA)
Integrated Risk Information System (IRIS) (U.S. EPA, 201 la) or on the Drinking Water
Standards and Health Advisories List (U.S. EPA, 2009). No RfD or RfC values are reported in
the Health Effects Assessment Summary Tables (HEAST) (U.S. EPA, 201 lb). The Chemical
Assessments and Related Activities (CARA) list does not include a Health and Environmental
Effects Profile (HEEP) for cyclohexene; there are no noncancer toxicity values (U.S. EPA,
1994). The toxicity of cyclohexene has not been reviewed by the Agency for Toxic Substances
and Disease Registry (ATSDR, 2011) or the World Health Organization (WHO, 2011). The
California Environmental Protection Agency (CalEPA, 2008, 2009) has not derived toxicity
values for exposure to cyclohexene. An 8-hour time-weighted average exposure limit of
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"3
300 ppm (1015 mg/m ) for cyclohexene has been derived by the American Conference of
Governmental Industrial Hygienists (ACGIH, 2008) and is considered a threshold exposure limit
by ACGIH. The same value has been adopted by the Occupational Safety and Health
Administration (OSHA, 2010) as a permissible exposure limit. A 10-hour time-weighted
"3
average exposure limit of 300 ppm (1015 mg/m ) has been recommended (i.e., the recommended
exposure limit) by the National Institute of Occupational Safety and Health (NIOSH, 2010).
The HEAST (U.S. EPA, 201 lb) does not report any values for cancer or a cancer
weight-of-evidence classification for cyclohexene. The International Agency for Research on
Cancer (IARC, 2011) has not reviewed the carcinogenic potential of cyclohexene. Cyclohexene
is not included in the 12th Report on Carcinogens (NTP, 2011). CalEPA (2008) has not prepared
a quantitative estimate of carcinogenic potential for cyclohexene.
Literature searches were conducted on sources published from 1900 through
November 2011 for studies relevant to the derivation of provisional toxicity values for
cyclohexene, CAS No. 110-83-8. Searches were conducted using U.S. EPA's Health and
Environmental Research Online (HERO) database of scientific literature. HERO searches the
following databases: AGRICOLA; American Chemical Society; BioOne; Cochrane Library;
DOE: Energy Information Administration, Information Bridge, and Energy Citations Database;
EBSCO: Academic Search Complete; GeoRef Preview; GPO: Government Printing Office;
Informaworld; IngentaConnect; J-STAGE: Japan Science & Technology; JSTOR: Mathematics
& Statistics and Life Sciences; NSCEP/NEPIS (EPA publications available through the National
Service Center for Environmental Publications [NSCEP] and National Environmental
Publications Internet Site [NEPIS] database); PubMed: MEDLINE and CANCERLIT databases;
SAGE; Science Direct; Scirus; Scitopia; SpringerLink; TOXNET (Toxicology Data Network):
ANEUPL, CCRIS, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK, FEDRIP,
GENE-TOX, HAPAB, HEEP, HMTC, HSDB, IRIS, ITER, LactMed, Multi-Database Search,
NIOSH, NTIS, PESTAB, PPBIB, RISKLINE, TRI, and TSCATS; Virtual Health Library; Web
of Science (searches Current Content database among others); World Health Organization; and
Worldwide Science. The following databases outside of HERO were searched for relevant
health information: ACGIH, AT SDR, CalEPA, U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA
HEEP, U.S. EPA OW, U.S. EPA TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 2 provides an overview of the relevant database for cyclohexene and includes all
potential repeated short-term-, subchronic-, and chronic-duration studies. Principal studies are
identified in bold and through the notation "PS". In this document, unless otherwise noted,
"statistical significant" denotes ap< 0.05.
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Table 2. Summary of Potentially Relevant Data for Cyclohexene (CASRN 110-83-8)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAEL3
BMDL/
BMCLa
LOAEL1
Reference
(Comments)
Notesb
Human
1. Oral (mg/kg-d)a
Acutec
ND
Short-termd
ND
Long-term6
ND
Chronicf
ND
2. Inhalation (mg/m3)a
Acutec
ND
Short-termd
ND
Long-term6
ND
Chronicf
ND
Animal
1. Oral (mg/kg-d)a
Subchronic
12/12, Crj:CD(SD)IGS
rat, gavage, 7 d/wk for
48 d in males or 43-53 d
in females
0, 50,150, or
500
(Adjusted)
Total bilirubin and total
bile acid
NDr
19.71
50
MHLW
(2001a)
PS, PR
Chronic
ND
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Table 2. Summary of Potentially Relevant Data for Cyclohexene (CASRN 110-83-8)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL1
Reference
(Comments)
Notesb
Developmental
12/12, Cij:CD(SD)IGS rat,
gavage, 7 d/wk for 48 d in
males or 43-53 d in
females, from 14 d prior to
mating through LD4
0, 50, 150, or
500
No effects observed on
developmental parameters
500
NDr
NDr
MHLW
(2001b)
PR
Reproductive
12/12, Cij:CD(SD)IGS rat,
gavage, 7 d/wk for 48 d in
males or 43-53 d in
females, from 14 d prior to
mating through LD4
0, 50, 150, or
500
(Adjusted)
No effects observed on
reproductive parameters
500
NDr
NDr
MHLW
(2001b)
PR
Carcinogenicity
ND
2. Inhalation (mg/m3)a
Subchronic
ND
Chronic
20/0, rat (strain not
specified), inhalation,
6 hr/d, 5 d/wk, 6 mo
0, 45, 90, 180,
or 360s
Increase in alkaline
phosphatase11
NDr
NDr
NDr
Laham
(1976a)
NPR

50/50, F344 rat,
inhalation, 6 hr/d,
5 d/wk, 104 wk
0, 360, 720, or
1440g
Increase incidence of
spongiosis hepatis
360
NDr
720
MHLW
(2003a)
PS,
NPR

50/50, Cij:BDFl mouse,
inhalation, 6 hr/d, 5 d/wk,
104 wk
0, 45, 90, or
180s
Data is lacking on many
endpoints, no critical effect
can be determined
NDr
NDr
NDr
MHLW
(2003b)
NPR

10/0, guinea pig,
inhalation, 6 hr/d, 5 d/wk,
6 mo
0, 45, 90, 180,
or 360s
No adverse effects reported
360
NDr
NDr
Laham
(1976b)
NPR
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Table 2. Summary of Potentially Relevant Data for Cyclohexene (CASRN 110-83-8)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL1
Reference
(Comments)
Notesb
Chronic
6/0, rabbit, inhalation,
6 hr/d, 5 d/wk, 6 mo
0, 45, 90, 180,
or 360s
No adverse effects reported
360
NDr
NDr
Laham
(1976c)
NPR
Developmental
ND
Reproductive
ND
Carcinogenicity
50/50, F344 rat, inhalation,
6 hr/d, 5 d/wk, 104 wk
0, 360, 720, or
1440s
No increases in tumors
NDr
NDr
NDr
MHLW
(2003c)
NPR
50/50, Cij:BDFl mouse,
inhalation, 6 hr/d, 5 d/wk,
104 wk
0, 45, 90, or
180s
No increases in tumors
NDr
NDr
NDr
MHLW
(2003d)
NPR
""Dosimetry: All exposure values of long-term exposure (4 weeks and longer) are converted from a discontinuous to a continuous (daily) exposure. Values for inhalation
(cancer and noncancer), and oral (cancer only) are further converted to an HEC/HED. Values from animal developmental studies are not adjusted to a continuous
exposure.
bNotes: PS = Principal study, PR = Peer reviewed, NPR = Not peer reviewed, NA = Not applicable.
0 Acute = Exposure for 24 hours or less (U.S. EPA, 2002).
dShort-term = Repeated exposure for >24 h <30 d (U.S. EPA, 2002).
"Long-term = Repeated exposure for >30 d <10% lifespan (based on typical lifespan of 70 years) (U.S. EPA, 2002).
fChronic = Repeated exposure for >10% lifespan (U.S. EPA, 2002).
8HECExresp = (ppm x MW ^ 24.45) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood:gas partition coefficient.
hAdversity of this endpoint cannot be determined.
ND = No data, NDr = Not determinable.
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HUMAN STUDIES
Oral Exposures
No suitable subchronic- or chronic-duration exposure studies are available.
Inhalation Exposures
No suitable subchronic- or chronic-duration exposure studies are available.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to cyclohexene have been evaluated in one
subchronic-duration study (i.e., MHLW, 2001a) and one reproductive/developmental screening
study (MHLW, 2001b), which were run concurrently.
Subchronic-Duration Studies
Ministry of Health, Labor, and Welfare (MHLW), 2001a
The subchronic component of the peer-reviewed rat study by MHLW (2001a) is
selected as the principal study for derivation of the subchronic and chronic p-RfD. MHLW
(2001a) conducted a subchronic oral toxicity study that also examined reproductive and
developmental effects that will be discussed separately (MHLW, 2001b). This study appears to
be proprietary (may have been part of a Japanese toxicity assessment conducted by MHLW) and
is in Japanese. OECD SIDS (2002) peer-reviewed and summarized the study (cited as MHLW,
2002) and EPA subsequently had the document translated. The internal and external peer
reviewers of this PPRTV document also concurred that the MHLW (2001a) study was suitable
for deriving a provisional toxicity value. This study was conducted as a combined repeated dose
toxicity study with reproduction/developmental toxicity screening according to OECD test
guideline 422 and was stated by OECD to be GLP compliant (no GLP statement was provided in
the study report). Crj :CD(SD)IGS rats (12 animals/sex/treatment group) were administered 0,
50, 150, or 500 mg/kg-day of cyclohexene (98.6% pure) in corn oil via gavage. Dose
formulations were tested for concentration and stability. Males were dosed for 48 days and
females for 43-53 days beginning 14 days before mating, throughout the mating and gestational
period, to Day 4 of lactation. Animals were observed for clinical signs of toxicity daily. Body
weight and food consumption were measured weekly and at necropsy. Urinalysis was conducted
on 5 males/treatment group at 43-48 days of treatment. At sacrifice (on Day 49 for males and
5 days after delivery for females), blood was collected for hematology and clinical chemistry in
all animals. The brain, liver, kidney, spleen, adrenal glands, thymus, testis, and epididymis were
weighed. Tissues and organs were examined histologically in at least the control and high-dose
group. Statistical analyses performed included Bartlett's test for homogeneity of variance,
Dunnett's multiple comparison test (if equal variance), and Steel's test for unequal variances.
The x and Fisher's exact probability tests were also used where appropriate.
Salivation was observed at 150 (for about 5 minutes in 3/12 males and 2/12 females) and
500 mg/kg-day (all animals for 30-60 minutes in males and 6 hours in females). Lacrimation
was observed in 2/12 males at 500 mg/kg-day and females at >150 mg/kg-day (1/12 for each
dose group). There were some small—but statistically significant—hematological changes at
500 mg/kg-day. Increased were the number of reticulocytes and bilirubin in males and
prothrombin time and total bile acids in females. Decreased was the level of APTT in males.
There were no treatment-related significant changes in body weight, or food consumption, in
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either sex or in the urinalysis findings for males (females not measured). There was a
dose-dependent decrease in triglyceride in males (see Table B. 1). Even though triglyceride in
the 500 mg/kg-day group males was 43% lower than in the controls, the results were not
statistically significant nor was this effect noted in the females. There was an increase in total
bilirubin in both sexes; reanalysis of the data indicates that there are statistically significant
increases at all doses in males and in high-dose females. Total bile acid was increased by >10%
in all dose groups. However, the results were highly variable and not dose dependent. Only the
150-mg/kg-day males and the 50- and 500-mg/kg-day females showed statistically significant
changes above the controls. High-dose males had a statistically significant increase in relative
kidney weight that was not accompanied by any histopathological changes and did not reach
1SD (standard deviation) above the control (see Table B.2). OECD SIDS (2002) reported a
NOAEL of 50 mg/kg-day for the repeated dose toxicity portion of the test based on transient
salivation observed in both sexes at 150 mg/kg-day. Transient salivation is not considered
sufficiently adverse to identify as a critical effect. Although the bile acid increase was not dose
dependent and was variable, the data taken together may indicate bile duct blockage. Bile duct
blockage is also consistent with the statistically significant increase in alkaline phosphatase in
rats noted by Laham (1976) following inhalation exposure. Based on the statistically significant
increase in total bile acid in females and total bilirubin in males at the lowest dose, no NOAEL
can be determined and the LOAEL is 50 mg/kg-day.
Chronic-Duration Studies
No studies were identified.
Reproductive/Developmental Studies
MHLW, 2001b
MHLW (2001b) conducted a subchronic oral combined reproductive/developmental
toxicity study that also examined subchronic oral effects that were discussed separately above
(MHLW, 2001a). This study appears to be proprietary (part of a Japanese toxicity assessment
that was conducted by the MHLW), and is in Japanese. OECD SIDS (2002) peer-reviewed and
summarized the study (cited as MHLW, 2002) and EPA subsequently had the report translated.
This study was conducted as a combined repeated dose toxicity study with
reproduction/developmental toxicity screening according to OECD test guideline 422 and was
stated by OECD to be GLP compliant (no GLP statement was provided in the study report).
Cij:CD(SD)IGS rats (12 animals/sex/treatment group) were administered 0, 50, 150, or
500 mg/kg-day of cyclohexene (98.6% pure) in corn oil via gavage. Males were dosed for
48 days and females for 43-53 days beginning 14 days before mating, throughout the mating and
gestational period, to Day 4 of lactation. Animals were observed for clinical signs of toxicity
daily. Body weight and food consumption were measured on a routine basis. The parameters
examined as part of the repeat dose portion of the study are reported above (MHLW, 2001a).
Reproductive and developmental parameters examined included successful copulation, number
of pregnant females, copulation index (number of pairs with successful copulation/number of
pairs mated x 100), fertility index (number of pregnant animals/number of animals with
successful copulation x 100), estrous cycle, number of dams delivering live pups, duration of
gestation, total number of corpora lutea, total number of implants, total number of pups born,
total number of live pups born, sex ratio, total number of dead pups, total number of cannibalized
pups, gestation index (number of females with live pups/number of pregnant females x 100),
implantation index (number of implants/number of corpora lutea x 100), delivery index (number
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of pups born/number of implants x 100), live birth index (number of live pups/number of pups
born x 100), and viability index on Day 4 (number of live pups on Day 4 after birth/number of
live pups born x 100). Pups were examined for external abnormalities and weighed on Days 0
and 4 after birth. Gross necropsy was also performed on pups in this study.
There were no statistically significant changes in body weight or in food consumption in
either males or females. No effects were reported on reproductive performance in the male and
female rats (see Table B.3). Similarly, there were no effects reported on the developmental
parameters examined. Although the mean estrous cycles were similar, it was stated that one
(8.3%) of 150 mg/kg-day females and two (16.7%) of 500 mg/kg-day females had an irregular
estrous cycle. This did not appear to affect reproduction. The reproductive and developmental
NOAEL is 500 mg/kg-day, the highest dose tested, in both sexes. No LOAEL can be determined
from the data.
Carcinogenicity Studies
No studies were identified.
Inhalation Exposures
The effects of inhalation exposure of animals to cyclohexene have been evaluated in five
chronic-duration studies (i.e., Laham, 1976a,b,c; MHLW, 2003a,b). Carcinogenicity data are
also provided by MHLW (2003c,d).
Subchronic Studies
No studies were identified.
Chronic Studies
Laham, 1976 a
Laham (1976a) conducted a chronic-duration inhalation study in which adult male rats
(strain not specified; 20/treatment group) were exposed to 0, 75, 150, 300, or 600 ppm of
cyclohexene (purity not reported) 6 hours/day, 5 days/week, for 6 months. The exposure
concentrations adjusted for continuous exposure and unit conversion are 0, 45, 90, 180, and
360 mg/m3. The only information, available for review is a published abstract that has not been
peer-reviewed. Cyclohexene levels were stated to be continuously monitored using an automatic
sampling system connected to a Carlo Erba gas chromatograph. The blood:gas partition
coefficient is assumed to equal one. Humidity, temperature, and pressure were also stated to be
monitored. None of the results, however, were provided. Body weight was obtained weekly.
Blood was collected for hematology (white blood cell counts [WBC], red blood cell counts
[RBC], platelets, hemoglobin, hematocrit, and differential WBC counts) before, during (timing
not specified), and after exposure. Clinical chemistry (glucose, blood urea nitrogen [BUN],
cholesterol, alanine aminotransferase [ALT], aspartate aminotransferase [AST], lactate
dehydrogenase [LDH], alkaline phosphatase, electrolytes, and other unspecified endpoints) and
gross pathology of hemopoietic organs were conducted at sacrifice after 6 months of exposure.
High-dose rats had significantly decreased body weight gain compared to controls, but no data
were provided. All exposure groups had a statistically significant higher alkaline phosphatase
level than the controls. However, all other parameters were within normal range (data were not
provided), no changes were observed in the bone marrow, and there is no indication that the liver
was examined; therefore, the biological significance of the elevated alkaline phosphatase cannot
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be determined. The data are not sufficient to determine adversity of the effects noted.
Therefore, no NOAEL/LOAEL can be determined. However, based on the increase in alkaline
"3
phosphatase a lowest-observed-effect-level (LOEL) of 45 mg/m is determined and a
no-observed-effect-level (NOEL) cannot be determined.
MHLW, 2003a
The nonpeer-reviewed inhalation study in rats by MHLW (2003a) is selected as the
principal study for derivation of the screening chronic p-RfC. There is no information to
indicate where the study was conducted and it is not considered to be peer-reviewed. The
internal and external peer reviewers of this PPRTV document concurred that the MHLW (2003 a)
study was suitable for deriving a screening provisional toxicity value. The study is in Japanese;
therefore, EPA had the study translated on February 17, 2011. Male and female F344/DuCij
(Fischer) rats (50/sex/treatment group) were administered 0, 600, 1200, or 2400 ppm
cyclohexene (purity not specified) via inhalation 6 hours/day, 5 days/week, for 104 weeks.
Duration adjusted HECs based on extra-respiratory effects with a blood gas partition coefficient
of 1 are 0, 360, 720, and 1440 mg/m3, respectively. Details of the inhalation procedure were not
provided. Animals were observed for general condition. Body weight and food consumption
were routinely measured. Blood counts, blood biochemistry, and urinalysis were measured at
unspecified times. Unspecified organs were stated to have been weighed. Histopathology was
stated to have been conducted, but details of the procedure were not provided. The results of
statistical tests were not specified, but tables indicate that Peto test, Cochran-Armitage test, and
Fisher test were conducted on tumor incidence data.
There were no treatment-related effects on survival. However, survival in high-dose
females was slightly lower than the controls (approximately 72% compared to 94%; data
digitized from the figure provided in the study report). The body weight was stated to be
decreased in high-dose males and females. Results were only provided in figure form, and
statistical significance was not specified. The difference from control, however, was less than
10%) (data digitized from the figure provided in the study report). Although hematology, clinical
chemistry, urinalysis, and organ weights were stated to be assessed, there were no data provided
on any of these endpoints. The study authors specify that there was an increase in the incidence
of spongiosis hepatis at >720 mg/m3 (presumably in both sexes), but specific incidence data were
not provided. Increases in the following lesions were also stated to occur: chronic kidney disease
in males, focal follicular cell hyperplasia in both sexes, and cerebellar granule cell degeneration
in both males and females; however, no details were provided in the report. Based on the
increases in the incidence of spongiosis hepatis, the NOAEL is 360 mg/m3 and the LOAEL is
"3
720 mg/m . However, Karbe and Kerlin (2002) did not consider it a preneoplastic lesion and
claim that it is a spontaneous liver lesion that occurs in aging rats (males more often than
females) with an unknown cause.
MHLW, 2003b
There is no information to indicate where this study was conducted. Although the web
site is in Japanese, EPA had the information translated on February 17, 2011. Male and female
Cij:BDFl mice (50/sex/treatment group) were administered 0, 75, 150, or 300 ppm cyclohexene
(purity not specified) via inhalation 6 hours/day, 5 days/week, for 104 weeks. Duration-adjusted
HECs based on extra-respiratory effects with a blood gas partition coefficient of 1 are 0, 45, 90,
"3
and 180 mg/m , respectively. Details of the inhalation procedure were not provided. Animals
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were observed for general condition. Body weight and food consumption were routinely
measured. Blood counts, blood biochemistry, and urinalysis were measured at unspecified times.
Unspecified organs were stated to have been weighed. Histopathology was stated to have been
conducted, but details of the procedure were not provided. The results of statistical tests were
not specified, but tables indicate that Peto test, Cochran-Armitage test, and Fisher test were
conducted on tumor incidence.
There were no treatment-related effects on survival, body weight, or food consumption.
Although hematology, clinical chemistry, urinalysis, and organ weights were stated to be
conducted, there were no data provided for any of these endpoints. Based on the lack of data
provided, no NOAEL or LOAEL can be determined.
Laham, 1976b,c
Laham (1976) conducted identical studies in guinea pigs (i.e., Laham, 1976b) and rabbits
(i.e., Laham, 1976c) as was conducted in rats (i.e., Laham, 1976a), which is detailed above.
Because the blood:gas partition coefficient is 1 for all these species, the dose conversion is also
the same. There were no adverse effects reported for either the guinea pigs or the rabbits.
Therefore, the NOAEL for the guinea pig (i.e., Laham, 1976b) and rabbit (i.e., Laham, 1976c)
"3
studies is 360 mg/m , the highest dose tested.
Developmental Studies
No studies were identified.
Reproductive Studies
No studies were identified.
Carcinogenicity Studies
MHLW, 2003c
There is no information to indicate where this study was conducted. Although the web
site is in Japanese, EPA had the information translated on February 17, 2011. Male and female
F344/DuCrj(Fischer) rats (50/sex/treatment group) were administered 0, 600, 1200, or 2400 ppm
cyclohexene (purity not specified) via inhalation 6 hours/day, 5 days/week, for 104 weeks.
Duration-adjusted HECs based on extra-respiratory effects with a blood gas partition coefficient
of 1 are 0, 360, 720, and 1440 mg/m3, respectively. Details of the inhalation procedure were not
provided. Animals were observed for general condition. Body weight and food consumption
were routinely measured. Nonneoplastic endpoints were measured and are detailed above under
chronic information. Histopathology was stated to have been conducted, but details of the
procedure were not provided. The results of statistical tests were not specified, but tables
indicate that Peto test, Cochran-Armitage test, and Fisher test were conducted on tumor
incidence data.
There were no treatment-related effects on survival. The body weight was stated to be
decreased in high-dose males and females, but is <10% different from the control (data digitized
from the figure provided in the study report). There was a slight increase in the combined
incidence of hepatocellular adenoma and carcinoma in males (see Table B.4). These results were
only statistically significant with the Peto test (tests for trend). They were not statistically
significant with Fisher test or the Cochran-Armitage trend test. Separately, the tumors
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(hepatocellular adenoma or hepatocellular carcinoma) were not statistically significantly
different from the control. Only one carcinoma is observed in the treated animals. The rest of
the combined incidence is composed of adenomas. The study authors specify that there was an
increase in the incidence of eosinophilic foci (a pretumorous lesion) in the livers of high-dose
males and an increased incidence of spongiosis hepatis at >720 mg/m (presumably in both
sexes), but specific incidence data were not provided. There were no increases in tumors found
in the female rat. The study authors concluded that cyclohexene was not carcinogenic in rats.
MHLW, 2003d
There is no information to indicate where this study was conducted. Although the web
site is in Japanese, EPA had the information translated on February 17, 2011. Male and female
Cij:BDFl mice (50/sex/treatment group) were administered 0, 75, 150, or 300 ppm cyclohexene
(purity not specified) via inhalation 6 hours/day, 5 days/week for 104 weeks. Duration adjusted
HECs based on extra-respiratory effects with a blood gas partition coefficient of 1 are 45, 90, and
"3
180 mg/m , respectively. Details of the inhalation procedure were not provided. Animals were
observed for general condition. Nonneoplastic endpoints were measured and are detailed above,
under chronic studies. Histopathology was stated to have been conducted, but details of the
procedure were not provided. The results of statistical tests were not specified, but tables
indicate that Peto test, Cochran-Armitage test, and Fisher test were conducted on tumor
incidence.
There were no treatment-related effects on survival, body weight, or food consumption.
There were no increases in tumors found in either sex of mice. In male mice, there was a
significant decrease in hepatocellular carcinomas and combined incidence of hepatocellular
adenomas and carcinomas in contrast to the male rat controls (see Table B.4).
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
The genotoxicity of cyclohexene has been studied in several in vitro test systems (i.e.,
Sycheva et al., 2000; BOZO Research Center, 2000a,b,c; De Mik and De Groot, 1978).
Table 3A summarizes the genotoxicity studies on cyclohexene. Table 3B provides information
on the toxicokinetics of cyclohexene (i.e., James et al., 1971; Leibman and Ortiz, 1970, 1971,
1978; Maples and Dahl, 1993). Table 3B also provides summaries of the two mechanistic
studies available for cyclohexene (i.e., Nesnow et al., 1985; Ortiz de Montellano and Mico,
1980).
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Table 3A. Summary of Cyclohexene Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Genotoxicity studies in prokaryotic organisms
Reverse mutation
Salmonella typhimurium
TA100, TA98
1000 ng/plate


Chlorination products of cyclohexene
were also tested and found to be
mutagenic to the TA100 strain without
metabolic activation as well as in a
micronucleus test with epithelial cells
from mouse urinary bladder and colon.
Sycheva et al.,
2000
Reverse mutation
Salmonella typhimurium
TA100, TA98, TA1535,
TA1537
1250 |ig/platc
(12.5 (ig/L)
- T
- T
Growth inhibition observed at
625 ng/plate or above with or without
activation.
BOZO Research
Center, 2000a°
Reverse mutation
Escherichia coli WP2 uvrA
5000 ng/plate
(without
activation)
1250 ng/plate
(with activation)
(12.5 and 50 ng/L
respectively)

- T
Growth inhibition observed at
1250 ng/plate or above with activation.
BOZO Research
Center, 2000b0
DNA damage
Escherichia coli MRE 162
(aerosolized into air)
1000 ppb
(2 mg/m3)

ND
Vaporized cyclohexene had no effect on
the survival of E. coli and did not cause
damage to the bacterial DNA (measured
as loss of reproduction and introduction
of breaks in the sedimented DNA).
Ozonized cyclohexene decreased
survival and induced many breaks in the
DNA of E. coli.
De Mik and
De Groot, 1978
SOS repair
induction
ND
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Table 3A. Summary of Cyclohexene Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
ND
Recombination
induction
ND
Chromosomal
aberration
ND
Chromosomal
malsegregation
ND
Mitotic arrest
ND
Genotoxicity studies in mammalian cells—in vitro
Mutation
ND
Chromosomal
aberrations
Chinese hamster lung
(CHL/IU) cells
400 mg/L
" T
" T
Structural chromosomal aberration and
polyploidy were not induced with either
short-term or continuous treatment.
Cell toxicity was observed at
400 iig/mL following continuous
treatment for 24 and 48 hr.
BOZO Research
Center, 2000c°
Sister chromatid
exchange (SCE)
ND
DNA damage
ND
DNA adducts
ND
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Table 3A. Summary of Cyclohexene Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without With
Activation Activation
Genotoxicity studies in mammals—in vivo
Chromosomal
aberrations
ND
Sister chromatid
exchange (SCE)
ND
DNA damage
ND
DNA adducts
ND
Mouse biochemical
or visible specific
locus test
ND
Dominant lethal
ND
Genotoxicity studies in subcellular systems
DNA binding
ND
aLowest effective dose for positive results, highest dose tested for negative results.
b+ = Positive, ± = Equivocal or weakly positive, - = Negative, T = Cytotoxicity, ND = No data.
°Original citation is in Japanese, but EPA had the reports translated in March of 2011.
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Table 3B. Other Studies"
Test
Materials and Methods
Results
Conclusions
References
Carcinogenicity other
than oral/inhalation
ND
Other toxicity studies
(exposures other than
oral or inhalation)
ND
Short-term studies
ND
Metabolism/
toxicokinetic
In vivo study: Rats (details unspecified) were
fed 3 mmol/kg cyclohexene and total liver
glutathione levels were measured 0.5, 1, 2, and
4 hr after dosing. In addition, urinary
excretion of mercapturic acids was measured
in rats and rabbits (details unspecified) dosed
with 2 mmol/kg cyclohexene by gavage using
paper chromatography separation, methylation,
and gas chromatography.
Ingestion of cyclohexene resulted in a rapid
drop in the total liver glutathione level in
rats. The main mercapturic acid urinary
metabolite following administration of
cyclohexene to rats and rabbits was found
to be 3-hydroxycyclohexylmercapturic
acid, with traces of cyclohexylmercapturic
acid and 2-hydroxycyclohexylmercapturic
acid also detected.
These results suggest that
cyclohexene undergoes
conjugation with glutathione
in rats and rabbits.
James et al., 1971
Metabolism/
toxicokinetic
In vitro study: Hepatic microsomes and
supernatant fractions of liver extracts (male
New Zealand rabbits pretreated with
phenobarbital) were incubated with 20 mM of
cyclohexene for up to 20 min. Reaction
mixtures were analyzed by both gas and thin
layer chromatography.
Cyclohexene oxide was detected with a
maximum concentration after 10 min
followed by a decline until no longer
detectable at the end of incubation.
Cyclohexene oxide is likely
an intermediate in the
oxidation of cyclohexene to
a glycol.
Leibman and Ortiz,
1970
Metabolism/
toxicokinetic
In vitro study: Hepatic microsomes and
supernatant fractions of liver extracts (male
Hotzmann rats and male New Zealand White
rabbits pretreated with phenobarbital) were
incubated with 10% cyclohexene in ethanol for
up to 1 hr. Reaction mixtures were analyzed
by both gas and thin layer chromatography.
Cyclohexene was oxidized to trans-
1,2-cyclohexanediol. There was no
evidence of the formation of cis-
1,2-cyclohexanediol.
Oxidation of cyclohexene by
liver microsomes results
predominantly or solely in
the diol product of the
/raws-configuration.
Leibman and Ortiz,
1971
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Table 3B. Other Studies"
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
In vitro study: Hepatic microsomes and
supernatant fractions of liver extracts (male
Hotzmann rats and male New Zealand White
rabbits pretreated with phenobarbital) were
incubated with 40 mM cyclohexene for up to
1 hr. Reaction mixtures were analyzed by both
gas and thin layer chromatography.
In vivo study: 2 male Holtzman rats were
administered 0.1 mL of cyclohexene by gavage
and their urine was analyzed (with and without
P-glucuronidase treatment).
In vitro study: 2-Cyclohexen-l-ol,
trails-eye 1 oliexancdio 1. and to a lesser
extent, cyclohexene oxide, were all
detected at the end of the incubation period.
The formation of 2-cyclohexen-l-ol
required a NADPH generating system.
Microsomes from rats pretreated with
phenobarbital induced cyclohexene
oxidation as much as 5 times greater than
microsomes from rats that were not
pretreated.
In vivo study: The 24 hr urine samples for
both rats contained 2-cyclohexen-l-one
(0.1% of the oral dose) but 2-cyclohexen-
l-ol was not detected.
Cyclohexene is
hydroxylated at the allylic
position in the presence of
liver microsomal
preparations and NADPH
through a typical drug
metabolizing,
mixed-function
oxygenase-catalyzed
reaction.
Leibman and Ortiz,
1978
Metabolism/
toxicokinetic
In vivo study: 15/0, F344/N rat, nose-only
inhalation study.
Rats were exposed to 600 ppm gaseous
cyclohexene for 20 or 360 min. Blood samples
collected at approximately 1.5, 2.5, 25, and
51.5 min were cryogenically distilled and then
analyzed using gas chromatography. Hepatic
cytochrome P-450 concentrations were
measured following sacrifice at 20 or 360 min.
During exposure, blood levels of
cyclohexene increased to approximately
2 |ig/g after 51.5 min. Blood levels of
cyclohexene oxide were below limit of
detection. Hepatic cytochrome P-450
levels in treated rats were unchanged from
controls.
Cyclohexene is absorbed
into the blood stream
following inhalation.
Metabolism of cyclohexene
in vivo to the epoxide is
slow. Exposure to 600 ppm
cyclohexene resulted in no
change in hepatic
cytochrome P-450.
Maples and Dahl,
1993
Mode of action/
mechanistic
In vitro study on the potential for several
chemicals to inhibit or enhance the
oncogenic/malignant cell transformations in
C3H10T1/2CL8 mouse embryo fibroblasts
(C3H cells).
Cyclohexene enhanced cell transformation
in mouse embryo fibroblasts.
Cyclohexene inhibited
epoxide-hydratase activity
allowing arene oxides to
accumulate in the cells,
which led to an
enhancement of malignant
cell transformations.
Nesnowetal., 1985
(abstract only)
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Table 3B. Other Studies"
Test
Materials and Methods
Results
Conclusions
References
Mode of action/
mechanistic
In vitro study: Hepatic microsomes (rats
pretreated with phenobarbital) were incubated
with 10 mM cyclohexene for up to 30 min.
The loss of cytochrome P-450 was measured
with spectroscopy.
In vivo study: 3-5 male Sprague Dawley rats
were injected intraperitoneally daily for 4 d
with 80 mg/kg phenobarbital followed by a
single intraperitoneal injection of 400 |iL/kg
cyclohexene. 4 hr later, the rats were
sacrificed and the livers excised to be tested
for porphyrin-substrate adducts (hepatic
pigments).
No cytochrome P-450 loss was observed
after incubation of hepatic microsomes
with cyclohexene.
Abnormal hepatic pigments were not found
after the administration of cyclohexene to
phenobarbital-pretreated rats.
These results suggest that
steric and electronic factors
are at play with cyclohexene
that can suppress the
destructive interaction with
cytochrome P-450 that other
olefins demonstrate.
Ortiz de Montellano
andMico, 1980
Immunotoxicity
ND
Neurotoxicity
ND
aND = no data.
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Tests Evaluating Carcinogenicity, Genotoxicity, and/or Mutagenicity
The genotoxicity of cyclohexene has been studied in several in vitro test systems (i.e.,
Sycheva et al., 2000; BOZO Research Center, 2000a,b,c; De Mik and De Groot, 1978). These
studies indicate that cyclohexene is not mutagenic or clastogenic in vitro. Studies investigating
the genotoxic potential of cyclohexene in vivo have not been identified. Table 3 A summarizes
the genotoxicity studies on cyclohexene.
Other Toxicity Studies (Exposures Other than Oral or Inhalation)
No studies were identified.
Short-Term Studies
No studies were identified.
Metabolism/Toxicokinetic Studies
Little information on the toxicokinetics of cyclohexene is available (James et al., 1971;
Leibman and Ortiz, 1970, 1971, 1978; Maples and Dahl, 1993). Results of the available studies
indicate that cyclohexene is absorbed to some extent following inhalation or oral exposure.
Metabolism of cyclohexene occurs in the liver via two metabolic pathways; namely, conjugation
with glutathione or oxidation at the allylic position. Cyclohexene oxide was shown to be an
intermediate in the oxidation of cyclohexene to trans- 1,2-cycloheanediol. Table 3B summarizes
the toxicokinetics/metabolism studies on cyclohexene.
Mode of Action/Mechanistic Studies
Two mechanistic studies are available for cyclohexene (i.e., Nesnow et al., 1985;
Ortiz de Montellano and Mico, 1980). Unlike ethylene and other olefins, cyclohexene was not
shown to cause a loss in hepatic cytochrome P-450 concentrations when tested in vitro. In an in
vitro cell transformation test, cyclohexene enhanced oncogenic cell transformation in mouse
embryo fibroblasts through the inhibition of epoxide-hydratase activity, which allowed arene
oxides to accumulate in the cells (Nesnow et al., 1985). Table 3B summarizes the mechanistic
studies on cyclohexene.
Immunotoxicity
No studies were identified.
Neurotoxicity
No studies were identified.
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DERIVATION OF PROVISIONAL VALUES
Table 4. Summary of Reference Values for Cyclohexene
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
POD
Method
PODhed
UFC
Principal Study
Subchronic p-RfD
(mg/kg-d)
Rat/M
Total bilirubin
5 x 1(T2
BMDLj sd
4.81
100
MHLW (2001a)
Chronic p-RfD
(mg/kg-d)
Rat/M
Total bilirubin
5 x 1(T3
BMDLj sd
4.81
1000
MHLW (2001a)
Subchronic p-RfC
(mg/m3)
NDr
Screening Chronic p-RfC
(mg/m3)
Rat/M+F
Spongiosis hepatis
1 x 10°
NOAEL
360
300
MHLW (2003a)
NDr = Not determinable.
Table 5. Summary of Cancer Values for Cyclohexene
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
NDr
p-IUR
NDr
NDr = Not determinable.
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
There is a single repeat-dosing study available, which includes
reproductive/developmental toxicity screening and an assessment of subchronic toxicity
(MHLW, 2001 a,b). The only finding in the study was observations of transient salivation and
changes in clinical chemistry parameters (e.g., triglyceride, total bilirubin, and total bile acid
levels). Although the transient salivation was considered an endpoint for developing a NOAEL
by OECD SIDS (2002), it is not a sufficient endpoint for deriving a p-RfD due to the lack of any
data indicating that the salivation was not just an effect of unpalatability. With the exception of
total bilirubin and total bile acids, the clinical chemistry parameters did not appear to have a dose
response and were variable. The increases in total bilirubin combined with increases in total bile
acid, however, indicate a possible blockage of the bile duct and was considered for POD
selection.
The study by MHLW (2001a) is selected as the principal study for derivation of the
subchronic p-RfD. The critical effect is a statistically significant elevation of total bilirubin in
male rats. The effect is linked to a possible blockage of the bile duct and is consistent with an
elevation of alkaline phosphatase observed by Laham (1976a) in rats following inhalation
exposure. Details of the MHLW (2001a) study are provided in the "Review of Potentially
Relevant Data" section. Benchmark dose (BMD) analyses were conducted on the data for total
bilirubin and total bile acids in both male and female rats. None of the female data had an
adequate fit for any of the models nor does the male total bile acid data. The only data with an
adequate fit is the total bilirubin in male rats. The remaining observed endpoints did not show a
clear dose-response (see Tables B.l. and B.2.). Among the available, acceptable studies, this
was the only study that identified an effect from cyclohexene exposure and also includes the
lowest POD for developing a subchronic p-RfD.
All available continuous models in the EPA Benchmark Dose Software (BMDS
version 2.1.2; U.S. EPA, 2010) were fit to the data on total bilirubin in male rats following
exposure to cyclohexene for approximately 48 days (see Table 6). In the absence of any cogent
basis for selecting a benchmark response (BMR) for the elevated total bilirubin data, a BMR of 1
standard deviation (SD) from the control mean can be used as the standardized reporting level
for comparisons for continuous data (U.S. EPA, 2000).
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Table 6. Total Bilirubin for Male Crj:CD(SD)IGS Rats Treated with Cyclohexene
for Approximately 48 Days—Used for BMD Analysis"
Average Daily Dose (mg/kg-d)b
Number of Subjects
Response0
0
12
0.03 ±0.01
50
11
0.04 ±0.01*
150
12
0.05 ±0.01**
500
12
0.05 ±0.01**
aMHLW (2001a).
bDose was as administered.
°Mean ± standard deviation.
* Statistically significant difference from control (p < 0.05) using two sample /-test was determined for this review
because the results reported by the study author were inconsistent (e.g., total bilirubin in males was significant in
the high-dose group, but not the mid-dose group even though all information was the same);
** p < 0.01.
Table 7 summarizes the BMD modeling results for the total bilirubin data in male
Cij :CD(SD)IGS rats. The curve and BMD output for the selected model are provided in
Appendix C. The BMD Exponential (M4) (constant variance) model with a BMDL of
19.71 mg/kg-day is selected because there is a good visual fit to the data and because it is the
only model that provides an adequate fit (using goodness-of-fit test; p > 0.1).
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Table 7. Model Predictions for Total Bilirubin in Male Crj:CD(SD)IGS Rats"
Model
Goodness of Fit
p-V alueb
AIC for Fitted
Model
bmd1sd
(mg/kg-day)
BMDL1sd
(mg/kg-day)
Conclusions
Hill
(constant variance)
N/A
-380.07
49.56
16.64
Fails p-valuc criteria
Exponential (M4)
(constant variance)
0.508
-381.63
39.96
19.71
Lowest AICC
Lowest BMDLC
Exponential (M5)
(constant variance)
N/A
-380.07
48.77
20.93
Fails p-valuc criteria
Linear
(constant variance)
0.001
-369.82
356.22
242.73
Fails p-valuc criteria
Polynomial
(constant variance)
0.001
-369.82
356.22
242.73
Fails />-value criteria
Power
(constant variance)
0.001
-369.82
356.22
242.73
Fails p-valuc criteria
Exponential (M2)
(constant variance)
0.001
-368.92
400.13
288.58
Fails />-value criteria
Exponential (M3)
(constant variance)
0.001
-368.92
400.13
288.58
Fails p-valuc criteria
aMHLW (2001a).
Values <0.10 fail to meet conventional goodness-of-fit criteria.
°Lowest values with acceptable Goodness of Fit.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose.
The POD in this study is a BMDLisd of 19.71 mg/kg-day based on elevated total
bilirubin in male rats (MHLW, 2001a). Comparatively, 50 mg/kg-day was a LOAEL for total
bilirubin in male rats and for total bile acids in females and a NOAEL for most other effects. No
dosimetric adjustments for duration of exposure are made because the doses in the principal
study were administered via gavage in mg/kg-day, 7 days/week for the study duration.
In EPA's Recommended Use of Body Weight3/4 as the Default Method in Derivation of
the Oral Reference Dose (U.S. EPA, 201 lc), the Agency endorses a hierarchy of approaches to
derive human equivalent oral exposures from data from laboratory animal species, with the
preferred approach being physiologically based toxicokinetic modeling. Other approaches may
include using some chemical-specific information, without a complete physiologically based
toxicokinetic model. In lieu of chemical-specific models or data to inform the derivation of
human equivalent oral exposures, EPA endorses body weight scaling to the 3/4 power (i.e.,
BW3 4) as a default to extrapolate toxicologically equivalent doses of orally administered agents
from all laboratory animals to humans for the purpose of deriving a RfD under certain exposure
conditions. More specifically, the use of BW3 4 scaling for deriving a RfD is recommended
when the observed effects are associated with the parent compound or a stable metabolite, but
not for portal-of-entry effects.
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The selected critical effect of elevated bilirubin is associated with the parent compound
or a stable metabolite. Therefore, scaling by BW3 4 is relevant for deriving human equivalent
doses (HEDs) for this effect.
Following U.S. EPA (201 lc) guidance, the POD based on elevated bilirubin in adult
animals is converted to a HED through application of a dosimetric adjustment factor (DAF)1
derived as follows:
DAF	= (BWa1/4 - BWh1/4)
where
DAF	= dosimetric adjustment factor
BWa	= animal body weight
BWh	= human body weight
Using a BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA, 1988), the
resulting DAF is 0.244. Applying this DAF to the elevated bilirubin, identified as the critical
effect in mature rats yields a BMDLni n as follows:
BMDLhed = BMDLisd x DAF
= 19.71 mg/kg-day x 0.244
= 4.81 mg/kg-day
The subchronic p-RfD for cyclohexene is derived as follows:
Subchronic p-RfD = BMDLhed ^ UFc
= 4.81 mg/kg-day -M00
= 5 x 10~2 mg/kg-day
Table 8 summarizes the uncertainty factors (UFs) for the subchronic p-RfD for
cyclohexene.
:As described in detail in Recommended Use of Body Weight3/4 as the Default Method in Derivation of the Oral
Reference Dose (U.S. EPA, 2011), rate-related processes scale across species in a manner related to both the direct
(BW171) and allometric scaling (BW3'4) aspects such that BW3'4 ^ BW11= BW"14, converted to a
DAF = BWa1/4 - BWhI/4.
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Table 8. Uncertainty Factors for Subchronic p-RfD of Cyclohexene
UF
Value
Justification
UFa
3
A UFa of 3 (10°5) was applied to account for uncertainty in characterizing the toxicodynamic
differences between rats and humans following oral cyclohexene exposure. The toxicokinetic
uncertainty was accounted for by calculation of a human equivalent dose (HED) through
application of a dosimetric adjustment factor (DAF) as outlined in the EPA's Recommended Use
of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA,
2011c).
ufd
3
A UFd of 3 is selected because there is limited reproduction and developmental data based on a
reproductive/developmental screening study (MHLW, 2001b).
UFh
10
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 to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a BMDL.
UFS
1
A UFS of 1 is applied because a subchronic-duration study was utilized.
UFC
100
Composite uncertainty factor for derivation of the subchronic p-RfD.
The confidence of the subchronic p-RfD for cyclohexene is low as explained in Table 9.
Table 9. Confidence Descriptors for Subchronic p-RfD for Cyclohexene
Confidence Categories
Designation"
Discussion
Confidence in study
M
The confidence in the study is medium. Although the Japanese study
was conducted according to OECD 422 guidelines with a wide array of
endpoints examined and translated by EPA, OECD SIDS (2002) provides
a secondary source peer review of the data.
Confidence in database
L
The confidence in the database is low because there is only one oral
repeat dose study in a single species available. Although the study
conducted reproductive and development screening, there are no
two-generation or full developmental studies available.
Confidence in subchronic
p-RfDb
L
The overall confidence is low because the confidence in the database is
low.
aL = Low, M = Medium, H = High.
bThe overall confidence cannot be greater than lowest entry in table.
Derivation of Chronic Provisional RfD (Chronic p-RfD)
There is a single repeat-dosing study available that includes reproductive/developmental
screening and subchronic portions (i.e., MHLW, 2001a,b). The subchronic portion of the study
by MHLW (2001a) is selected as the principal study for derivation of the chronic p-RfD. The
critical effect is total bilirubin in male rats. Details on the MHLW (2001a,b) study are provided
in the "Review of Potentially Relevant Data" section. The same justification and BMD analysis
applies, as was conducted for the subchronic p-RfD, with details provided in the "Derivation of
Subchronic Provisional RfD (Subchronic p-RfD)" section.
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The POD in this study is a BMDLisd of 19.71 mg/kg-day based on elevated total
bilirubin in male rats (MHLW, 2001a). No dosimetric adjustments are made because the doses
in the principal study were administered via gavage in mg/kg-day, 7 days/week for the study
duration. Following U.S. EPA (201 lc) guidance, the POD based on elevated bilirubin in adult
animals was converted to a HED through application of a DAF as described above. The chronic
p-RfD for cyclohexene is derived as follows:
BMDLhed = BMDLisdx DAF
= 19.71 mg/kg-day x 0.244
= 4.81 mg/kg-day
Chronic p-RfD = BMDLhed ~=~ UFc
= 4.81 mg/kg-day 1000
= 5 x 10~3 mg/kg-day
Table 10 summarizes the UFs for the chronic p-RfD for cyclohexene.
Table 10. Uncertainty Factors for Chronic p-RfD of Cyclohexene
UF
Value
Justification
ufa
3
A UFa of 3 (10°5) was applied to account for uncertainty in characterizing the toxicodynamic
differences between rats and humans following oral cyclohexene exposure. The toxicokinetic
uncertainty was accounted for by calculation of a human equivalent dose (HED) through application of
a dosimetric adjustment factor (DAF) as outlined in the EPA's Recommended Use of Body Weight3/4
as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 201 lc).
ufd
3
A UFd of 3 is selected because there is limited reproduction and developmental data based on a
reproductive/developmental screening study (MHLW, 2001b).
UFh
10
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 to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a BMDL.
UFS
10
A UFS of 10 is applied because a subchronic-duration study was utilized.
UFC
1000
Composite uncertainty factor used in the derivation of the chronic p-RfD.
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The confidence of the chronic p-RfD for cyclohexene is low as explained in Table 11.
Table 11. Confidence Descriptors for Chronic p-RfD for Cyclohexene
Confidence Categories
Designation"
Discussion
Confidence in study
M
The confidence in the study is medium. Although the Japanese study was
conducted according to OECD 422 guidelines with a wide array of
endpoints examined and translated by EPA, OECD SIDS (2002) provides
a secondary source peer review of the data.
Confidence in database
L
The confidence in the database is low because there is only one repeat
dose oral study in a single species available. Although the study
conducted reproductive and development screening, there are no
two-generation or full developmental studies available.
Confidence in
subchronic p-RfDb
L
The overall confidence is low because the confidence in the database is
low.
aL = Low, M = Medium, H = High.
bThe overall confidence cannot be greater than lowest entry in table.
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)
No subchronic p-RfC value can be derived because no subchronic inhalation studies were
identified.
Derivation of Chronic Provisional RfC (Chronic p-RfC)
No chronic p-RfC value can be derived because the principal study is not peer reviewed.
However, Appendix A of this document contains a screening value that might be useful in
certain instances. Please see the attached Appendix for details.
Justification
The only information available for inhalation toxicity was provided in a conference
abstract or details on a web site (i.e., Laham, 1976a,b,c; MHLW, 2003). These publications are
not considered to be peer reviewed. The abstract by Laham (1976a,b,c) details similar studies
conducted in three species (rat, guinea pigs, and rabbits). There was a statistically significant
increase in alkaline phosphatase level compared to the controls in rats reported, however, the
data were not provided. The increase in alkaline phosphatase is consistent with the increase in
bilirubin and total bile acids seen after oral exposure (MHLW, 2001a) as an indicator of bile duct
blockage. The Laham (1976a,b,c) abstract, however, is not considered suitable for deriving the
chronic p-RfC, due to lacking information. MHLW (2003) reports on a 2-year study in rats and
mice. There were no noncancer effects reported for the chronic-duration exposure in mice.
Although the web site does not provide sufficient data, it appears that the respiratory tract was
examined as incidences of lung tumors were reported. In rats, an increased incidence of
"3
spongiosis hepatis was stated to occur at concentrations >720 mg/m , however, no incidence data
are provided. In addition, the hematology and clinical chemistry results are not provided.
Taking all the data into consideration, the liver appears to be a target organ. Although there is
some concern that spongiosis hepatis is a preneoplastic lesion, Karbe and Kerlin (2002) suggest
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that it is a sign of hepatotoxicity. They examined 12 oncogenicity studies where spongiosis
hepatis occurred and found that it was more predominant in the males and that it was associated
more with hepatotoxicity than carcinogenicity. Therefore, this lesion can be considered as a
critical effect for p-RfC derivation. A NOAEL of 360 mg/m3 is used to derive the screening
chronic p-RfC from the MHLW (2003a) study (see Appendix A). Because the study is not peer
reviewed and there are no data available on all the endpoints stated to have been tested, the
p-RfC is relegated to a screening value.
CANCER WEIGHT-OF-EVIDENCE (WOE) DESCRIPTOR
Table 12 identifies the cancer WOE descriptor for cyclohexene.
Table 12. Cancer WOE Descriptor for Cyclohexene
Possible WOE
Descriptor
Designation
Route of Entry (Oral,
Inhalation, or Both)
Comments
"Carcinogenic to
Humans "
NS
NA
There are no human data available.
"Likely to Be
Carcinogenic to
Humans "
NS
NA
There is not enough evidence to support this
statement.
"Suggestive Evidence
of Carcinogenic
Potential"
NS
NA
There is not enough evidence to support this
statement.
"Inadequate
Information to Assess
Carcinogenic
Potential"
Selected
Both
No oral carcinogenicity studies were identified.
There was a slight increase in combined
hepatocellular adenomas and carcinomas at the
highest dose in male rats, however, no
concentration was significant and tumors
analyzed separately were not significant. Male
mice had a significant decrease in liver tumors.
Female rats and mice were not affected.
"Not Likely to Be
Carcinogenic to
Humans "
NS
NA
There is not enough evidence to support this
statement.
NS = Not selected; NA = Not applicable.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
The lack of oral data on the carcinogenicity of cyclohexene precludes the derivation of
quantitative estimates for oral (p-OSF) exposure.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
MHLW (2003) conducted a 2-year inhalation carcinogenicity study in rats and mice.
While there was a statistically significant dose-related trend for increased incidence of combined
hepatocellular adenomas and carcinomas in male rats noted with the Peto trend test, the results
were not significant with the Cochran- Armitage trend test, nor were any of the concentrations
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statistically different from the control. This incidence was low and primarily composed of
adenomas, which did not achieve statistical significance alone. Additionally, there was a lack of
a clear dose response in the data (the low dose incidence is less than the controls). Male rats also
had increased incidence of preneoplastic lesions in the liver, but the incidences were not
provided. However, male mice had a statistically significant decrease in the incidence of
combined hepatocellular adenomas and carcinomas and no effects on liver tumors were noted in
females of either species. The increase in liver tumors in male rats was given consideration for
deriving a p-IUR. However, the low incidence and the lack of a clear dose response were not
conducive to BMD modeling. When the data are applied to the multistage linear model with a
10% extra risk, the BMD is approximately 1700 mg/m3, which is above the highest dose tested
(i.e., 1440 mg/m ). This is likely due in part because there was no statistically significant
increase in the response and because the incidence at the highest dose was only 10%, and not
increased above the control by 10% (control had a 4% incidence in combined hepatocellular
adenomas and carcinomas). Because the incidence was low, lacked a dose response, and was not
supported by data in male mice or females of either species, the available data are inadequate to
derive a p-IUR.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For the reasons noted in the main document, it is inappropriate to derive a provisional
chronic p-RfC for cyclohexene. 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." Screening values 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 values should understand that there is considerably
more uncertainty associated with their derivation than for a PPRTV. Questions or concerns
about the appropriate use of screening values should be directed to the Superfund Health Risk
Technical Support Center.
DERIVATION OF SCREENING PROVISIONAL INHALATION REFERENCE
CONCENTRATIONS
Derivation of Screening Chronic Provisional RfC (Screening Chronic p-RfC)
The study by MHLW (2003a) is selected as the principal study for derivation of the
screening chronic p-RfC. The critical effect is spongiosis hepatis observed in male and female
rats (MHLW, 2003a). This study was only provided on a web site and was not peer reviewed
nor was there information available on GLP compliance. Details are provided in the "Review of
Potentially Relevant Data" section. Benchmark dose (BMD) analysis is not possible as no data
were provided. Among the available, acceptable studies, the MHLW (2003a) study represents
the lowest POD for developing a chronic p-RfC.
The POD in this study is a NOAEL of 360 mg/m3.
Cyclohexene is likely a category 2 gas. This is based on the moderate solubility and the
fact that there is some absorption into the blood stream after inhalation exposure (Maples and
Dahl, 1993). Because there is no indication of any adverse respiratory effects, but there is an
indication of possible adverse systemic effects, the following dosimetric adjustments have been
made for inhalation with a NOAEL for extra-respiratory effects:
NOAELhec, exresp = PPm x (ppm conversion) x (average daily dose) x
(blood gas partition coefficients)
= ppm x (MW - 24.45) x [(hours exposed - 24) x
(days per week exposed - 7 days in a week)] x
(blood gas partition coefficients)
= 600 x (82.15 -24.45) x [(6-24) x (5-7)] x (1)
= 360 mg/m3
The chronic p-RfC for cyclohexene, based on a NOAELhec,exresp of 360 mg/m in male
rats, is derived as follows:
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Screening Chronic p-RfC = NOAELhec,exresp + UFc
= 360 mg/m3 300
= 1 x 10° mg/m3
= 1 mg/m3
Table A. 1 summarizes the uncertainty factors for the screening chronic p-RfC for
cyclohexene.
Table A.l. Uncertainty Factors for Screening Chronic p-RfC of Cyclohexene
UF
Value
Justification
ufa
3
A UFa of 3 is applied for animal-to-human extrapolation to account for the toxicodynamic portion of
the UFa because the toxicokinetic portion (100 5) has been addressed in dosimetric conversions.
ufd
10
A UFd of 10 is selected because there are no acceptable two-generation reproduction studies or
developmental studies by this route.
UFh
10
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 to humans.
ufl
1
A UFl of 1 is applied because the POD was developed using a NOAEL.
UFS
1
A UFS of 1 is applied because a chronic study was utilized.
UFC
300
Composite uncertainty factor used in the derivation of the screening chronic p-RfC.
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APPENDIX B. DATA TABLES
Table B.l. Select Clinical Chemistry Parameters in Male and Female Crj:CD(SD)IGS Rats
After 43-53 Days Oral (Gavage) Exposure to Cyclohexene"
Parameter
Average Daily Dose (mg/kg-d)b
0
50
150
500
Male
Sample size
12
11
12
12
Triglyceride (mg/dL)
39.2 ± 22.4°
36.8 ± 18.8 (94%)
27.7 ± 16.7(71%)
22.5 ± 7.7 (57%)
Total bilirubin (mg/dL)
0.03 ±0.01
0.04 ±0.01 (133%)*
0.05 ±0.01 (167%)**
0.05 ±0.01 (167%)**
Total bile acid (|imol/L)
18.8 ± 15.0
20.8 ± 16.6 (111%)
39.9 ±21.0 (212%)*
32.6 ± 25.5 (173%)
Female
Sample size
10
10
10
10
Triglyceride (mg/dL)
45.4 ±24.9
66.6 ± 85.8 (147%)
37.8 ± 9.0 (83%)
39.4 ± 13.9 (87%)
Total bilirubin (mg/dL)
0.04 ±0.01
0.05 ± 0.02 (125%)
0.05 ± 0.02 (125%)
0.06 ±0.01 (150%)*
Total bile acid (|imol/L)
19.3 ±8.6
49.2 ± 28.8 (255%)*
31.2 ± 19.7 (162%)
82.2 ±81.1 (426%)*
aMHLW (2001a).
bDose was as administered.
°Mean ± standard deviation (% of control); % is calculated.
Statistically different from the control *p < 0.05; **p < 0.01 using two sample t-test for the purpose of this review
because the results reported by the study author were inconsistent (e.g., total bilirubin in males was significant in the
high-dose group, but not the mid-dose group even though all information was the same).
Table B.2. Select Organ Weights in Male Crj:CD(SD)IGS Rats After 43-53 Days Oral
(Gavage) Exposure to Cyclohexene"
Parameter
Average Daily Dose (mg/kg-d)b
0
50
150
500
Sample size
12
11
12
12
Absolute kidney weight (g)
3.21 ± 0.33°
3.09 ±0.27 (96%)
3.20 ±0.27 (100%)
3.31 ±0.34 (103%)
Relative kidney weight (g%)
0.652 ±0.057
0.619 ±0.031
(95%)
0.667 ±0.059
(102%)
0.705 ± 0.053
(108%)*
aMHLW (2001a).
bDose was as administered.
°Mean ± standard deviation (% of control); % is calculated.
Statistically different from the control *p < 0.05.
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Table B.3. Reproductive/Developmental Screening Parameters in Male and Female
Crj:CD(SD)IGS Rats After 43-53 Days Oral (Gavage) Exposure to Cyclohexene"
Parameter
Exposure Group (mg/kg-d)b
0
50
150
500
Number of pairs mated
12
12
12
12
Number of pairs copulated
12
11
12
12
Copulation index (%)
100.0
91.7
100.0
100.0
Number of pregnant females
11
10
10
10
Fertility index (%)
91.7
90.9
83.3
83.3
Number of dams delivering live pups
11
10
10
10
Gestation index (%)
100.0
100.0
100.0
100.0
Duration of gestation0
22.5 ±0.5
22.2 ±0.4
22.3 ±0.5
22.5 ±0.5
Number of corpora lutea per dam0
19.2 ±2.6
17.4 ±3.3
18.4 ±3.2
20.1 ±3.8
Number of implants per dam0
13.7 ±3.0
14.4 ± 1.6
14.3 ± 1.5
14.3 ± 1.6
Implantation index (%)°
73.2 ±20.3
84.1 ±9.7
79.0 ± 10.6
72.9 ± 13.1
Number of live pups born per dam0
12.8 ±3.5
13.4 ± 1.6
13.5 ±2.1
12.5 ±2.2
Number of dead pups per litter0
0.3 ±0.6
0.1 ±0.3
0.1 ± 0.3
0.3 ±0.9
Delivery index (%)°
93.2 ± 11.9
93.8 ±6.1
97.3 ±4.7
90.0 ± 10.3
Live birth index (%)°
98.0 ±4.7
99.3 ±2.3
96.9 ±9.7
97.0 ±9.5
Sex ratio0
0.80 ±0.23
1.32 ±0.68
1.14 ± 1.60
0.81 ±0.56
Number of live pups on
Day 4 per litter0
Males
5.5 ±2.2
6.9 ±2.1
6.7 ±2.6
5.0 ±2.1
Females
6.8 ±2.8
6.2 ± 1.9
6.7 ±2.4
7.2 ±2.0
Viability index on Day 4°
Males
90.9 ±30.2
95.3 ± 10.0
100.0 ±0.0
96.7 ± 10.5
Females
88.6 ±29.8
100.0 ±0.0
98.3 ±5.3
98.3 ±5.3
aMHLW (2001b).
bDose was as administered.
°Mean ± standard deviation.
Statistically different from the control *p < 0.05.
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Table B.4. Liver Tumors in Male and Female F344 Rats and CrjrBDFl Mice After a
2-Year Inhalation Exposure"
Parameter
Exposure group, ppm (HEC, mg/m3)b
0
600 (360)
1200 (720)
2400 (1440)
Male F344 rats
Sample size
50
50
50
50
Hepatocellular adenoma
2 (4%)°
1 (2%)
4 (8%)
4 (8%)
Hepatocellular carcinoma
0 (0%)
0 (0%)
0 (0%)
1 (2%)
Hepatocellular adenoma/hepatocellular
carcinoma
2 (4%)
1 (2%)
4 (8%)
5 (10%)d
Female F344 rats
Sample size
50
50
50
50
Hepatocellular adenoma
1 (2%)
1 (2%)
1 (2%)
0 (0%)
Hepatocellular carcinoma
1 (0%)
0 (0%)
0 (0%)
0 (0%)
Parameter
Exposure group, ppm (HEC, mg/m3)b
0
75 (45)
150 (90)
300 (180)
Male Crj:BDFl mice
Sample size
50
50
50
50
Hepatocellular adenoma
8 (16%)
6 (12%)
9 (18%)
5 (10%)
Hepatocellular carcinoma
12 (24%)
7 (14%)
5 (10%)
3 (6%)*e
Hepatocellular adenoma/hepatocellular
carcinoma
20 (40%)
13 (26%)
12 (24%)
8 (16%)**e
Female Crj:BDFl mice
Sample size
50
50
50
50
Hepatocellular adenoma
3 (6%)
1 (2%)
3 (6%)
1 (2%)
aMHLW (2003c,d).
bDoses are converted from ppm to mg/m3 using the following equation: HEC = ppm x (molecular weight ^ 24.45) x
(hours of exposure per day 24) x (days dosed total days) x blood gas partition coefficient.
°Number of animals with tumors (%).
dSignificant increasing trend with the Peto test, but not with the Cochran-Armitage test.
"Significant decreasing trend with the Cochran-Armitage test, but not with the Peto test.
Statistically different from controls *p < 0.05; **p <0.01; Fisher test performed by study authors.
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APPENDIX C. BMD OUTPUTS
Mill AY 2001a_Total bilirubin Male_M4 ExpCV_l
Exponential Model 4 with 0.95 Confidence Level
Exponential
0.055
0.05
0.045
0.04
0.035
0.03
0.025
BMDL
BMD
0
100
200
300
400
500
dose
09:50 05/10 2011
Figure C.l. Exponential (M4) (Continuous Variance) BMD Model for Total Bilirubin Data
(MHLW, 2001a)
Text Output for Exponential (M4) (Continuous Variance) BMD Model for Total Bilirubin
Data (MHLW, 2001a)
Exponential Model. (Version: 1.7; Date: 12/10/2009)
Input Data File: C:/1/MHLW 2001a Total bilirubin Male ExpCV 1.(d)
Gnuplot Plotting File:
Tue May 10 09:50:46 2011
[add notes here]
The form of the response function by Model:
Model 2:	Y[dose] = a * exp{sign * b * dose}
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Model 3: Y[dose] = a * exp{sign *	(b * dose)Ad}
Model 4: Y[dose] = a * [c-(c-l) *	exp{-b * dose}]
Model 5: Y[dose] = a * [c-(c-l) *	exp{-(b * dose)Ad}]
Note: Y[dose] is the median response for exposure = dose;
sign = +1 for increasing trend in	data;
sign = -1 for decreasing trend.
Model 2 is nested within Models 3 and 4.
Model 3 is nested within Model 5.
Model 4 is nested within Model 5.
Dependent variable = Mean
Independent variable = Dose
Data are assumed to be distributed: normally
Variance Model: exp(lnalpha +rho *ln(Y[dose]))
rho is set to 0.
A constant variance model is fit.
Total number of dose groups = 4
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
MLE solution provided: Exact
Initial Parameter Values
Variable	Model 4
(S) = Specified
lnalpha	-9.29929
rho ( S)	0
a	0.0285
b	0.00546459
c	1.84211
d	1
Parameter Estimates
Variable	Model 4
lnalpha	-9.28998
rho	0
a	0.0297272
b	0.0152693
c	1.70773
d	1
Table of Stats From Input Data
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Dose	N	Obs Mean	Obs Std Dev
0
12
o
o
CO
O
o
I—1
50
11
o
o
o
o
I—1
150
12
0. 05
o
o
I—1
500
12
0. 05
o
o
I—1
Estimated Values of Interest
Dose	Est Mean	Est Std	Scaled Residual
0	0.02973	0.00961	0.09834
50	0.04096	0.00961	-0.3316
150	0.04864	0.00961	0.4916
500	0.05076	0.00961	-0.2724
Other models for which	likelihoods are calculated:
Model A1:	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)}	= exp(lalpha + log(mean(i)) * rho)
Model R:	Yij	= Mu + e(i)
Var{e(ij)}	= SigmaA2
Likelihoods of Interest
Model	Log(likelihood)	DF	AIC
A1	195.0333	5	-380.0665
A2	195.0334	8	-374.0668
A3	195.0333	5	-380.0665
R	181.669	2	-359.338
4	194.8145	4	-381.629
Additive constant for all log-likelihoods =	-43.19. This constant
added to the
above values gives the log-likelihood including the term that does not
depend on the model parameters.
Explanation of Tests
Test 1: Does response and/or variances differ among Dose levels? (A2 vs.
R)
Test 2: Are Variances Homogeneous? (A2 vs. A1)
Test 3: Are variances adequately modeled? (A2 vs. A3)
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Test 6a: Does Model 4 fit the data? (A3 vs 4
Tests of Interest
Test
2*log(Likelihood Ratio)
D. F.
p-value
Test 1
Test 2
Test 3
Test 6a
26.73
0. 0002897
0. 0002897
0.4376
6
3
3
1
0. 0001628
1
1
0.5083
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.
The p-value for Test 3 is greater than .1. The modeled
variance appears to be appropriate here.
The p-value for Test 6a is greater than .1. Model 4 seems
to adequately describe the data.
Benchmark Dose Computations:
Specified Effect = 1.000000
Risk Type = Estimated standard deviations from control
Confidence Level = 0.950000
BMD
39.9627
BMDL
19.7087
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