DRAFT - DO NOT CITE OR QUOTE EPA/635/R-07/004
www.epa.gov/iris
TOXICOLOGICAL REVIEW
OF
CHLORDECONE (KEPONE)
(CAS No. 143-50-0)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
January 2008
NOTICE
This document is an External Review draft. This information is distributed solely for the
purpose of pre-dissemination peer review under applicable information quality guidelines. It has
not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
-------
DISCLAIMER
This document is a preliminary review draft for review purposes only. This information
is distributed solely for the purpose of pre-dissemination peer review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not
represent and should not be construed to represent any Agency determination or policy. Mention
of trade names or commercial products does not constitute endorsement or recommendation for
use.
JANUARY 2008 ii DRAFT - DO NOT CITE OR QUOTE
-------
CONTENTS —TOXICOLOGICAL REVIEW OF CHLORDECONE (CAS No. 143-50-0)
LIST OF TABLES v
LIST OF FIGURES vii
LIST OF ABBREVIATIONS AND ACRONYMS viii
FOREWORD x
AUTHORS, CONTRIBUTORS, AND REVIEWERS xi
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS 3
3. TOXICOKINETICS 5
3.1. ABSORPTION 5
3.2. DISTRIBUTION 7
3.3. METABOLISM 10
3.4. ELIMINATION 12
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 15
4. HAZARD IDENTIFICATION 17
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 17
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 18
4.2.1. Subchronic Studies 19
4.2.1.1. Oral Exposure Studies 19
4.2.1.2. Inhalation Exposure Studies 19
4.2.2. Chronic Studies 20
4.2.2.1. Oral Exposure Studies 20
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES 33
4.3.1. Reproductive Toxicity Studies 33
4.3.2. Developmental Toxicity Studies 44
4.3.3. Screening Studies 46
4.4. OTHER STUDIES 47
4.4.1. Acute Toxicity Studies 47
4.4.2. Potentiation of Halomethane Toxicity 47
4.4.3. Neurotoxicity Studies 49
4.4.4. Endocrine Disruption Studies 49
4.4.5. Immunological Studies 51
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION 55
4.5.1. Genotoxicity 55
4.5.2. Tumor Promotion and Mechanistic Studies 55
4.5.3. Structural Analog Data—Relationship to Mirex 58
JANUARY 2008 iii DRAFT - DO NOT CITE OR QUOTE
-------
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 61
4.6.1. Oral Exposure 64
4.6.2. Mode-of-Action Information—Glomerular Lesions 67
4.7. EVALUATION OF CARCINOGENICITY 68
4.7.1. Summary of Overall Weight of Evidence 68
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 70
4.7.3. Mode-of-Action Information 74
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 75
4.8.1. Possible Childhood Susceptibility 75
4.8.2. Possible Gender Differences 76
5. DOSE-RESPONSE ASSESSMENTS 78
5.1. ORAL REFERENCE DOSE(RfD) 78
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification78
5.1.2. Methods of Analysis 83
5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 85
5.1.4. RfD Comparison Information 86
5.1.5. Previous RfD Assessment 89
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 89
5.3. CANCER ASSESSMENT 90
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF 91
HAZARD AND DOSE RESPONSE 91
6.1. HUMAN HAZARD POTENTIAL 91
6.2. DOSE RESPONSE 92
6.2.1. Noncancer 92
6.2.2. Cancer 95
7. REFERENCES 97
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND A-l
PUBLIC COMMENTS AND DISPOSITION A-l
APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE RfD B-l
JANUARY 2008 iv DRAFT - DO NOT CITE OR QUOTE
-------
LIST OF TABLES
Table 3-1. Whole blood chlordecone level by group of exposed persons 6
Table 3-2. Distribution of chlordecone in man 7
Table 4-1. Incidence and time to tumor of hepatocellular carcinoma in rats 23
Table 4-2. Summary of endocrine and reproductive system tumor incidence among rats
exposed to chlordecone 24
Table 4-3. Incidence and time to tumor of hepatocellular carcinoma in mice 28
Table 4-4. Percent body weight gain and percent survival of chlordecone-exposed
rats and mice 28
Table 4-5. Testicular atrophy in male rats receiving chlordecone in the diet for 3 months 30
Table 4-6. Incidence of histopathologic liver lesions (fatty changes and hyperplasia)
and renal glomerulosclerosis in male and female Wistar rats following
administration of chlordecone in the diet for 1-2 years 31
Table 4-7. Effects of dietary chlordecone on reproduction in male and female mice
(of mixed parentage) treated for 1 month prior to mating and for 100 days
following the initiation of mating 34
Table 4-8. Effects of dietary chlordecone (0 or 40 ppm) on reproduction in
BALB/cJaxGnMc mice during 100 days of treatment (preceded by 2 months
of pre-mating treatment) and during 100 days of a crossover-mating period
following the termination of treatment 35
Table 4-9. Effects of dietary chlordecone for 1 month prior to mating on reproductive
indices of male and female laboratory mice of mixed breeds 36
Table 4-10. Effects of dietary chlordecone (0 or 5 ppm) 1 month prior to mating and
5 months during mating on reproduction inBALB/c mice 37
Table 4-11. Effects of chlordecone on estrous cyclicity and ovulation in CD-I mice
exposed to chlordecone by gavage 5 days per week for up to 6 weeks 40
Table 4-12. Abundance of various-sized follicles and the condition of large-sized
follicles in the ovaries of female CD-I mice exposed to chlordecone by
gavage 5 days per week for 4 weeks 41
Table 4-13. Effects of chlordecone on adult female offspring of Sprague-Dawley
JANUARY 2008 v DRAFT - DO NOT CITE OR QUOTE
-------
rat dams administered chlordecone by gavage on gestation days 14-20 42
Table 4-14. Sperm parameters in male Sprague-Dawley rats following
administration of chlordecone in the diet for 90 days 43
Table 4-15. Maternal and fetal effects following gavage dosing of pregnant rat dams
with chlordecone on gestation days 7-16 45
Table 4-16. Maternal and fetal effects following gavage dosing of pregnant mouse
dams with chlordecone on gestation days 7-16 46
Table 4-17. Physiochemical properties of chlordecone and mirex 59
Table 4-18. Summary of noncancer results of repeat-dose studies for oral exposure
of experimental animals to chlordecone 62
Table 5-1. Incidence of histopathologic renal lesions (glomerulosclerosis grades
1, 2, or 3 combined) in male or female Wistar rats following administration
of chlordecone in the diet for 1-2 years 83
Table 5-2. Possible PODs with applied UFs and resulting RfDs 88
Table B-l. Incidence of histopathologic renal lesions (glomerulosclerosis grades
1, 2, or 3 combined) in female Wistar rats following administration of
chlordecone in the diet for 2 years B-l
Table B-2. BMD modeling results for the incidence of histopathologic renal
lesions (glomerulosclerosis) in female Wistar rats, following administration
of chlordecone in the diet for 2 years B-2
Table B-3. Incidence of testicular atrophy in male rats receiving chlordecone in the
diet for 3 months B-5
Table B-4. BMD modeling results for the incidence of testicular atrophy in male
Wistar rats, following administration of chlordecone in the diet for 3 months B-6
Table B-5. Incidence of histopathologic liver lesions (fatty changes and hyperplasia)
in Wistar rats, following administration of chlordecone in the diet
for 1-2 years B-9
Table B-6. BMD modeling results for the increased incidence of liver lesions
in rats (both sexes combined), following administration of chlordecone
in the diet for 1-2 years B-10
JANUARY 2008 vi DRAFT - DO NOT CITE OR QUOTE
-------
LIST OF FIGURES
Figure 2-1. The structure of chlordecone 3
Figure 3-1. A metabolic scheme for chlordecone 11
Figure 4-1. Dosing regimen for male rats in the study by NCI (1976b) 21
Figure 4-2. Dosing regimen for female rats in the study by NCI (1976b) 21
Figure 4-3. Dosing regimen for male mice in the study by NCI (1976b) 26
Figure 4-4. Dosing regimen for female mice in the study by NCI (1976b) 26
Figure 5-1. RfD comparison array for alternate points of departure 88
Figure 6-1. RfD comparison array for alternate points of departure 94
Figure B-l. Observed and predicted incidence of histopathologic renal lesions
(glomerulosclerosis grades 1, 2, or 3 combined) in female Wistar rats
following administration of chlordecone in the diet for 1-2 years B-2
Figure B-2. Observed and predicted incidence of testicular atrophy in male Wistar
rats, following administration of chlordecone in the diet for 3 months B-6
Figure B-3. Observed and predicted incidence of liver lesions in male and female
Wistar rats following administration of chlordecone in the diet for 1-2 years... B-10
JANUARY 2008 vii DRAFT - DO NOT CITE OR QUOTE
-------
LIST OF ABBREVIATIONS AND ACRONYMS
AIC Akaike's Information Criterion
ALT alanine aminotransferase
AST aspartate aminotransferase
ATSDR Agency for Toxic Substances and Disease Registry
BMD benchmark dose
BMDio benchmark dose associated with a 10% extra risk
BMDLio benchmark dose lower 95% confidence limit
BMDS Benchmark Dose Software
BMR benchmark response
BUN blood urea nitrogen
CHO Chinese hamster ovary
conA concanavalin A
CYP cytochrome
CYP450 cytochrome P450
DEN diethynitrosamine
ELISA enzyme-linked immunosorbent assay
FSH follicle-stimulating hormone
GOT y-glutamyl transpeptidase
HDL high density lipoproteins
HSDB Hazardous Substances Data Bank
IRIS Integrated Risk Information System
LD50 median lethal dose
LOAEL lowest-observed-adverse-effect level
LSPC Life Science Products Company
NCI National Cancer Institute
NIOSH National Institute for Occupational Safety and Health
NK natural killer
NLM National Library of Medicine
NOAEL no-observed-adverse-effect level
PBTK physiologically based toxicokinetic
PFC plaque-forming cell
PHA phytohemagglutinin
POD point of departure
PVE persistent vaginal estrus
RfC reference concentration
RfD reference dose
SALP serum alkaline phosphatase
s.c. subcutaneous
SCK serum creatine kinase
SER smooth endoplasmic reticulum
SGOT serum glutamic oxaloacetic transferase
SGPT serum glutamic pyruvic transferase
SRBC sheep red blood cell
JANUARY 2008
Vlll
DRAFT - DO NOT CITE OR QUOTE
-------
STM Salmonella typhimurium mitogen
TD toxicodynamic
UF uncertainty factor
U.S. EPA U.S. Environmental Protection Agency
U.S. DHHS U.S. Department of Health and Human Services
JANUARY 2008 ix DRAFT - DO NOT CITE OR QUOTE
-------
FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to
chlordecone. It is not intended to be a comprehensive treatise on the chemical or toxicological
nature of chlordecone.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of the data and related uncertainties. The discussion is intended to convey the
limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the
risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
JANUARY 2008 x DRAFT - DO NOT CITE OR QUOTE
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Kathleen Newhouse, M.S.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
CONTRIBUTING AUTHORS
Debdas Mukerjee, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
Andrew Rooney, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Mark Follansbee, Ph.D.
Syracuse Research Corporation
N. Syracuse, NY
Julie Stickney, Ph.D.
Syracuse Research Corporation
N. Syracuse, NY
David Wohlers, Ph.D.
Syracuse Research Corporation
N. Syracuse, NY
INTERNAL EPA REVIEWERS
Jamie Strong, Ph.D.
National Center for Environmental Assessment
Washington, DC
TedBerner, M.S.
National Center for Environmental Assessment
Washington, DC
JANUARY 2008 xi DRAFT - DO NOT CITE OR QUOTE
-------
Chao Chen, Ph.D.
National Center for Environmental Assessment
Washington, DC
Prasada Kodavanti, Ph.D.
National Health and Environmental Effects Research Laboratory
Research Triangle Park, NC
Harlal Choudhury, Ph.D.
National Center for Environmental Assessment
Cincinnati, OH
Lynn Flowers, Ph.D.
National Center for Environmental Assessment
Washington, DC
JANUARY 2008 xii DRAFT - DO NOT CITE OR QUOTE
-------
1. INTRODUCTION
This document presents background information and justification for the Integrated
Risk Information System (IRIS) Summary of the hazard and dose-response assessment of
chlordecone. IRIS Summaries may include oral reference dose (RfD) and inhalation reference
concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity
assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The
inhalation RfC considers toxic effects for both the respiratory system (portal of entry) and for
effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference
values are generally derived for chronic exposures (up to a lifetime), but may also be derived for
acute (<24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of
lifetime) exposure durations, all of which are derived based on an assumption of continuous
exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are
derived for chronic exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, the oral slope factor is an upper bound on the
estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is an upper
bound on the estimate of risk per ug/m3 air breathed.
Development of these hazard identification and dose-response assessments for
chlordecone has followed the general guidelines for risk assessment as set forth by the National
Research Council (1983). EPA guidelines and technical reports that may have been used in the
development of this assessment include the following: Guidelines for Mutagenicity Risk
Assessment (U.S. EPA, 1986a), Guidelines for the Health Risk Assessment of Chemical Mixtures
(U.S. EPA, 1986b), Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991),
Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for
Neurotoxicity Risk Assessment (U.S. EPA, 1998a), Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 2005a), Supplimental Guidance for Assessing Susceptibility from Early-Life
JANUARY 2008 1 DRAFT - DO NOT CITE OR QUOTE
-------
Exposure to Carcinogens (U.S. EPA, 2005b), Recommendations for and Documentation of
Biological Values for Use in Risk Assessment (U.S. EPA, 1988), (proposed) Interim Policy for
Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods
for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry
(U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA,
1995), Science Policy Council Handbook: Peer Review (U.S. EPA, 1998b, 2000a, 2005c),
Science Policy Council Handbook: Risk Characterization (U.S. EPA, 2000b), Benchmark Dose
Technical Guidance Document (U.S. EPA, 2000c), Supplimentary Guidance for Conducting
Helath Risk Assessment of Chemical Mixtures (U.S. EPA, 2000d) and A Review of the Reference
Dose and Reference Concentration Processes (U.S. EPA, 2002).
The literature search strategy employed for this compound was based on the CASRN and
at least one common name. Any pertinent scientific information submitted by the public to the
IRIS Submission Desk was also considered in the development of this document. The relevant
literature was reviewed through May 2007.
JANUARY 2008 2 DRAFT - DO NOT CITE OR QUOTE
-------
2. CHEMICAL AND PHYSICAL INFORMATION RELEVANT TO ASSESSMENTS
Chlordecone is a tan to white crystalline odorless solid (NIOSH, 2004). The structure of
chlordecone is shown in Figure 2-1. Synonyms include Kepone, decachlorooctahydro-1,3,4-
metheno-2H-cyclobuta[cd]-pentalen-2-one, and GC-1189 (O'Neil, 2001). Selected chemical and
physical properties of chlordecone are listed below.
Cl
ct
'k
cr
— 7
^
^r^
X!
Cl
\
Cl
Figure 2-1. The structure of chlordecone.
CAS number:
Molecular weight:
Chemical formula:
Melting point:
Vapor pressure:
Density:
Water solubility:
Other solubilities:
Partition coefficient:
143-50-0 (Lide, 2000)
490.64 (O'Neil, 2001)
CioClioO (O'Neil, 2001)
Decomposes at 350°C (Lide, 2000)
2.25 x 10~7 mm Hg at 25°C (Kilzer et al., 1979)
1.61 g/mL at 25°C (Lide, 2000)
2.70 mg/L at 25°C (Kilzer et al., 1979)
Slightly soluble in hydrocarbon solvents; soluble in
alcohols, ketones, acetic acid (O'Neil, 2001)
log Kow = 5.41 (Hansch et al., 1995)
Chlordecone production begins with the condensation of hexachlorocyclopentadiene
with sulfur trioxide under heat and pressure (NLM, 2004a; ATSDR, 1995). Antimony
pentachloride is used as a catalyst. The product of this reaction is hydrolyzed and then
neutralized (ATSDR, 1995; IARC, 1979). Chlordecone is obtained by centrifugation or filtration
and hot air drying. Chlordecone is also a contaminant in mirex formulations and is a degradation
product of mirex (Bus and Leber, 2001).
JANUARY 2008
DRAFT - DO NOT CITE OR QUOTE
-------
Chlordecone was first produced in the United States in the early 1950s (IARC, 1979). It
was introduced commercially in 1958 (Bus and Leber, 2001). Approximately 3.6 million pounds
of chlordecone were produced in the United States between 1951 and 1975 (ATSDR, 1995).
Chlordecone production in the United States ended in 1975 after intoxication from severe
industrial exposure was observed in employees who worked at the only chlordecone
manufacturing plant in the country (Bus and Leber, 2001). Typical signs of chlordecone
intoxication include nervousness, headache, and tremor (Cannon et al., 1978). Its registration
was cancelled in 1978 (Metcalf, 2002; IARC, 1979).
Chlordecone was primarily used as an insecticide (IARC, 1979). Specific applications
have included control of the banana root borer, application on non-fruit-bearing citrus trees to
control rust mites, control of wireworms in tobacco fields, control of apple scab and powdery
mildew, control of the grass mole cricket, and control of slugs, snails, and fire ants (NLM,
2004a; ATSDR, 1995). The registration of pesticide products containing chlordecone was
cancelled by the US EPA in 1976 (US EPA, 1976).
Chlordecone is resistant to degradation in the environment. It is not expected to react
with hydroxyl radicals in the atmosphere or to hydrolyze or photolyze (NLM, 2004a).
Chlordecone in the air is likely to be removed by deposition of particles (NLM, 2004a). Studies
have shown that microorganisms degrade chlordecone slowly (NLM, 2004a). Chlordecone is
expected to adsorb to soil and to stick to suspended solids and sediments in water (NLM, 2004a).
Small amounts of chlordecone will evaporate from soil or water surfaces (NLM, 2004a).
Chlordecone has a high potential for bioaccumulation in fish and other aquatic organisms
(ATSDR, 1995).
JANUARY 2008 4 DRAFT - DO NOT CITE OR QUOTE
-------
3. TOXICOKINETICS
The available data for humans and animals indicate that chlordecone is well absorbed
following oral exposure. Once absorbed, it is widely distributed and eventually concentrates in
the liver. It is metabolized by humans and some animal species to chlordecone alcohol.
Glucuronide conjugates of chlordecone and chlordecone alcohol, as well as unconjugated
chlordecone, are slowly excreted in the bile and eliminated in the feces. Fecal excretion is
limited by enterohepatic recirculation.
3.1. ABSORPTION
Chlordecone absorption in humans has been demonstrated by the measurement of
chlordecone concentrations in blood, subcutaneous fat, and other body fluids and tissues
following subchronic occupational exposure, presumably through ingestion, inhalation, and
dermal contact (Taylor, 1982; Adir et al., 1978; Cannon et al., 1978; Cohn et al., 1978). Workers
with neurological symptoms of chlordecone toxicity (i.e., tremors, ataxia) had whole blood
concentrations ranging between 0.009 and 11.8 ppm. Chlordecone blood concentrations for
workers without neurological symptoms were between 0.003 and 4.1 ppm. Chlordecone was
also detected in the blood of Hopewell community residents living near a pesticide plant with
concentrations ranging from 0.005 to 0.0325 ppm. Potential exposure routes for community
residents included inhalation of chlordecone associated with fine particulate matter and ingestion
of contaminated soil and drinking water. Neurological symptoms were reported for some
residents living near the plant site. In general, the highest blood chlordecone concentrations
were observed in affected workers, and lower concentrations were measured in unaffected
workers and community residents (Table 3-1) (Cannon et al., 1978).
No data were available in laboratory animals to evaluate chlordecone absorption
following inhalation exposure. Quantitative data on absorption of orally administered
chlordecone are limited; however, studies on the distribution and excretion of chlordecone in
rats, mice, gerbils, and pigs following oral administration of chlordecone indicate that this
chemical is readily absorbed from the gastrointestinal tract in animals (Hewitt et al., 1985;
Aldous et al., 1983; Fujimori et al., 1982a; Wang et al., 1981; Kavlock et al., 1980; Egle et al.,
1978). One study (Egle et al., 1978) attempted to estimate oral absorption quantitatively. Male
Sprague-Dawley rats received a single oral dose of 40 mg/kg-day C[14]-labeled chlordecone in
corn oil solution. The percentage of radioactivity excreted in the feces was measured over time.
Approximately 10% of the dose was detected in the feces on the first day after dosing,
suggesting that 90% of the orally administered dose was absorbed from the corn oil vehicle.
Animal studies suggest that chlordecone is absorbed only to a limited extent through the
skin (Heatherington et al., 1998; Shah et al., 1987). The in vivo percutaneous absorption of
JANUARY 2008 5 DRAFT - DO NOT CITE OR QUOTE
-------
chlordecone was evaluated in young (33 days old) and adult (82 days old) F344 rats (Shah et al.,
1987). Acetone solution that contained C[14]-labeled chlordecone was applied to the shaved
backs of animals, with the treatment area constituting 2 to 3% of the total body surface area.
Table 3-1. Whole blood chlordecone level by group of exposed persons
Group
Affected LSPCa workers
Unaffected LSPC workers
Family members, LSPC workers
Alliedb chlordecone workers
Neighborhood workers
Sewage treatment plant workers
Cab drivers
Truck drivers
Hopewell community residents0
No.
tested
57
49
32
39
32
10
5
2
214
No. with
detectable
level
57
48
30
30
23
6
1
1
40
% with
detectable
level
100
99
94
77
72
60
20
50
19
Range of
detectable level,
ppm
0.009-11.8
0.003-4.1
0.003-0.39
0.003-0.45
0.003-0.031
0.004-0.014
0.003
0.004
0.005-0.0325
Mean of
detectable
level, ppm
2.53
0.60
0.10
0.06
0.011
0.006
0.003
0.004
0.011
aLSPC = Life Science Products Company.
bAllied Chemical Corporation.
'Excludes chlordecone factory workers.
Source: Cannon etal. ( 1978).
Urine and feces were collected over a 72-hour period, after which animals were sacrificed
to determine the recovery of radioactivity and the percutaneous absorption of chlordecone.
Three dose levels were used to compare dermal absorption in young and adult rats (three rats per
dose group). No age-related differences in dermal absorption of chlordecone were noted in this
study. Dermal absorption decreased over the dose range in both young and adult rats. In adults,
9% of the applied dose was absorbed at the lowest dose (0.2587 |imol/cm2), 6% absorption
occurred at the medium dose (0.536 jimol/cm2), and 1% absorption occurred at the highest dose
tested (2.679 jimol/cm2). In young rats, 10% of the applied dose was absorbed at the lowest dose
(0.3357 |imol/cm2), 7% absorption occurred at the medium dose (0.536 |imol/cm2), and 2%
absorption was seen at the highest dose tested (2.679 jimol/cm2). The nonlinear relationship
between in vivo dermal absorption and dose described by Shah et al. (1987) was confirmed by
Heatherington et al. (1998) in young and adult rats. In adults, 8% of the applied dose was
absorbed at the lowest dose (0.29 jimol/cm2), 6% absorption was seen at the medium dose (0.535
|imol/cm2), and 1% absorption occurred at the highest dose tested (2.68 |imol/cm2). In young
rats, 9% of the applied dose was absorbed at the lowest dose (0.340 jimol/cm2), 7% absorption
JANUARY 2008
DRAFT - DO NOT CITE OR QUOTE
-------
occurred at the medium dose (0.535 jimol/cm ), and 2% absorption was seen at the highest dose
tested (2.68 |imol/cm2).
The time course of chlordecone dermal absorption was studied in young and adult rats by
using a serial sacrifice study design (Heatherington et al., 1998). Young and adult F344 rats
were dermally exposed to 0.285 |imol/cm2 chlordecone by using the procedure described above
for the Shah et al. (1987) study. Rats were sacrificed at 6, 24, 48, 72, and 120 hours
posttreatment. No significant age-related differences were noted in the time course for dermal
penetration of chlordecone. In adult rats, the average cumulative absorption was 0.4, 3, 6, 9, and
14% measured at 6, 24, 48, 72, and 120 hours, respectively. In young rats, the average
cumulative absorption was 0.6, 4, 7, 10, and 14% measured at 6, 24, 48, 72, and 120 hours,
respectively. In vitro test systems using static and flow through diffusion cells were also
employed by Heatherington et al. (1998). Only 1% of the applied chlordecone dose penetrated
excised dorsal skin from young and adult rats under in vitro conditions. Based on the in vivo
dermal absorption data obtained, a biophysically based percutaneous absorption model was
developed to describe the movement of chlordecone through the skin. This model was
embedded in a whole animal physiologically based toxicokinetic (PBTK) model that was
employed to predict tissue concentrations of chlordecone following dermal exposure (see Section
3.5).
3.2. DISTRIBUTION
In 32 workers exposed to chlordecone for a period that ranged from 3 to 16 months, high
concentrations of chlordecone were found in blood, liver, and subcutaneous fat. Modest amounts
of chlordecone were detected in muscle, gall bladder, bile, and stool, while only trace amounts
were detected in aqueous body fluids such as cerebrospinal fluid, urine, saliva, and gastric juice
(Cohn et al., 1978). The ratio of the chlordecone concentration in fat as compared to the
chlordecone concentration in the blood was 7:1, which is relatively low for a lipophilic
organochlorine pesticide. The liver to blood concentration ratio in exposed workers was reported
to be 15:1 (Table 3-2).
Table 3-2. Distribution of chlordecone in man
Tissue
Whole blood
Liver
Subcutaneous fat
Muscle
Gallbladder bile
Number of
patients
32
10
29
5
6
Concentration
range (jig/g)
0.6-32.0
13.3-173.0
1.7-62.1
1.2-11.3
2.5-30.0
Partition
Tissue:blood
1.0
15.0
6.7
2.9
2.5
Range
4.6-31
3.8-12
1.8-4.5
1.4-4.1
Source: Cohn etal. (1978).
JANUARY 2008
DRAFT - DO NOT CITE OR QUOTE
-------
The preferential uptake and slow elimination of chlordecone from the liver was
confirmed in laboratory animals (Belfiore et al., 2007; Hewitt et al., 1985; Egle et al., 1978).
Chlordecone concentrations in rat plasma, kidney, liver, and adipose tissue were determined at
various time points following a single oral dose of 50 mg/kg (Hewitt et al., 1985). Chlordecone
concentrations persisted in rat tissues throughout the 32-day study period. The highest tissue
concentrations were observed in the liver, and this organ had the slowest elimination rate.
Between days 8 and 32, liver concentrations were reduced by 73%, while plasma, kidney, and
adipose levels were reduced 90, 88, and 81%, respectively. The distribution of chlordecone was
also studied in rats receiving a single oral dose of 40 mg/kg-day C[14]-labeled chlordecone in
corn oil solution (Egle et al., 1978). Initially, the highest levels of radioactivity were found in
the adrenal glands followed by liver, lung, and fat. By 3 days following dosing, the highest
concentration was in the liver, and this continued throughout the 182-day study period.
Chlordecone is eliminated more slowly from the liver as compared with other tissues. The liver
to blood ratio increased from 28:1 on day 1 to 126:1 on day 84. The fat to blood ratio reached a
maximum of 31:1 on day 7 and declined thereafter, while other organ to blood ratios remained
constant. Belfiore et al. (2007) measured chlordecone concentrations in rat liver, fat, blood,
kidney, and muscle at 1, 14, or 30 days following a single oral dose of 40 mg/kg. The highest
tissue concentrations were observed in the liver, followed by the kidney. The slowest
elimination rate was seen in the liver, with chlordecone concentrations reduced 25% between day
1 and day 30. At day 30, levels were reduced by 65, 69, 73, and 75% in blood, fat, muscle, and
kidney, respectively. Liver to blood ratios increased from 71:1 on day 1 to 150:1 on day 30.
The preferential retention of chlordecone by the liver is related to chlordecone binding to
plasma proteins and lipoproteins. Serum gel filtration indicated that chlordecone was
predominantly bound to albumin and lipoproteins in exposed workers. Electrophoresis of
normal human plasma following the addition of C[14]-labeled chlordecone demonstrated 80%
binding to lipoproteins, with most of this binding associated with high-density lipoproteins
(HDLs) (Skalsky et al., 1979). The preferential binding of chlordecone to albumin and HDL was
demonstrated in human, rat, and pig plasma (Soine et al., 1982). In human plasma, the in vitro
distribution of C[14]-labeled chlordecone was 46% protein, 30% HDL, 20% low density
lipoprotein, and 6% very low density lipoprotein. Similar distributions were seen for pig plasma
and for in vitro and in vivo distribution studies in rat plasma. Albumin was identified as the
major component of the protein fraction that binds chlordecone. Experiments in isolated
perfused pig liver demonstrated that an increase in HDL can affect the distribution of
chlordecone, favoring chlordecone uptake and retention in the liver and decreased chlordecone
elimination in the bile (Soine et al., 1984). Chlordecone and cholesterol have been shown to
compete for similar intracellular binding and transport proteins, which are inducible by
chlordecone pretreatment (Gilroy et al., 1994; Carpenter and Curtis, 1991, 1989).
JANUARY 2008 8 DRAFT - DO NOT CITE OR QUOTE
-------
The brain and plasma levels of chlordecone in mice were measured after daily oral dosing
with 10 or 50 mg/kg-day (Wang et al., 1981). At the lower dose, the plasma level of chlordecone
increased steadily throughout the 12-day treatment period, while the brain chlordecone level
reached a plateau on day 10. Brain and plasma levels decayed biphasically following
administration of 50 mg/kg-day chlordecone for 1 or 2 days. Brain and plasma concentrations
were correlated with loss of motor control at both administered dose levels. Chlordecone was
distributed to discrete areas of the mouse brain following a single gavage dose of 50 mg/kg
(Fujimori et al., 1982a). The striatum and the medulla/pons had significantly higher chlordecone
levels than the cortex, midbrain, or cerebellum.
The distribution of chlordecone following dermal absorption was studied by
Heatherington et al. (1998) in young and adults rats (see Section 3.1 for study design
information). Less than 15% of the applied dose was absorbed within 120 hours. Organ
concentrations increased slowly over time, with the highest concentrations observed in the liver
followed by (in decreasing order) kidney, carcass, skin, and blood. Kinetic differences in liver
accumulation of chlordecone were suggested between young and adult rats, but all other organ
concentrations were comparable. Tissue levels did not appear to have reached steady-state
conditions by 120 hours of dermal exposure to chlordecone.
Kavlock et al. (1980) studied the distribution of chlordecone in fetal and neonatal rats.
Pregnant rats were given an oral dose of 5 mg/kg chlordecone on days 15, 18, or 20 of gestation.
For the prenatal study, animals were killed at 4, 24, or 48 hours after dosing, and maternal and
fetal tissues were obtained for chlordecone analysis. In the postnatal study, the dams were given
chlordecone at a dose of either 1 or 10 mg/kg-day on days 2 through 5 of the lactation period.
Maternal milk was obtained following an injection of oxytocin on days 5, 9, and 15 of gestation.
Pups were sacrificed for chlordecone tissue analysis on days 3, 5, 7, 9, 12, 15, and 17 of
lactation. Chlordecone crossed the placenta and was observed in fetal tissues as early as 4 hours
after maternal dosing. The maximum concentrations of chlordecone on the placenta were 3.5
and 4.00 ppm. Maternal tissue levels were 4 to 5 times higher than fetal concentrations,
indicating that the placental barrier retards the distribution of chlordecone to the fetus.
Chlordecone levels in the fetus were highest in the liver, followed by the brain, heart, and
kidneys. Chlordecone excretion into milk was an important pathway for elimination in nursing
dams. Neonatal organ concentrations of chlordecone increased steadily over the lactation period.
Tissue uptake for neonates was highest in the liver, followed by the brain and the eyes. Day 5
liver and brain levels rose from 2 to 23 jig and 16 to 150 jig, respectively, in pups nursed by
10 mg/kg-day dosed dams. Tissue concentrations were correlated with chlordecone levels in
milk.
The tissue distribution of chlordecone was investigated in rats following pretreatment
with phenobarbital, an inducer of hepatic metabolism (Aldous et al., 1983). Repeat doses of
phenobarbital (65 mg/kg) were administered intraperitoneally to adult male Sprague-Dawley rats
JANUARY 2008 9 DRAFT - DO NOT CITE OR QUOTE
-------
6, 12, and 24 hours prior to gavage administration of C[14]-labeled chlordecone. Phenobarbital
pretreatment resulted in an increase in the specific activity in the liver and uniformly reduced the
specific activity in other tissues. In phenobarbital pretreated rats, 87% of the C[14]-labeled
chlordecone was found in the liver, compared to 55% in control rats not receiving phenobarbital.
Fecal and urinary excretion of chlordecone was reduced. A single dose of phenobarbital (12 or
24 hours prior to chlordecone administration) similarly altered the distribution of chlordecone;
however, changes were more marked with multiple dose administration.
3.3. METABOLISM
Based on available data, a proposed metabolic scheme for chlordecone is shown in Figure
3-1. Although chlordecone is not extensively metabolized in mammals, chlordecone alcohol is
formed in humans and some laboratory animal species by reduction of the hydrated carbonyl
group (Fariss et al., 1980; Blanke et al., 1978). A cytosolic aldo-keto reductase enzyme appears
to be responsible for the formation of chlordecone alcohol (Molowa et al., 1986). Chlordecone
alcohol is excreted in bile primarily as a glucuronide conjugate, while chlordecone is excreted
into bile mostly in the unconjugated form (Fariss et al., 1980).
The metabolism of chlordecone to chlordecone alcohol occurs in humans, gerbils, and
pigs but not to a significant extent in rats, mice, guinea pigs, or hamsters (Houston et al., 1981;
Fariss et al., 1980; Blanke et al., 1978). Species differences were also observed in phase II
conjugation reactions, with chlordecone conjugation occurring in humans but not in gerbils or
rats (Houston et al., 1981). In humans, a reduced form of chlordecone was first identified in the
stool of pesticide workers experiencing symptoms of chlordecone toxicity, including
nervousness, headache, and tremor (Blanke et al., 1978). Fariss et al. (1980) utilized human bile
samples for further analysis of chlordecone and possible metabolites. Human bile was obtained
from exposed workers by either aspirated duodenal contents (six workers) or bile collected
directly from a T-tube that was implanted during gallbladder surgery (one worker). The initial
analysis of human bile using gas-liquid chromatography revealed significant amounts of free
chlordecone and small amounts of free chlordecone alcohol in exposed workers. Subsequent
treatment of bile samples with P-glucuronidase prior to the analysis resulted in large amounts of
measurable chlordecone alcohol. It was estimated that >90% of the chlordecone alcohol in
human bile is present as a glucuronide conjugate, while <10% of the chlordecone parent
compound is conjugated prior to biliary excretion. The ratio of chlordecone to chlordecone
alcohol following P-glucuronidase, sulfatase, and acid hydrolysis treatments was between 1:2
and 1:4 in human bile. In contrast, rat bile contained only trace amounts of chlordecone alcohol,
with a corresponding chlordecone to chlordecone alcohol ratio of 155:1.
JANUARY 2008 10 DRAFT - DO NOT CITE OR QUOTE
-------
CHLORDECONE
CHLORDECONE
HYDRATE
CHLORDECONE
ALCOHOL
UDP
a-D-glucuronic acid
UDP
a-D-glucuronic acid
CHLORDECONE
GLUCURONIDE
(mono ordi)?
CHLORDECONE
ALCOHOL
GLUCURONIDE
Figure 3-1. A proposed metabolic scheme for chlordecone.
Molowa et al. (1986) characterized a unique cytosolic aldo-keto reductase enzyme
responsible for the conversion of chlordecone to chlordecone alcohol. Chlordecone reductase
activity was detected in the liver cytosol of rabbits, gerbils, and humans but was absent in rats,
mice, hamsters, and guinea pigs. Pretreatment of gerbils with a single oral dose of chlordecone
(20 mg/kg) resulted in a 38% increase in the specific activity of chlordecone reductase 7 days
later. Soine et al. (1983) also demonstrated the metabolism of chlordecone to chlordecone
alcohol in the pig. Pigs were given an intraperitoneal dose of either 40 or 80 mg/kg-day, and
chlordecone and chlordecone alcohol concentrations in the blood and gallbladder bile were
measured at regular intervals over a 35-day study period. At the end of the study, hepatic bile,
liver, and feces were also analyzed for chlordecone and chlordecone alcohol levels. The plasma
half-life of chlordecone in the pig was determined to be 12 days at the higher dose and 22 days at
the lower dose. Chlordecone metabolites were generally not detected in the plasma; however,
JANUARY 2008
11
DRAFT - DO NOT CITE OR QUOTE
-------
free chlordecone, free chlordecone alcohol, and conjugated chlordecone alcohol were measured
in gallbladder bile at both doses. Conjugated chlordecone was only observed in gallbladder bile
at the high-dose level. The induction of chlordecone reductase in the pig was suggested by the
observed increase in the chlordecone alcohol to chlordecone ratio in the gallbladder bile over the
time course of the study. On the last day of the study, 20% of chlordecone was conjugated in the
plasma and bile, while only 3% of chlordecone was conjugated in the liver and feces.
Chlordecone alcohol was not detected in the plasma or the liver but was 85% conjugated in the
bile and 15% conjugated in the feces.
Chlordecone has been shown to induce the cytochrome (CYP) 450 (CYP450) mixed
function oxidase enzyme system in male and female rats (Gilroy et al., 1994; Hewitt et al., 1985;
Mehendale et al., 1978, 1977). Mehendale et al. (1978, 1977) exposed male and female rats to 0,
50, 100, or 150 ppm chlordecone in the diet for 16 days. A dose-related decrease in body weight
gain was observed, while liver weights were unaltered by chlordecone treatment. Enzyme
activities that were increased by chlordecone treatment at each dose level include aniline,
pentobarbital and hexobarbital hydroxylation, and aminopyrine and ethylmorphine
demethylation. CYP450, cytochrome c reductase, and aniline binding were all increased, while
cytochrome b5 and NADPH dehydrogenase activity were unaffected by chlordecone treatment.
Hewitt et al. (1985) demonstrated increases in microsomal CYP450 and NADPH cytochrome c
reductase following a single oral dose of 50 mg/kg (days 2 to 32). Cytochrome b5 was also
increased, but not until 24 to 32 days after chlordecone administration. A single oral dose of 15
mg/kg to Sprague-Dawley rats resulted in an increase in CYP450 and ethoxyresorufin-O-
deethylase and ethoxycoumarin-O-deethylase enzyme activities (Gilroy et al., 1994). Weanling
pups of Sprague-Dawley rat dams exposed to chlordecone from day 2 of gestation to day 21
postpartum (0, 0.1, 1, or 1.5 mg/kg-day) exhibited a dose-related increase in metabolism and
excretion of lindane (Chadwick et al., 1979).
Chlordecone was shown to selectively induce CYP2B2 in adult rat hepatocyte cultures
(Kocarek et al., 1991). Chlordecone selectively increased the mRNA for CYP2B2, and both
chlordecone and chlordecone alcohol induced the immunoreactive protein levels for CYP2B2.
Chlordecone did not affect the mRNA or immunoreactive protein levels for CYP2B1 in isolated
rat hepatocytes. In addition to its selective induction of CYP2B2, chlordecone also suppressed
the induction of CYP2B1 and CYP2B2 when coincubated with phenobarbital in hepatocyte
culture. Mechanistic studies suggest that selective induction of CYP2B2 is not due to the
estrogenic properties of chlordecone, while the ability to suppress phenobarbital induction may
relate to the gem-diol configuration of chlordecone (Kocarek et al., 1994).
3.4. ELIMINATION
Chlordecone and chlordecone alcohol are eliminated from the body primarily through
biliary excretion into feces. In humans, chlordecone is eliminated slowly from the blood.
JANUARY 2008 12 DRAFT - DO NOT CITE OR QUOTE
-------
Estimates of the chlordecone serum half-life (ti/2) in chemical plant workers ranged from 63 to
128 days (Adir et al., 1978). Analysis of excretory fluids in exposed pesticide workers showed
that, while chlordecone was undetectable in sweat and present only in minor quantities in urine,
saliva, and gastric juice, concentrations in gallbladder bile were approximately equivalent to
chlordecone concentrations in blood (Cohn et al., 1978). The excretion rate of chlordecone into
hepatic bile was estimated from either aspirated duodenal contents (six workers) or bile collected
directly from a T-tube that was implanted during gallbladder surgery (one worker) (Cohn et al.,
1978). The biliary excretion rates varied widely among workers (~1 to 10 mg/day); however, the
daily excretion amount expressed as a percent of the total body content was relatively constant
(0.29 to 0.85%). For workers who underwent duodenal aspiration, only 5 to 10% of the
chlordecone that entered the duodenal lumen via the bile was detected in the feces. Similarly,
the rate of chlordecone excreted in bile collected from a surgically implanted T-tube was 19
times greater than the rate of elimination in the stool. These results suggest that enterohepatic
recycling plays an important role in the slow excretion of chlordecone. In order to prevent the
reabsorption of chlordecone into the gastrointestinal tract, cholestyramine was investigated as a
possible treatment for chlordecone intoxication. Cholestyramine is an anion-exchange resin that
binds chlordecone but is not absorbed in the gastrointestinal tract. Treatment with
cholestyramine reduced the average ti/2 in the blood of workers from 165 days to 80 days (Cohn
etal., 1978).
Gastrointestinal secretion of chlordecone also appears to play a role in fecal excretion in
humans (Boylan et al., 1979). Diversion of the bile stream from the intestine was accomplished
in a chlordecone-exposed worker with a surgically implanted T-tube. Chlordecone excretion in
stool increased eightfold when bile was diverted from the gut. This nonbiliary mechanism for
fecal excretion does not appear to be related to salivary or gastric juice, because chlordecone
concentrations in these fluids were minimal in exposed workers. Chlordecone is apparently
transferred from the bloodstream to gastrointestinal lumen via a secretory process governed by
diffusion (Bungay et al., 1979). High concentrations of chlordecone in the lumen inhibit
gastrointestinal secretion. Experimental data in rats confirmed the presence of a nonbiliary
pathway for fecal excretion of chlordecone. Bungay et al. (1979) evaluated the transport of
chlordecone in and out of the gut and utilized a PBTK model to describe the results (see Section
3.5). The transport of chlordecone into and out of the gut was studied following intravenous
administration to the bile duct of cannulated rats and oral administration to intact rats. The
primary route of elimination of chlordecone is in the feces.
Animal studies evaluated the elimination of chlordecone following oral exposure. Egle et
al. (1978) studied chlordecone excretion in male Sprague-Dawley rats receiving a single oral
dose of 40 mg/kg-day C[14]-labeled chlordecone in corn oil solution. The percentage of
radioactivity excreted in the feces was measured over time. Approximately 30% of the
administered chlordecone was excreted within the first 7 days, after which the rate of excretion
JANUARY 2008 13 DRAFT - DO NOT CITE OR QUOTE
-------
steadily declined. After 12 weeks, 65.5% of the dose had been excreted into the feces and after
26 weeks, the cumulative excretion in feces was only 69.8%. A small amount of the
administered chlordecone was excreted in the urine. Only 1.6% of the administered dose was
found in the urine by 12 weeks, one-third of which was excreted into urine in the first 24 hours.
Chlordecone was measured in expired air on days 1 and 9 after dosing, and less than 1% of the
administered dose was detected in expired air.
Heatherington et al. (1998) studied the excretion of chlordecone following dermal
absorption in young and adult rats (see Section 3.1 for study methods). Higher concentrations of
chlordecone were detected in the urine of young rats as compared with adults. Chlordecone
elimination was primarily in the feces, with limited urinary excretion. Feces to urine ratios 120
hours following dermal application of chlordecone were 3:1 and 3:8 in young and adult rats,
respectively.
Chlordecone treatment has been shown to decrease the biliary excretion of other
chemicals (Curtis and Mehendale, 1979). Male Sprague-Dawley rats were fed diets containing
0, 10, 50, or 150 ppm chlordecone for 15 days. Food consumption and body weight data were
used to estimate daily dose levels of 0, 0.69, 3.2, and 8.0 mg/kg-day. Clinical signs of
chlordecone toxicity were not apparent in the 10 or 50 ppm groups, but hyperexcitability and
tremors were observed at 150 ppm. Decreased body weight gain was observed at the two highest
dose levels. Biliary function was evaluated in bile-duct-cannulated intact animal preparations.
The highest dose of chlordecone reduced the biliary excretion of the polar metabolites of
imipramine (31% of control) and phenolphthalein glucuronide (27% of control). These
decreases occurred despite an increase in cumulative bile flow at the 150 ppm dose level.
Oligomycin-sensitive mitochondrial ATPase activity was inhibited by chlordecone in this study;
however, the dose-response data do not suggest a direct correlation between enzyme inhibition
and hepatobiliary dysfunction. Chlordecone was also shown to inhibit oligomycin-sensitive
Mg2+-ATPase activity in the rat bile canaliculi-enriched fraction of the liver; however, it is not
known whether this inhibition represents a causal factor in hepatobiliary dysfunction or simply
an indication of membrane perturbation (Curtis, 1988).
Teo and Vore (1991) studied the effect of chlordecone on bile acid secretory function
(i.e., bile flow, bile acid concentration, bile acid secretory rate) in the isolated perfused rat liver.
Rats were given an oral dose of 18.75 mg/kg-day chlordecone for 3 days prior to measurement of
bile secretory parameters. Chlordecone treatment resulted in an increase in bile flow while
decreasing bile acid concentration and bile acid secretory rate. These results suggest that
chlordecone acts primarily at the bile canalicular membrane to decrease biliary excretion.
Rochelle et al. (1990) demonstrated that chlordecone perturbs the membrane and inhibits the
active transport of glutamate at the bile canalicular membrane. Hepatobiliary dysfunction does
not appear to be related to the concentration of chlordecone associated with the liver plasma
membrane (Rochelle and Curtis, 1994); however, inhibition and recovery of 5'-nucleotidase
JANUARY 2008 14 DRAFT - DO NOT CITE OR QUOTE
-------
activity in the liver plasma membrane suggest that biochemical alterations in membrane function
may be involved.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
Physiologically-based toxicokinetic (PBTK) models have been used to describe the
hepatic sequestration of chlordecone (Belfiore et al., 2007), movement of chlordecone in and out
of the gut (Bungay et al., 1979), percutaneous absorption and disposition of chlordecone
(Heatherington et al., 1998), and toxic interactions between chlordecone and carbon tetrachloride
(el-Masri et al., 1995) in laboratory animals. PBTK models are not available to describe
toxicokinetic processes in humans.
Belfiore et al. (2007) developed a PBTK model to describe sequestration of chlordecone
in the liver of rats. Male Sprague-Dawley rats received a one-time treatment of 40 mg/kg-day of
chlordecone in corn oil by gavage. Rats were sacrificed at 1, 14, or 30 days following dosing,
and liver, fat, kidney, and muscle specimens were removed and assayed for chlordecone
concentration. Data from this time course and from distribution studies in the literature (Hewitt
et al., 1985 Egle et al., 1978) were used to develop and validate a toxicokinetic model to describe
the preferential sequestration of chlordecone in the liver. A model was constructed in which
liver, fat, and slowly perfused and rapidly perfused tissues were flow limited. Metabolism was
not included due to the low biotransformation rate for chlordecone. The model fit to the
experimental data was greatly improved by adding blood and liver binding coefficients derived
from data from Soine et al. (1984, 1982). This model provides additional support for the hepatic
sequestration of chlordecone in Sprague-Dawley rats; however, several factors limit its use in the
derivation of reference values. It is not known how the measured blood, fat, or liver tissue levels
would correlate other organ compartments not included in the model. This model also does not
provide information on inhalation exposure that would be needed for route-to-route extrapolation
and thus cannot be used for the derivation of an RfC. Additionally, the model is not
parameterized for humans, so it cannot be used to evaluate interspecies toxicokinetic differences.
Bungay et al. (1979) conducted experiments comparing intravenous administration of
chlordecone in bile-duct-cannulated rats and oral administration in intact rats. The data were
used in the gut portion of a whole body PBTK model. The gastrointestinal tract was divided into
six segments, and the lumens of these segments were connected in series in the model. Flow
rates were measured in each segment, and the net secretion or absorption was determined for
each compartment. Diffusional processes were assumed to govern chlordecone exchange
between blood, gut tissue, and the lumen. In the rat, the PBTK model yields a maximum
clearance estimate for gut secretion of 25 mL/hour. Measurement of biliary clearance in bile-
duct-cannulated rats was 5 mL/hour, suggesting a total maximum clearance rate of 30 mL/hour.
Assuming that the permeability of the gut to chlordecone is similar in rats and humans, a
maximum human clearance rate of 1000 mL/hour was calculated by the authors by using a body-
JANUARY 2008 15 DRAFT - DO NOT CITE OR QUOTE
-------
weight scaling factor (body-weight ratio raised to the 2/3 power). The chlordecone clearance
rate estimated for pesticide workers not receiving cholestyramine treatment (Cohn et al., 1978)
was only 40 mL/hour due to the presence of chlordecone in the lumens and the inhibition of
diffusion from the gut.
A PBTK model was developed to describe the percutaneous absorption and disposition of
chlordecone in young and adult rats (Heatherington et al., 1998). The experimental data for the
dose effect and time course of chlordecone dermal absorption are described in Section 3.1. The
distribution and excretion data for this study are reported in Sections 3.2 and 3.3. A
biophysically based percutaneous absorption model was developed based on in vivo dermal
absorption data. The absorption model consisted of five first-order rate constants describing the
movement of chlordecone by diffusion from the site of application to the stratum corneum,
where it undergoes partitioning with the viable epidermis, followed by entry into the blood and
distribution throughout the body. The rate constants for movement among compartments were
based on chlordecone physical and chemical characteristics, skin physiology, and experimental
data. The absorption model was significantly limited by its inability to describe the nonlinear
dose effect of percutaneous exposure (i.e., decreasing percent absorption with increasing dose).
Therefore, the data for only one dose level could be used for PBTK disposition modeling (i.e.,
time course data for 0.285 |imol/cm2). The absorption model was embedded in the whole body
PBTK model to describe the distribution and excretion of chlordecone in young and adult rats.
The distribution of chlordecone from blood to various tissue compartments was described. The
PBTK model took into account chlordecone binding to albumin and lipoproteins in the blood,
preferential uptake by the liver, and the predominant fecal excretion pathway for chlordecone.
Once optimized using the experimental data for chlordecone, the PBTK model was used to
predict partition coefficients and excretion rates. Tissue concentrations at varying dose levels
were reasonably well estimated if the nonlinear dermal absorption at high doses and the
nonlinear uptake of bound chlordecone into the liver were considered.
el-Masri et al. (1995) utilized PBTK and toxicodynamic (TD) modeling to evaluate the
toxic interaction between chlordecone and carbon tetrachloride. Chlordecone significantly
potentiates the hepatotoxicity and lethality of carbon tetrachloride by interfering with the
regeneration process in the liver (see Section 4.4.2). A PBTK model for carbon tetrachloride
was adapted and verified using experimental data. The PBTK model was then linked with a TD
model based on the mechanistic data for the interaction between chlordecone and carbon
tetrachloride in liver cells. The combined model yielded a time course simulation of mitotic,
injured, and pyknotic cells following treatment with carbon tetrachloride alone or in combination
with chlordecone. The PBTK/TD model was coupled with Monte Carlo simulation techniques to
predict the acute lethality of carbon tetrachloride under various exposure conditions. Predictions
of lethality were in agreement with experimentally derived values except at very high doses
where neurotoxicity led to significant mortality.
JANUARY 2008 16 DRAFT - DO NOT CITE OR QUOTE
-------
4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
Information regarding the health effects of chlordecone in humans comes from studies of
a single group of 133 men exposed occupationally to chlordecone at a facility in Hopewell,
Virginia (Taylor, 1985, 1982; Guzelian, 1982a; Guzelian et al., 1980; Sanborn et al., 1979;
Cannon et al., 1978; Martinez et al., 1978; Taylor et al., 1978). Of the 133 men, 76 experienced
neurological symptoms, especially nervousness, headaches, and tremors, sometimes persisting as
long as 9-10 months after cessation of exposure and the start of treatment (Cannon et al., 1978).
In addition, some of the men experienced oligospermia. Sperm count and motility had returned
to normal by 5 to 7 years following the cessation of chlordecone exposure and treatment with
cholestyramine to reduce chlordecone blood levels (Taylor, 1982). Some workers exposed to
high levels of chlordecone developed skin rashes, enlarged livers, and joint pain. Liver
enlargement developed in 20 out of 32 workers with high blood levels of chlordecone (>0.6
ug/mL) after an average duration period of 5-6 months, although evidence of significant liver
toxicity was not found (Guzelian, 1982a; Guzelian et al., 1980; Taylor et al., 1978). Normal
results were obtained in all patients for serum bilirubin, albumin, globulin, prothrombin time,
cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and y-glutamyl
transpeptidase (GGT), and serum alkaline phosphatase was only minimally elevated in seven
patients. Sulfobromophthalein retention, a measure of liver clearance, was normal in a subset of
18 workers tested (Guzelian et al., 1980). Liver biopsy samples taken from 12 workers with
hepatomegaly showed histological changes in the liver that were characterized as nonadverse in
nature. These included proliferation of the smooth endoplasmic reticulum (SER) and
cytoplasmic accumulation of lipofuscin. No evidence of liver neoplasia, fibrosis, cholestasis, or
hepatocellular necrosis was found. Neurological symptoms were reported in workers exposed to
high doses of chlordecone for a period of months to years (Taylor, 1985, 1982; Guzelian, 1982a;
Guzelian et al., 1980; Sanborn et al., 1979; Cannon et al., 1978; Martinez et al., 1978; Taylor et
al., 1978). These symptoms included tremor, headache, irritability, poor recent memory, rapid
random eye movements, muscle weakness, gait ataxia, incoordination, and slurred speech. The
effects persisted for as long as 9-10 months after cessation of exposure and the start of treatment
(Cannon et al., 1978). Martinez et al. (1978) reported that nerve conduction velocity tests,
electroencephalography, radioisotope brain scans, computerized tomography, and analyses of
cerebral spinal fluid content from these workers were all normal. Sural nerve and skeletal
muscle biopsies in workers with detectable neurological impairment exhibited a reduction in the
number of unmyelinated axons and a disruption in Schwann cell metabolism (Martinez et al.,
1978).
JANUARY 2008 17 DRAFT - DO NOT CITE OR QUOTE
-------
The factory did not follow good industrial hygiene practices. Substantial inhalation,
dermal, and oral exposures could have occurred to the workers (Guzelian, 1982a; Guzelian et al.,
1980; Cannon et al., 1978). Because of uncertainties regarding exposure routes and levels at the
facility and concomitant exposure to the precursors used to manufacture chlordecone, no-
observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs)
could not be established for the adverse effects observed on the nervous systems, livers, and
reproductive systems of these men. Liver biopsy samples taken from 12 workers with
hepatomegaly resulting from intermediate- or chronic-duration exposures to high levels of
chlordecone showed no evidence of significant liver toxicity or cancer (Guzelian et al., 1980);
however, conclusions from this study are limited by the very small number of workers sampled,
uncertainties concerning exposure dose and route, the relatively brief duration of exposures, and
the absence of a sufficient latency period for tumor development. The average exposure of the
subjects was 5-6 months, and they were examined immediately after exposure (Cannon et al.,
1978). A review of biological and epidemiological evidence of cancer found no population-
based studies on cancer in humans related to chlordecone exposure (Ahlborg et al., 1995). These
case reports of occupationally exposed workers at the pesticide plant (who were repeatedly
exposed to high but unmeasured levels for less-than-lifetime durations) indicate that primary
target organs for chlordecone toxicity in humans are the nervous system, reproductive organs,
skin, and liver.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
Animal studies show effects similar to those reported in occupationally exposed humans:
neurological effects, oligospermia, hepatomegaly, and skin rashes, as well as kidney lesions that
were not reported in humans. Chlordecone is moderately lethal by single exposures; oral median
lethal dose (LDso) values range from 71 mg/kg body weight for rabbits to 250 mg/kg body
weight for dogs (Larson et al., 1979a). The oral LD50 value for rats is 125 mg/kg body weight
(Gaines, 1969). In experimental animals, the effects of chlordecone following short-term
exposures generally include nervous system effects (tremor and hyperexcitability), liver
hypertrophy (with induction of mixed-function oxidases), and structural and ultrastructural
changes in the liver, thyroid, adrenal glands, and testes (ATSDR, 1995; U.S. EPA, 1986c; WHO,
1984). In subchronic studies with experimental animals, chlordecone produced tremors and
other neurological symptoms, liver hypertrophy with induction of mixed function oxidases,
hepatobiliary dysfunction, and centrilobular hepatocellular necrosis. Chlordecone also interferes
with reproductive processes in both males (oligospermia) and females (disruption of estrous
cyclicity), and it is fetotoxic in experimental animals. Chronic testing of chlordecone in
laboratory animals is limited to two studies: NCI (1976a) and Larson et al. (1979a). In a dietary
cancer bioassay with chlordecone, NCI (1976a) found a statistically significant increase in the
JANUARY 2008 18 DRAFT - DO NOT CITE OR QUOTE
-------
incidence of and a reduction in the time to detection of hepatic tumors among male (marginal)
and female Osborne-Mendel rats and male and female B6C3F1 mice. The Larson et al. (1979a)
study also reported hepatic proliferative lesions, but the determination of whether these
represented tumors was equivocal. No data are available concerning the toxicity of chlordecone
in animals following inhalation exposure. Studies demonstrating adverse effects in experimental
animals following oral exposures are presented below. No studies were available for inhalation
or dermal routes of exposure.
4.2.1. Subchronic Studies
4.2.1.1. Oral Exposure Studies
Huang et al. (1980) administered chlordecone by gavage to adult male ICR mice (15 per
dose group) at 0 (corn oil vehicle), 10, 25, or 50 mg/kg-day. Animals were gavaged daily for up
to 24 days. Tremor and hyperexcitability were observed in all mice receiving chlordecone; time
to onset was dose dependent. Loss of body weight (accompanied by reduced food and water
consumption) was also apparent in chlordecone-exposed animals, with the greatest loss of body
weight coincident with the onset of tremor. The authors speculated that the reduction in body
weight among treated mice was due to paralysis and loss of motor control, which resulted in a
decreased ability to eat. Upon termination of chlordecone administration, a diminution of tremor
and corresponding recovery of body weight were observed in surviving animals. Chlordecone-
treated mice showed a high degree of mortality. Time to onset of first death and slope of the
mortality curves (cumulative mortality per day) were dose dependent. Mortality among mice
exposed to 50 mg/kg-day began on day 4, and all were dead by day 6. For the 25 mg/kg-day
group, mortality began on day 6 and reached 100% by day 11. For the 10 mg/kg-day group,
mortality began on day 12 and reached nearly 90% by day 24 of treatment. The control group
had no deaths. The cumulative oral LDso was estimated by the authors to be between 180 and
200 mg/kg. Due to the high incidence of mortality among even the lowest dose group, these
doses are considered frank effect levels and cannot be used to establish effect levels for
quantitative risk assessment. In a follow-up study, Fujimori et al. (1982b) administered
chlordecone by gavage to male ICR rats at 0 (corn oil vehicle), 10, 25, or 50 mg/kg-day for 9
consecutive days. The results of this study also demonstrated a dose response in the time to
onset of chlordecone impairment of motor coordination (days 2, 4, and 9 for 50, 25, and 10
mg/kg-day dose groups). The authors examined dopamine and serotonin levels and speculated
that the dopaminergic pathway may be involved (though not necessarily the sole player) in
mediating chlordecone-induced tremor and neurotoxicity.
4.2.1.2. Inhalation Exposure Studies
No inhalation exposure studies were found in the literature.
JANUARY 2008 19 DRAFT - DO NOT CITE OR QUOTE
-------
4.2.2. Chronic Studies
4.2.2.1. Oral Exposure Studies
Chu et al. (1981a) fed male Sprague-Dawley rats (10 per group) diets containing 0 or 1
ppm of chlordecone (0 or 0.07 mg/kg-day reported by the authors) for 21 months. Corn oil was
used to dissolve the chemicals, and control diets contained 4% corn oil. Survival and weight
gain were similar in treated and control rats. Hematology and clinical chemistry were also
unaffected by treatment. Histopathological findings included apparent increases in the incidence
of lesions in the liver (5/6 [83%] vs. 3/7 [43%] in controls) and thyroid (4/6 [67%] vs. 1/7 [14%]
in controls). The differences in incidences were not statistically significant (Fisher's exact test
conducted for this review), although the power of the statistical test to detect a difference at such
small sample sizes is low. The lesion in the liver was described as pericentral cytoplasmic
vacuolation with mild anisokaryosis, while the thyroid lesion was described as a mild
degenerative and proliferative change in the epithelium. Severity of both lesions was reported to
be increased in chlordecone-treated rats in comparison with controls, although the nature and
extent of these differences were not described. This study was a follow-up to an earlier short-
term (28 day) study with the same exposure protocol (Chu et al., 1980). Twenty-eight days of
oral exposure to chlordecone produced no statistically significant alteration in hepatic
microsomal or serum enzyme activities. The 28-day study found chlordecone-induced lesions in
the liver (3/10 exhibited multiple lymphoid aggregates, perivenous cytoplasmic ballooning, and
perinuclear halos in the portal area) and kidney (2/10 showed eosinophilic inclusions in proximal
tubules) but not the thyroid. Interpretation of these findings is difficult due to the small number
of rats used, use of a single exposure dose, and occurrence of the reported effects among control
rats.
Osborne-Mendel rats and B6C3F1 mice were exposed to technical-grade chlordecone in
the diet for 80 weeks (NCI, 1976a,b). The test material was reported to contain no more than 2%
impurities other than water. Chlordecone was added to finely ground rat chow in acetone (to aid
uniform dispersion of the chemical); the diets were mixed for homogeneity and to allow the
acetone to evaporate. Corn oil (2%) was added to the diet as a dust suppressant. Dietary
concentrations of chlordecone began at 0, 15, 30, or 60 ppm for male rats and 0, 30, or 60 ppm
for female rats. Treatment groups comprised 50 rats per sex; however, only 10 animals per sex
were used in matched control groups. Pooled control groups (from the same laboratory with
birth dates within 3-4 months of the animals in the matched control and exposed groups)
contained 105 male rats and 100 female rats. Overt clinical signs of toxicity observed in the
treated animals indicated that the initial doses exceeded the maximum tolerated dose in the high
exposure groups; consequently, concentrations of chlordecone in feed were reduced (to one-third
to one-sixth of the original concentration) during the experiment (after durations ranging from as
JANUARY 2008 20 DRAFT - DO NOT CITE OR QUOTE
-------
short as 42 days in high-dose female rats to as long as 386 days in high-dose male rats). The
specific dosing regimens for male and female rats are illustrated in Figures 4-1 and 4-2.
Male Rats
A High Dose
0 Mid Dose
Q Low Dose
Days on
Study
*Lines represent changes in the dose levels made throughout the study period. The undulating line for the mid
dose from day 485 until the end of the study represents a recovery period of a week between doses for the last
75 days of the study. Additionally, the low dose group was added in the middle of the study period.
Figure 4-1. Dosing regimen for male rats in the study by NCI (1976b).
Female Rats
60 -.^..
55 _
50 -
45 -
40 -
30 -
25 -
20 -
15 -
10 -
5 -
A High Dose
I I Low Dose
Days on 0 42
Study
*Lines represent changes in the dose levels made throughout the study period.
Figure 4-2. Dosing regimen for female rats in the study by NCI (1976b).
JANUARY 2008
21
DRAFT - DO NOT CITE OR QUOTE
-------
The initial group of high-dose male rats was discontinued due to excess toxicity;
however, nine rats were transferred to the lower dose group in the study. A new dose group of
male rats was started 8 months after the beginning of the study. Time-weighted-average dietary
concentrations were reported by the authors (and confirmed for this review) to be 0, 8, or 24 ppm
for male rats and 0, 18, or 26 ppm for female rats. Doses estimated from U.S. EPA (1988)
reference values for body weight and food consumption were calculated1: 0, 0.6, or 1.7 mg/kg-
day for male rats and 0, 1.4, or 2.0 mg/kg-day for female rats. Following the 80-week exposure,
surviving rats were sacrificed at 112 weeks. The following tissues were taken from sacrificed
animals, and those dying early, for histological examination: brain, pituitary, lymph nodes,
thyroid, parathyroid, salivary glands, lung, heart, diaphragm, stomach, duodenum, jejunum or
ileum, large intestine, pancreas, adrenal glands, kidney, liver, skin, gonads, bladder, prostate or
uterus, and femur with marrow.
Clinical signs of chlordecone toxicity, including tremor and dermatological changes,
were indicated in the NCI (1976a) report, although incidence by dose was not reported (NCI,
1976a,b). Survival was reduced for high-dose male and female rats (NCI, 1976a). Percentages
of male rats surviving to study termination (112 weeks) were 63% for pooled controls, 90% for
matched controls, 60% for the low-dose group, and 42% for the high-dose group; for female rats,
the respective percentages were 61%, 70%, 56%, and 40%. The decreases in survival occurred
primarily during the second year of the study, although some early mortality was observed
among high-dose male rats (four animals in the first 4 months). Many of the treated rats also
showed decreases in food consumption and body weight gain (NCI, 1976b). In male rats, body
weight gain at 79 weeks was 82% and 79% of control for the low- and high-dose groups,
respectively. Body weight gain in female rats at 79 weeks was 76% and 66% of control for the
low- and high-dose groups, respectively. Some high-dose males were observed to have bleeding
of the eyes and nose during the first 4 months of the study, and, by week 5 of the study, most
high-dose females showed generalized tremors. By week 28, many low-dose females were also
experiencing tremors. The incidence of tremors and other clinical signs (rough hair coat,
dermatitis, anemia) was low to moderate during the remainder of the first year but gradually
increased during the second year of the study. The authors reported that rats surviving to study
termination were generally in very poor physical condition, though more specific data regarding
occurrence of clinical signs were not reported.
In rats, the incidence of noncancer lesions was reported in summary tables included in the
microfiche for the bioassay (NTP, 1976b). These tables showed chronic kidney inflammation in
low-dose male rats and high-dose female rats but did not confirm the presence of extensive
noncancer liver lesions in male or female rats. Liver tumors described as hepatocellular
Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food consumption rates
of 0.036 kg/day (males) and 0.030 kg/day (females) and reference body weights of 0.514 kg (males) and 0.389 kg
(females) were used (U.S. EPA, 1988).
JANUARY 2008 22 DRAFT - DO NOT CITE OR QUOTE
-------
carcinomas were observed in high-dose female rats at an incidence that was significantly
elevated compared with the pooled control incidence (0/100, 0/10, 1/49, and 10/45 in the pooled
control, matched control, low-dose, and high-dose groups, respectively). Incidences of male rats
with hepatocellular carcinomas were 0/105, 0/10, 1/50, and 3/44, respectively. The incidence of
carcinomas in high-dose males was significant (p = 0.049) in comparison with pooled controls.
The incidence of hepatocellular carcinomas was not statistically significant in comparison with
matched controls for rats of either sex. A significant dose-response trend was observed for the
incidence of hepatocellular carcinoma in both male and female rats (Cochran-Armitage test
conducted for this review). Hepatocellular carcinomas were described as large, poorly
circumscribed masses that were well differentiated without vascular invasion or metastases.
Liver tumors described as neoplastic nodules were also found but not at elevated incidences in
exposed groups compared with control groups. Neoplastic nodule incidences were reported to be
0/10, 2/50, and 0/44 in the matched control, low-dose, and high-dose male groups and 1/10, 0/49,
and 2/45 in female rats. The incidence and time to tumor data for hepatocellular carcinoma in
rats in the NCI (1976a) report are summarized in Table 4-1.
Table 4-1. Incidence and time to tumor of hepatocellular carcinoma in rats
Osborne-Mendel rats
Male (0, 0.6, or 1.7 mg/kg-day)a
Time to first tumor (weeks)
Female (0, 1.4, or 2.0 mg/kg-day)a
Time to first tumor (weeks)
Exposure group
Matched
control
0/10
NAd
0/10
NA
Pooled
control
0/105
NA
0/100
NA
Low dose
1/50
112 weeks
1/49
87 weeks
High dose
3/44b'c
108 weeks
10/45C
83 weeks
aDoses were calculated for this review using the allometric equation for food consumption by laboratory animals
with time-weighted concentrations from NCI (1976a) and reference body weights from U.S. EPA (1988).
bMarginal increase (p = 0.049) compared with pooled controls.
Statistically significant increase in incidence as compared with pooled controls, using one-tail (p < 0.05) Fisher's
exact test for 2 x 2 contingency table (NCI, 1976a).
dNA = not available.
Source: NCI(1976a).
In addition to the liver, the rats developed tumors in other organs of the endocrine system
(NCI, 1976a). Table 4-2 shows the incidence of these tumors by organ and tumor type. The
incidence rate for all tumor types combined for each of these systems (endocrine or reproductive)
was not statistically increased as compared with controls (Fisher's exact test conducted for this
review). Individual tumor types were also not significantly increased, and no dose-response
trend was observed (Cochran-Armitage test conducted for this review).
JANUARY 2008
23
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-2. Summary of endocrine and reproductive system tumor incidence
among rats exposed to chlordecone
Number of rats
Number of rats with any type of
tumor3
Endocrine Organs
Reproductive Organs
1 Reproductive Organs
Pituitary chromophobe
adenoma
Pituitary adenocarcinoma
Thyroid follicular-cell
carcinoma
Thyroid follicular-cell
adenoma
Thyroid C-cell adenoma
Thyroid C-cell carcinoma
Parathyroid adenoma
Pancreatic islet cell
adenoma
Adrenal cortical adenoma
Mammary gland
fibroadenoma
Mammary gland adenoma
Mammary gland fibroma
Mammary gland
adenocarcinoma
Mammary gland
fibrolipoma
Uterus endometrial/
stromal polyp
Uterus malignant
lymphoma
Uterus squamous cell
carcinoma
Ovary arrhenoblastoma
Ovary granulosa-cell
tumor
Cervix uteri squamous cell
carcinoma
Males
Control
10
3 (30%)
2 (20%)
-
-
-
-
-
-
-
-
-
-
1 (10%)
-
-
-
—
—
-
—
-
0.6
mg/kg-day
50
24 (48%)
12 (24%)
-
3 (6%)
2 (4%)
3 (6%)
1 (2%)
-
1 (2%)
1 (2%)
1 (2%)
-
-
-
-
-
—
—
-
—
-
1.7
mg/kg-day
44
16 (36%)
5(11%)
1 (2%)
-
-
-
-
1 (2%)
1 (2%)
-
1 (2%)
1 (2%)
-
-
-
-
—
—
-
—
-
Females
Control
10
7 (70%)
3 (30%)
-
-
-
-
-
-
-
-
4 (40%)
2 (20%)
-
-
-
-
—
—
-
—
-
1.4
mg/kg-day
49
29 (59%)
13 (26%)
-
-
1 (2%)
2 (4%)
-
-
1 (2%)
-
4 (8%)
1 (2%)
-
2 (4%)
-
3 (6%)
1 (2%)
—
1 (2%)
—
1 (2%)
2.0
mg/kg-day
45
31(69%)
4 (9%)
-
1 (2%)
-
1 (2%)
1 (2%)
-
-
2 (4%)
1 (2%)
-
-
-
1 (2%)
1 (2%)
—
—
-
1 (2%)
-
"Some animals had multiple tumors.
Source: NCI (1976a).
JANUARY 2008
24
DRAFT - DO NOT CITE OR QUOTE
-------
In mice, dietary concentrations of chlordecone began at 0 or 40 ppm (two groups at this
concentration) for males and 0, 40, or 80 ppm for females. Treatment groups comprised 50 mice
per sex; however, only 10 female mice and 19 male mice were used as matched controls. Pooled
control groups (from the same laboratory with birth dates within 3-4 months of the animals in
the matched control and exposed groups) contained 49 male mice and 40 female mice. Overt
clinical signs of toxicity observed in the high-dose male and female mice indicated that the
maximum tolerated dose was exceeded in those exposure groups; consequently, concentrations
of chlordecone in feed for all dose groups were reduced (to one-fourth to one-half of the original
concentration) during the experiment. The specific dosing regimen for male and female mice
was described in the microfiche for the bioassay (NCI, 1976b) and is illustrated in Figures 4-3
and 4-4. The initial high-dose group of male mice was discontinued due to excess toxicity, and a
new group was started 7 months later after the beginning of the study. Time-weighted-average
dietary concentrations were reported by the authors (and confirmed for this review) to be 0, 20,
or 23 ppm for male mice and 0, 20, or 40 ppm for female mice. Doses estimated from U.S. EPA
(1988) reference values for body weight and food consumption were calculated2: 0, 3.4, or 3.9
mg/kg-day for male mice and 0, 3.5, or 7.0 mg/kg-day for female mice. Following the 80-week
exposure, surviving mice were sacrificed at 90 weeks. Histological examination was similar to
that described previously for rats.
Survival was reduced for male mice at both the high and low dose; though survival rates
in female mice at both dose levels were comparable with those of controls (NCI, 1976a). The
percentages of male mice surviving to study termination at 90 weeks were 92% for pooled
controls, 90% for matched controls, 58% for the low-dose group, and 50% for the high-dose
group. The percentages of survival for female mice were 85% for pooled controls, 90% for
matched controls, 84% for the low-dose group, and 84% for the high-dose group. The decreases
in survival occurred primarily during the second year of the study, although some early mortality
was observed. Decreases in food consumption and body weight gain were less pronounced in
mice as compared to rats (NCI, 1976b). In male mice, body weight gain at 81 weeks was 93%
and 88% of control for the low- and high-dose groups, respectively. Body weight gain in female
mice at 81 weeks was 94% and 88% of control for the low- and high-dose groups, respectively.
A comparison of survival rates and body weight gain for animals in the NCI study is presented in
Table 4-4.
2
Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food consumption rates
of 0.0064 kg/day (males) and 0.0061 kg/day (females) and reference body weights of 0.0373 (males) and 0.0353 kg
(females) were used (U.S. EPA, 1988)
JANUARY 2008 25 DRAFT - DO NOT CITE OR QUOTE
-------
80 -
70 -
60 -
50 -
40 -
30 _
20 -
10 _
Davs on
Study
Male Mice
A
A High Dose
9 New High Dose
Low Dose
D
0 134 230 295 320
*Lines represent changes in the dose levels made throughout the study period.
Figure 4-3. Dosing regimen for male mice in the study by NCI (1976a,b).
END
Female Mice
so -
70 -
60 -
50 -
40 -
30 -
20 -
10 -
D
A High Dose
Q] Low Dose
„
°
*Lines represent changes in the dose levels made throughout the study period.
Figure 4-4. Dosing regimen for female mice in the study by NCI (1976a,b).
END
JANUARY 2008
26
DRAFT - DO NOT CITE OR QUOTE
-------
Clinical signs of chlordecone toxicity were reported in mice; however, the incidence by
dose was not reported (NCI, 1976a,b). High-dose female mice developed tremors during the
first week of the study that persisted to study termination. Tremors were also observed in some
high-dose male mice, and about 20% of high-dose males were highly excitable during the second
year of the study. Abdominal distention was first observed in high-dose males at week 45 and
high-dose females at week 68, presumably associated with hepatic hypertrophy. Palpable
abdominal masses were found in high- and low-dose males during the second year of the study.
Alopecia, rough hair coats, and tail sores were seen primarily in males and were thought to be
due to fighting. More specific data regarding occurrence of clinical signs were not reported.
In mice, statistically significant elevated incidences of hepatocellular carcinomas were
found in both exposed groups compared with matched and pooled control incidences (NCI,
1976a). Incidences for matched control, low-, and high-dose groups were 6/19, 39/48, and 43/49
for male mice and 0/10, 26/50, and 23/49 for female mice. The incidence in control male mice
was reported as abnormally high. Two of the pooled control male mice had hepatocellular
carcinomas. Combining the matched and pooled control male mouse groups resulted in an
overall incidence of 8/49 for control male mice. Hepatocellular carcinomas in mice were
described as varying from demarcated nodules to large masses that were well differentiated
without vascular invasion or metastases. Extensive liver hyperplasia also was found in both
sexes in both low- and high-dose mouse groups. Incidences for liver hyperplasia were not
specified, but the report noted that "a few matched controls of each sex also had liver hyperplasia
although the incidence was quite low as compared to the treated groups." No tumors of other
endocrine organs were reported, aside from one ovary cystadenoma in a single high-dose female
(1/49 or 2% incidence rate). No elevated incidences of tumors at other tissue sites were found in
exposed mice compared with controls. The incidence and time-to-tumor data for hepatocellular
carcinoma in the NCI (1976a) report are summarized in Table 4-3. No exposure-related
noncancer lesions were mentioned other than the liver atypia and nodular and diffuse hyperplasia
(NCI, 1976a,b). Induction of noncancerous liver lesions (i.e., hyperplasia) was observed at all
dose levels for each sex and species. Thus, freestanding LOAELs identified for this study are
0.6, 1.4, 3.4, and 3.5 mg/kg-day for male rats, female rats, male mice, and female mice,
respectively.
JANUARY 2008 27 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-3. Incidence and time to tumor of hepatocellular carcinoma in mice
Mouse/B6C3Fl
Male (0, 3.4, or 3.9 mg/kg-day)a
Time to first tumor (weeks)
Female (0, 3.5, or 7.0 mg/kg-day)a
Time to first tumor (weeks)
Exposure group
Matched
control
6/19(31%)
87 weeks
0/10 (0%)
NAC
Pooled
control
8/49 (16%)
87 weeks
0/40 (0%)
NA
Low-dose
group
39/48b(81%)
70 weeks
26/50b (52%)
87 weeks
High-dose
group
43/49b (88%)
62 weeks
23/49b (47%)
76 weeks
aDoses were calculated for this review using the allometric equation for food consumption by laboratory animals
with time-weighted concentrations from NCI (1976a) and reference body weights from U.S. EPA (1988).
bStatistically significant increase in incidence as compared to matched or pooled controls, using one-tail (p < 0.05)
Fisher's exact test for 2 x 2 contingency table (NCI, 1976a).
°NA = not available.
Source: NCI(1976a).
The NCI (1976a) study provides evidence of carcinogenicity in Osborne-Mendel rats and
B6C3F1 mice; however, decreases in survival rates and decreased body weight gain indicate that
excessively high doses were utilized in all animal groups except the low- and high-dose female
mice (see Table 4-4).
Table 4-4. Percent body weight gain and percent survival of chlordecone-
exposed rats and mice
Male rats
Female rats
Male mice
Female mice
Time-weighted-average
daily dose (mg/kg-day)
0 (room controls)
0 (matched controls)
0.6
1.7
0 (room controls)
0 (matched controls)
1.4
2.0
0 (room controls)
0 (matched controls)
3.4
3.9
0 (room controls)
0 (matched controls)
3.5
7.0
Survival
(%)
63
90
60
42
61
70
56
40
92
90
58
50
85
90
84
84
Body weight
gain (%)
82
79
76
66
93
88
94
88
Liver tumor
incidence (%)
0/105
0/10
1/50 (2)
3/44 (7)a
0/100
0/10
1/49 (2)
10/45 (22)a
8/49 (16)
6/19 (31)
39/48 (81)a
43/49 (88)a
0/40
0/10
26/50 (52)a
23/49 (47)a
Time to 1st
tumor (weeks)
NAb
NA
112
108
NA
NA
87
83
87
87
70
62
NA
NA
87
76
""Statistically significant increase in incidence as compared with matched per pooled controls, using one-tail Fisher's
exact test (p < 0.05).
bNA = not available.
Source: NCI(1976a).
JANUARY 2008
28
DRAFT - DO NOT CITE OR QUOTE
-------
In another chronic study, groups of 40 male and 40 female Wistar rats were fed diets
containing 0, 5, 10, 25, 50, or 80 ppm of chlordecone for up to 2 years (Larson et al., 1979a).
Larson et al. (1979a) added chlordecone to warmed corn oil before combining it with the food.
From food consumption and body weight data graphically presented in Larson et al. (1979a) for
5-8 time points measured throughout the study, time-weighted-average food consumption rates
were estimated for the 5 through 80 ppm groups as 49, 53, 59, 73, and 80 g food/kg body weight-
day for males and 56, 55, 69, 83, and 93 g food/kg body weight-day for females. Using average
food consumption rates and averaged body weights (between males and females), doses were
estimated to be 0, 0.3, 0.5, 1.6, 3.9, and 7.0 mg/kg-day. In a separate phase of the experiment,
groups of 40 males and 40 females were exposed to 0 or 1 ppm for up to 2 years. Because food
consumption data were not reported for the 1 ppm group, an estimated dose of 0.06 mg/kg-day
was calculated by assuming food consumption equal to the 5 ppm group. Groups of five rats per
sex per dose were sacrificed at 3 and 12 months. Another three to five rats per sex per group
were sacrificed after 12 months of exposure and a 4-week recovery period. Remaining rats were
sacrificed at 24 months. From samples collected at 3-month intervals, hematocrit, hemoglobin,
and total and differential white cell counts were measured in blood, and reducing substances and
protein were measured in urine. Additional blood studies were performed at 3 months for
platelet count, prothrombin clotting time, and serum calcium. Oxygen consumption was
measured by spirometry at 9 months. Organ-to-body-weight ratios (liver, kidneys, heart, spleen,
and testes) were determined in sacrificed rats. The following tissues were taken from sacrificed
rats for histopathological study: brain, spinal cord, heart, lung, liver, kidney, spleen, gut, urinary
bladder, bone marrow, skeletal muscle, skin, pancreas, thyroid, adrenal, pituitary, and gonad.
Tremors developed in the 3.9 and 7.0 mg/kg-day groups within a few weeks of the start
of the study and became progressively more severe with time (Larson et al., 1979a). Slight
tremors were noted in some rats at 1.6 mg/kg-day after 3 months, becoming moderate in severity
after 5-6 months, but then regressing. Tremors were not observed at 0.5 mg/kg-day or below.
The incidence of tremors was not reported. All rats in the 3.9 and 7.0 mg/kg-day groups died
during the first 6 months. Long-term survival was reduced in the 1.6 mg/kg-day females
(measured at 1 and 2 years, data not shown). Body weights were depressed after 3 weeks of
study in males at >1.6 mg/kg-day and in females at >0.3 mg/kg-day. Food consumption (per
body weight) tended to increase with concentration of chlordecone in the feed. Metabolic rate
(measured by oxygen consumption) increased with dose in both males and females, although
statistical significance was achieved only in males at 1.6 mg/kg-day (the highest dose with
survivors remaining when tested at 9 months). Hematology analyses revealed no differences
related to treatment. Apparent increases in urinary protein concentrations or proteinuria (a
clinical indicator of glomerular dysfunction) were reported in both male and female rats exposed
to >0.3 mg/kg-day for 6-24 months, though statistical analysis was not performed on these data
because of incomplete data reporting. Proteinuria was not observed in rats exposed to 0.06
JANUARY 2008 29 DRAFT - DO NOT CITE OR QUOTE
-------
mg/kg-day in a separate phase of the experiment (the time of analysis and other details were not
reported). Relative liver weight increased with dose at 3, 12, and 24 months in both male and
female rats. The difference from controls was statistically significant at >1.6 mg/kg-day in males
and >0.5 mg/kg-day in females. Relative testes weights were significantly decreased in the 3.9
and 7.0 mg/kg-day groups at the 3-month sacrifice. Relative weight changes in the kidneys and
other organs were not remarkable. Absolute organ weights were not reported.
Histopathological examination of five rats (randomly selected) from each sex at each
feeding level at 13 weeks revealed minimal congestion of the liver at 0.5 mg/kg-day and more
degenerative changes in the liver at higher doses (Larson et al., 1979a). There was a trend in
dose response for degenerative liver changes. Swollen liver cells were noted in 4/5 males and
5/5 females in the 3.9 mg/kg-day group and 5/5 males and 3/5 females in the 7.0 mg/kg-day
group (compared with 0/10 males and 0/10 females in the control groups [both male and female
rat studies combined]). The liver-to-body-weight ratios were significantly increased in the 3.9
and 7.0 mg/kg-day groups for both sexes. Histological examination also uncovered a dose-
related increase in the incidence and severity of testicular atrophy at 13 weeks, though not at 1-2
years. The study authors did not speculate as to why testicular atrophy was observed after 13
weeks, but not at the chronic time point. Interim (3-month) gross and histopathologic
examinations performed on 10 control males and 5 chlordecone-treated males per group revealed
statistically significantly increased incidences of chlordecone-induced testicular atrophy (Table
4-5). The atrophy was described as minimal in the control male and generally increased in
severity with increasing chlordecone concentration. Also, the testes-to-body-weight ratios in
males were significantly decreased in the 3.9 and 7.0 mg/kg-day groups. The study identified a
NOAEL of 0.5 mg/kg-day and a LOAEL of 1.6 mg/kg-day for testicular atrophy in male rats
exposed to chlordecone in the diet for 13 weeks.
Table 4-5. Testicular atrophy in male rats receiving chlordecone in the diet
for 3 months
Dietary level (ppm)
Average dose3 (mg/kg-day)
Incidence of testicular atrophyb
0
0
1/10
5
0.3
0/5
10
0.5
1/5
25
1.6
4/5c
50
3.9
4/5c
80
7.0
5/5c
"Average dose to rats, based on graphically depicted food consumption data presented by the authors.
bStatistically significant dose-response trend according to the Cochran-Armitage trend test (p < 0.01) performed for
this review.
Statistically significantly different from controls according to Fisher's exact test (p < 0.05) performed for this
review.
Source: Larson et al. (1979a).
JANUARY 2008
30
DRAFT - DO NOT CITE OR QUOTE
-------
At the 12-month sacrifice, congestion of the liver was reported for treated groups, but
details were not reported. No treatment-related lesions were observed after 12 months of
treatment and a 4-week recovery period.
Histopathological examination of rats sacrificed after 2 years and rats that died during the
second year showed exposure-related lesions only in the liver and kidney (Larson et al., 1979a).
Incidence data for liver and kidney effects are presented in Table 4-6. The principal renal lesion
was glomerulosclerosis, or scarring of the system of capillaries that comprise the glomeruli. The
increased incidence of glomerulosclerosis was statistically significant (Fisher's exact test
performed for this review) in the 0.3, 0.5, and 1.6 mg/kg-day females compared with controls.
The background incidence of glomerulosclerosis in male rats was high (56% as compared to
12% in female rats) and, as such, male rat incidence data for glomerulosclerosis did not achieve
statistical significance. Incidences of liver lesions (predominately fatty changes and hyperplasia)
in male and female rats were also statistically increased by chlordecone administration. The
hepatic lesions in three females in the 0.5 mg/kg-day group and one female and two males in the
1.6 mg/kg-day group were described by the authors as being possibly "carcinomatous in nature";
however, the authors reported that an independent review by four pathologists found the
evidence for carcinogenic responses in this study to be equivocal. Thus, this study provides
equivocal evidence of chlordecone carcinogenicity in Wistar rats.
Table 4-6. Incidence of histopathologic liver lesions (fatty changes and
hyperplasia) and renal glomerulosclerosis in male and female Wistar rats
following administration of chlordecone in the diet for 1-2 years
Endpoinf
Liver lesions'3
Male rats
Female rats
Glomerulosclerosisb
Male rats
Female rats
Dose (mg/kg-day)
0
1/22
2/34
12/22
4/34
0.06
1/11
1/13
3/11
2/13
0.3
2/6
2/17
4/6
8/17c
0.5
2/9
4/12c
6/9
8/12c
1.6
3/4c
1/4
3/4
3/4c
aThe number of animals reported relates to the number of animals analyzed between 1 and 2 years. Due to interim
measurements, the approximate number of animals per sex per dose group after 12 months is 25.
bThe dose-response trend was also statistically significant for each data set according to the Cochran-Armitage trend
test performed for this review.
Statistically different from control groups according to Fisher's exact test (p < 0.05) performed for this review.
Source: Larson et al. (1979a).
This study identified 5 ppm (0.3 mg/kg-day) as a LOAEL and 1 ppm (0.06 mg/kg-day) as
a NOAEL for kidney effects (proteinuria and increased incidence of glomerulosclerosis) in
female rats. Also observed were increased incidences of hepatic lesions; these increases were
statistically significant (Fisher's exact test performed for this review) starting at 1.6 mg/kg-day in
JANUARY 2008
11
DRAFT - DO NOT CITE OR QUOTE
-------
males and at 0.5 mg/kg-day in females. Higher doses (3.9 and 7.0 mg/kg-day) produced overt
clinical signs (tremors) and mortality in the rats.
Larson et al. (1979a) also conducted a long-term study in dogs. Groups of two male and
two female purebred beagle dogs were fed diets containing 0, 1,5, or 25 ppm of chlordecone for
up to 128 weeks, beginning at an age of about 6 months. Although not specified, Larson et al.
(1979a) likely added chlordecone to warmed corn oil before combining it with the food (the
protocol used in the rat study in the same report). Two dogs in the 25 ppm group were sacrificed
at the end of week 124; the remaining dogs were sacrificed during week 128. Organ-to-body
weights were determined, and 17 tissues were taken for histopathological examination: brain,
spinal cord, heart, lung, liver, kidney, spleen, gut, urinary bladder, bone marrow, skeletal muscle,
skin, pancreas, thyroid, adrenal, pituitary, and gonad. The same hematological and urine
endpoints as those described for the rat studies were determined in samples collected before
exposure and at 3-month intervals during exposure. Using reference body weights and food
consumption rates of 10.5 kg and 0.2 kg dry food/day, respectively, for beagle dogs (U.S. EPA,
1988), doses were estimated to be 0, 0.02, 0.1, and 0.5 mg/kg-day (the authors did not report
food consumption data, body weight data, or estimated dose levels for the dogs). Three dogs
died during the study, showing severe dermatitis that did not appear to be related to exposure
(one control dog during week 71, one 0.02 mg/kg-day dog during week 48, and one 0.1 mg/kg-
day dog during week 50). Body weight gain in the 0.5 mg/kg-day group was reported to be
lower than the weight gain in the control dogs during the second year of exposure, but the
magnitude of the decrease was not reported and the data were not shown. Decreased food
efficiency (kg body weight gain/kg food consumed) was suggested by measurements of food
consumption, but again the data were not shown. The only statistically significant changes
associated with exposure to chlordecone were a moderate (37%) increase in relative liver weight
in dogs from the 0.5 mg/kg-day group (males and females combined) and slight changes (less
than about 25%) in relative kidney (increase), heart (increase), and spleen (decrease) weight in
the same group. Absolute organ weights were not reported. No exposure-related changes were
reported for clinical signs of toxicity; hematological, histopathological, or urinalysis endpoints;
sulfobromophthalein retention; or serum cholinesterase. Interpretation of this study is limited by
the small number of dogs tested, the deaths of three dogs during the study for reasons not
apparently related to treatment, and the reporting of results, including failure of the researchers to
present data to support the reported decrease in body weight in dogs from the 0.5 mg/kg-day
group during the second year of the study. Nevertheless, the statistically significant changes in
organ-to-body-weight ratios support occurrence of an adverse effect on body weight, and the
increase in relative liver weight is consistent with other studies demonstrating hepatic toxicity
with chlordecone exposure. Therefore, upon review, the results of this study suggest a LOAEL
of 25 ppm (0.5 mg/kg-day) and aNOAEL of 5 ppm (0.1 mg/kg-day), based on decreased body
JANUARY 2008 32 DRAFT - DO NOT CITE OR QUOTE
-------
weight and organ-to-body-weight changes (without histological changes) in beagle dogs fed
chlordecone in the diet for up to 128 weeks.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES
4.3.1. Reproductive Toxicity Studies
Information on reproductive effects in humans is restricted to findings of oligospermia,
reduced sperm motility, and decreased libido in a group of men who were occupationally
exposed to chlordecone for periods up to 1.5 years (Taylor, 1985, 1982; Guzelian, 1982a; Taylor
et al., 1978). Sperm concentration and motility had returned to normal upon follow-up 5 years
following cessation of chlordecone exposure. Even though two of seven workers sired children,
there is no indication of the true denominator of how many were trying to conceive and/or the
fertility rate. In one worker, low sperm count persisted (Taylor, 1985). No information is
available concerning chlordecone-induced reproductive effects in women.
Reproductive toxicity has been assessed in some animal studies but not in adequately
designed multiple generation studies. Available animal data suggest that chlordecone is a male
reproductive toxicant, causing alteration of sperm parameters at low doses and testicular atrophy
at higher doses. Persistent vaginal estrus is reported to occur in exposed females and decreased
reproductive success has been demonstrated. No animal studies are available to assess the
developmental or reproductive toxicity of chlordecone by the inhalation route of exposure.
Huber (1965) performed a series of experiments designed to assess reproductive toxicity
in mice exposed to chlordecone in the diet. In a pilot reproduction test (group A), 3-month-old
male and female mice of mixed parentage (eight pairs per group) were administered chlordecone
(technical-grade chlordecone, 93.6% purity) in the diet at concentrations of 0, 10, 30, or 37.5
ppm for 1 month prior to mating and during the 100 days following individual pairing within
each exposure group. Corresponding chlordecone doses of 0, 1.9, 5.6, and 7.0 mg/kg-day were
estimated for males and females combined by using reference values for food consumption and
body weight from U.S. EPA (1988). The 100-day treatment period allowed sufficient time for
mating pairs to produce two litters. Individual males were housed with individual females
except during the period of gestation and weaning of offspring. Reproductive parameters
assessed included number of pairs producing first and second litters, average number of young
per litter, percent survival of offspring, and the average time required to produce the offspring
(expressed as pair days per litter [number of pairs * 100 days/number of litters produced] and
pair days per offspring [number of pairs x 100 days/number of offspring). Vaginal smears were
taken daily for 3-4 weeks for analysis of the estrous cycle following the termination of the
reproduction phase. Smears were taken in one group after their reproduction test and in another
group prior to mating.
In the chlordecone-treated groups, the number of pairs producing first and second litters,
the average number of young per litter, and the percent survival of offspring appeared to be
JANUARY 2008 33 DRAFT - DO NOT CITE OR QUOTE
-------
lower compared with controls. The average time required to produce offspring during the
treatment period was greater in chlordecone-treated pairs than controls. However, except for the
quantal data presented for pairs producing litters, the data presented for the continuous
parameters (average number of offspring, pair days per litter, and percent survival of offspring)
did not include a measure of the variance and thus were not adequate for statistical analysis,
though visual evaluation of the data appear to indicate a clear reduction in reproductive success
at doses greater than 5.6 mg/kg-day. Statistical analysis of the number of pairs producing second
litters (Fisher's exact test performed for this review) revealed a significant reduction in the 5.6
and 7.0 mg/kg-day exposure groups relative to controls (Table 4-7).
In another phase (group B) of the study, 4-month-old BALB/cJaxGnMc mice (14 pairs
per group) were administered chlordecone in the diet at concentrations of 0 or 40 ppm for 2
months before mating and during a 100-day reproduction period (Huber, 1965). Otherwise, the
study design was the same as that used for group A. The corresponding chlordecone dose was
7.6 mg/kg-day (estimated for males and females combined, using reference values for food
consumption and body weight from U.S. EPA [1988]). Following the termination of treatment, a
second reproduction phase was performed for 100 days and consisted of crossover matings
(control males with control females, control females with chlordecone-treated males, and
chlordecone-treated females with control males).
Table 4-7. Effects of dietary chlordecone on reproduction in male and
female mice (of mixed parentage) treated for 1 month prior to mating and
for 100 days following the initiation of mating
Dietary level
(ppm)
0.0
10.0
30.0
37.5
Average
dose3
(mg/kg-day)
0.0
1.9
5.6
7.0
Pairs
producing
first litter
7/8
6/8
4/8
3/8
Pairs
producing
second
litter
5/8
4/8
0/8b
0/8b
Average
number
offspring
per litter
7.7
7.1
4.7
4.0
Percent
survival of
offspring
89
87
26
42
Pair days
per litter
67
80
200
267
Pair days
per
offspring
8.7
11.3
42.1
66.7
aAverage doses to male and female mice (combined), based on reference values for subchronic body weight and
food consumption taken from U.S. EPA (1988).
bStatistically different from control groups according to Fisher's exact test (p < 0.05), performed for this review.
Source: Huber (1965).
The results are summarized in Table 4-8. During the initial reproduction period, each of
the control (0 ppm) pairs produced two litters. No offspring were produced by the pairs of mice
treated with 7.6 mg/kg-day of chlordecone. The ability to produce offspring was restored during
the posttreatment reproduction period. Results of crossover matings indicate that female mice
were slightly more affected by chlordecone than males; however, information concerning the
statistical significance of the findings was not provided by the author.
JANUARY 2008 34 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-8. Effects of dietary chlordecone (0 or 40 ppm) on reproduction in
BALB/cJaxGnMc mice during 100 days of treatment (preceded by 2 months
of pre-mating treatment) and during 100 days of a crossover-mating period
following the termination of treatment
Pairs with first litter
Pairs with second litter
Offspring per litter
Offspring survival (%)
Pair days per litter
Pair days per offspring
Reproduction period during
chlordecone treatment
Controls
14/14
14/14
7.1
89
50
7
Treated pairs
0/14
0/14
Crossover reproduction period following
termination of chlordecone treatment
Controls
5/5
4/5
7.2
87.3
55.6
7.6
Control male
X
treated
female
8/10
5/10
4.5
76.1
76.9
17.3
Control
female
X
treated male
10/10
6/10
5.6
88.3
62.5
11.5
Source: Huber (1965).
Huber (1965) also assessed the effect of chlordecone on estrous cyclicity in virgin female
mice (20 per group) given either 0 or 40 ppm of chlordecone in the diet for 120 days. After 21
and 120 days of treatment, daily vaginal smears were taken for 3 to 4 weeks. In the 40 ppm
females, persistent estrus appeared within 8 weeks of treatment initiation. Seventy-one percent
of the smears taken in the 40 ppm females for 4 weeks after termination of chlordecone treatment
were in estrus versus only 24% in controls. Huber (1965) also noted persistent estrus in 30 and
37.5 ppm female mice from group A following the reproduction test and 40 ppm female mice
from group B prior to mating. The occurrence of persistent estrus is an indication that the treated
female mice were under a prolonged stimulation of follicular stimulating hormone (FSH) and
estrogen with insufficient luteinizing hormone stimulation. The 30 ppm treatment level
represents a LOAEL for this effect.
In summary, the multiple dose reproduction test (Huber, 1965), in which male and female
mice were given chlordecone in the diet for 1 month prior to mating and for 100 days following
the initiation of mating, resulted in adverse reproductive effects. The 1.9 mg/kg-day dose
represents a NOAEL and the 5.6 mg/kg-day dose represents a LOAEL (as determined for this
review), based on a statistically significantly reduced number of mouse pairs producing a second
litter.
Male and female laboratory mice (7-16 pairs per group) of mixed breeds were
administered chlordecone (purity unspecified) in the diet at concentrations of 0, 10, 17.5, 25, 30,
or 37.5 ppm for 1 month and then were sex paired within the same exposure grouping and placed
on a normal diet throughout mating and production of offspring (Good et al., 1965).
Corresponding chlordecone doses of 0, 1.9, 3.3, 4.7, 5.6, or 7.0 mg/kg-day were estimated for
males and females combined by using reference values for food consumption and body weight
JANUARY 2008
35
DRAFT - DO NOT CITE OR QUOTE
-------
from U.S. EPA (1988). Reproductive indices (number of litters produced, average number of
young per litter, pair days per litter, and pair days per young produced) were assessed for
approximately 5 months following the initiation of mating. As shown in Table 4-9, the results
suggest a dose-related effect on reproductive success (decreases in number of litters and average
number of young per litter, increases in pair days per litter and per young). Though the data
presented in the study were not adequate for statistical analysis (no measures of variance were
provided for the reproductive parameters), visual evaluation of the data appears to indicate a
clear reduction in reproductive success at doses >5.6 mg/kg-day.
Table 4-9. Effects of dietary chlordecone for 1 month prior to mating on
reproductive indices of male and female laboratory mice of mixed breeds
Dietary level
(ppm)
0
10
17.5
25
30
37.5
Average
dose"
(mg/kg-day)
0.0
1.9
3.3
4.7
5.6
7.0
Number of
pairs
9
13
16
11
7
10
Number of
litters
15
26
25
12
2
2
Number of
offspring per
litter
7.93
7.62
7.0
6.08
3.0
5.0
Pair days per
litter
65.3
54.46
72.16
100.42
241.5
555.0
Pair days per
offspring
8.3
7.15
13.09
16.51
80.5
111.0
"Average doses to male and female mice (combined), based on reference values for subchronic body weight and
food consumption taken from U.S. EPA (1988).
Source: Good etal. (1965).
In separate experiments by Good et al. (1965), impaired reproductive success, expressed
as significantly (p < 0.05) reduced production of a second litter, was observed in mice that were
administered chlordecone (purity unspecified) in the diet at a concentration of 5 ppm for 1 month
prior to mating and for up to 5 months following initiation of mating (shown in Table 4-10). The
corresponding chlordecone dose of 0.94 mg/kg-day was estimated for males and females
combined by using reference values for food consumption and body weight from U.S. EPA
(1988). The authors reported that continued treatment of offspring of chlordecone-treated mice
with either control or 5 ppm chlordecone diets resulted in significantly reduced production of a
first litter (p < 0.05), compared with untreated offspring of untreated parental mice, though
reduced production of the second litter did not achieve statistical significance. The results of
these studies identified a LOAEL of 0.94 mg/kg-day for impaired reproductive success; a
NOAEL was not identified.
JANUARY 2008
36
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-10. Effects of dietary chlordecone (0 or 5 ppm) 1 month prior to
mating and 5 months during mating on reproduction in BALB/c mice
Number of pairs
Number of litters
Number of offspring
% producing 1st litter
% producing 2nd litter
First litter size
Second litter size
Pair days per litter
Pair days per offspring
First generation
Control
24
40
275
96
78
6.2
7.3
70.1
10.2
Treated
36
52
314
81
50a
6.2
5.7
86
14.2
Second generation
Control
21
21
123
71
29
5.6
6.5
120
21
Offspring of
treated mice
on control
diet
23
9
42
30a
9
4.3
6.0
307
66
Treated
20
10
40
25a
15
4.4
5.3
240
60
"Reported as significant atp < 0.05, using binomial distribution.
Source: Good etal. (1965).
As in the previous data reported by Good et al. (1965), reproductive parameters,
including litter size, pair days/litter, and pair days/young produced, were all reported as averages
for the treatment or control group without any measure of variance given (i.e., standard
deviation). Therefore the degree of variability for the reported reproductive parameters is
unclear. Additionally, there appears to be reduced fertility of the BALB/c untreated controls just
one generation apart. For instance, 96 and 78% of untreated control animals produced first and
second litters, respectively, whereas only 71 and 29% of their untreated progeny produced first
and second litters. These inconsistencies limit confidence in this study and the reproducibility of
the data.
In a reproductive and neurodevelopmental toxicity study, female F344 rats (10 per group)
were fed diets containing 0, 1, or 6 ppm of chlordecone (purity unspecified) for 60 days prior to
mating (with nonexposed male rats) through lactation day 12 (Squibb and Tilson, 1982).
Corresponding doses of 0, 0.07, and 0.4 mg/kg-day were estimated by using reference values for
food consumption from U.S. EPA (1988) and the average of reported body weights of the dams
prior to mating and on the day after parturition. Chlordecone treatment did not produce adverse
effects on litter size or sex ratio of the offspring. Litters were culled to three male and three
female offspring per dam on postpartum day 3. Pup body weights were similar to those of
controls at 1, 7, 14, and 30 days of age, but after 100 days body weight was significantly reduced
in male pups at 6 ppm (19% decrease relative to controls) and female pups at 1 ppm (27%
decrease relative to controls) and 6 ppm (27% decrease relative to controls). Pups were exposed
to higher concentrations of chlordecone exposure during the first 2 weeks of life (i.e., during
lactation), without any significant effects on body weight. A pharmacokinetic elimination study
JANUARY 2008
37
DRAFT - DO NOT CITE OR QUOTE
-------
in rats (Egle et al., 1978) demonstrated that 65.5% of an orally administered dose of chlordecone
had been excreted into the feces by 12 weeks. The chlordecone body burden must therefore be
assumed to be much lower at 100 days, when compared with earlier time points. No clear dose-
response relationship was demonstrated in this study for decreased pup body weight; thus, the
significance of the body weight reductions at 100 days postpartum is uncertain.
One male and one female pup from each litter were chosen at random for behavioral and
pharmacological challenge testing (10 males and 10 females from each dose group) (Squibb and
Tilson, 1982). The results of behavioral testing, conducted at 30 and 100 days, were primarily
negative. Exposed offspring showed no statistically significant changes (compared with
controls) in forelimb or hind-limb grip strength, spontaneous motor activity, startle
responsiveness (air puff or acoustic stimulus), or tail-flick frequency in response to thermal
stimulation. Positive results were found for one test in male offspring exposed to 6 ppm that
took significantly longer time to reorient themselves to a vertical position in an assay for
negative geotaxis at 100 days of age. The effect was not seen at 30 days in males and was not
seen at either time point in female offspring.
The results of pharmacological challenge tests were mixed (Squibb and Tilson, 1982).
Motor activity induced by subcutaneously injected 1 mg/kg apomorphine (a dopamine receptor
agonist) at 114 days of age was significantly increased in male offspring of the 6 ppm group 30
minutes after dosing and male offspring of the 1 and 6 ppm groups 60 minutes after dosing. This
effect was not seen in females. There was no effect on motor activity induced by d-amphetamine
(a presynaptic releaser of both dopamine and norepinephrine) at 134 days in either male or
female offspring. This study found little evidence of an effect of chlordecone on
neurodevelopment in rats. The weight of evidence of behavioral tests was negative, and the one
positive finding (increased negative geotaxis latency in males) was not supported by results in
females. Similarly, the positive result in the challenge test with apomorphine in males was not
supported by the results in females. Further, in the absence of any clear neurological or
behavioral response, it is uncertain that a potential alteration in dopaminergic function associated
with chlordecone exposure should be considered adverse.
Adult Sherman strain male and female rats (22-25 rats/sex/group) were fed diets
containing 0 or 25 ppm commercial grade chlordecone (80.6% purity) for 3 months, during
which time they were housed individually and observed for clinical signs of neurotoxicity
(Cannon and Kimbrough, 1979). At the end of the treatment period, selected control and
chlordecone-treated male and female rats were subjected to gross and histopathologic
examinations. The remaining rats 20/sex/group were pair mated, control males with
chlordecone-treated females, control females with chlordecone-treated males, and control
females with control males, during a breeding period of approximately 2 months. The
production of offspring was used as an indicator of reproductive toxicity.
JANUARY 2008 38 DRAFT - DO NOT CITE OR QUOTE
-------
According to the study authors, chlordecone intake ranged from 1.62 to 1.71 mg/kg-day
in 25 ppm females and from 1.17 to 1.58 mg/kg-day in 25 ppm males. Body tremors were seen
in chlordecone-treated rats after 4 weeks of treatment and appeared to be most marked in treated
females. At the end of the exposure period, chlordecone-treated male and female rats exhibited
depressed body weight, and gross and microscopic signs of adverse hepatic effects. The adrenals
showed hyperplasia of the zona fasciculata and zona reticularis with marked hypertrophy of the
cortex. The study authors noted gross and histopathologic signs of adverse adrenal effects in
treated females. Twelve of the 20 pairs of control females and chlordecone-treated males
produced offspring compared with 13 of 20 pairs in the controls. However, no offspring were
produced among the 20 pairs of control males and chlordecone-treated females. Mating of
chlordecone-treated females and control males was repeated 9 weeks after exposure cessation.
Reproductive function appeared to be partially restored with 9 of 20 pairs producing litters,
indicating some reversibility of the observed reproductive deficit in chlordecone-treated females.
This study identified a LOAEL of 1.6-1.7 mg/kg-day for impaired reproductive success in
female rats.
Groups of sexually mature virgin female CD-I mice were administered chlordecone by
gavage (in sesame oil) at doses of 0, 0.062, 0.125, or 0.25 mg/day (0, 2, 4, or 8 mg/kg-day), 5
days per week for 2, 4, or 6 weeks (Swartz et al., 1988). A positive control group received
17|3-estradiol at a dose of 0.1 mg/day. Some mice from each group were assessed for production
of oocytes (intraperitoneal administration of pregnant mare's serum gonadotropin followed 48
hours later by human chorionic gonadotropin) during the second, fourth, and sixth week of
chlordecone treatment. As shown in Table 4-11, persistent vaginal estrus was noted in most
chlordecone-treated mice and positive controls as early as 2 weeks following the initiation of
treatment. By week 4, all chlordecone-treated mice exhibited persistent vaginal estrus versus 0/9
vehicle controls.
After 4 and 6 weeks of treatment, ovulation in the highest chlordecone treatment group
(8 mg/kg-day) resulted in statistically significantly lower numbers of ovulated oocytes relative to
vehicle controls. This study identified a LOAEL of 2 mg/kg-day for persistent vaginal estrus in
virgin female CD-I mice.
JANUARY 2008 39 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-11. Effects of chlordecone on estrous cyclicity and ovulation in CD-I
mice exposed to chlordecone by gavage 5 days per week for up to 6 weeks
Test
PVEa'b
Week 2
WeekS
Week 4
Ovulation0
Week 2
Week 4
Week 6
Vehicle controls
0/9
0/9
0/9
19.9 ± 2.4 (15)d
28.4 ± 2.9 (22)
23.7 ±2.4 (16)
Positive
controls
(17p-estradiol)
7/8
8/8
7/8
30.2 ±11. 8 (6)
29.7 ±2.2 (11)
22.1 ±2.5 (9)
Chlordecone dose (mg/kg-day)
2
6/8
6/8
8/8
26.7 ±3.2 (10)
22.9 ±4.3 (7)
32.4 ±3. 8 (7)
4
5/9
7/9
9/9
19.2 ±3.2 (10)
27.1 ±5.0 (6)
21.0 ±5. 8 (7)
8
8/9
9/9
9/9
17.7 ±4.5 (15)
14.1±2.4(22)e
14.5±3.5(16)e
aPVE = persistent vaginal estrus, defined as the presence of epithelial cells (without leukocytes) in vaginal smears.
bAll treatment groups for this endpoint significantly different from vehicle controls (p < 0.05), using the Fisher's
exact test.
0 Average number of oocytes in the oviducts at sacrifice.
dNumber of animals.
Statistically significantly different from vehicle controls (p < 0.05) using the Student's /-test.
Source: Swartz et al. (1988).
Swartz and Mall (1989) administered chlordecone (98% purity) to groups of female CD-I
mice via gavage (in sesame oil) at doses of 0 or 0.25 mg/day (8 mg/kg-day), 5 days per week for
4 weeks. A positive control group received 17|3-estradiol at a dose of 0.1 mg/day. Animals were
sacrificed 24 hours following the final treatment, and the ovaries were fixed and sectioned. The
abundance of small-, medium-, and large-sized follicles was determined in every tenth section.
Significantly fewer small- and medium-sized follicles were found in chlordecone-treated mice
relative to vehicle controls (Table 4-12). Based on observations that many of the large-sized
follicles in the ovaries of chlordecone-treated mice appeared to be atretic, all histological
sections of the ovaries were examined for the presence and condition of large-sized follicles.
The number of large-sized follicles in chlordecone-treated mice did not differ significantly from
controls; however, a significantly lower abundance of healthy large-sized follicles was noted
(Table 4-12). This study identified a LOAEL of 8 mg/kg-day for adverse effects on follicle size
and condition.
JANUARY 2008
40
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-12. Abundance of various-sized follicles and the condition of large-
sized follicles in the ovaries of female CD-I mice exposed to chlordecone by
gavage 5 days per week for 4 weeks
Treatment
Controls
17p-Estradiol
Chlordecone
Number of small-, medium-, and large-sized
follicles"
Small
279.2 ±39.6
368.0 ±47.5
190.1±32.8C
Medium
116.2 ±7.8
231.9 ±41.0°
103.8 ± 11.8
Large
21.3 ±2.5
28.0 ±8.3
27.5 ±3.2
Number of healthy and atretic large-sized
folliclesb
Total
58.1 ±7.3
69.6 ±6.7
58.7 ±5.8
Healthy
28.4 ±6.0
25.4 ±2.7
18.5±1.9C
Atretic
29.7 ±3.4
44.2±4.3C
40.1 ±5.1
aMean ± SEM, based on evaluations of every 10th section.
bMean ± SEM, based on evaluations of all sections.
Statistically significantly different from vehicle controls (p < 0.05) using the Student's Mest.
Source: Swartz and Mall (1989).
Gellert and Wilson (1979) administered chlordecone (purity unspecified; vehicle: 5%
ethanol in sesame oil) to pregnant Sprague-Dawley rats by gavage at doses of 0 or 15 mg/kg-day
on gestation days 14-20. Untreated controls were included in the study, as well as groups of
dams that were administered other pesticides. The study report did not specify the number of
rats in each treatment group. The pregnant rats were allowed to deliver and raise their offspring.
At 21 days of age, the offspring were sexed and weaned. At approximately 6 months of age,
estrous cyclicity of female offspring was assessed via daily vaginal smears for about 2 weeks.
Persistent vaginal estrus (PVE) was defined as 4 or more consecutive days with only cornified or
nucleated cells in the vaginal smear. At sacrifice immediately following assessment for estrous
cyclicity, the rats were weighed and blood was collected for analysis of serum estradiol.
Ovaries, uteri, and adrenals were weighed, and ovaries were histologically examined for the
presence of corpora lutea. Animals with visible corpora were considered to be ovulatory. At 6
months of age, the male offspring of the treated dams were subjected to fertility testing by
placing them with two experienced female rats for a period of 2 weeks. The resulting offspring
of these matings were counted and sexed. At sacrifice, adrenals, testes, and ventral prostates of
the Fl generation were individually weighed, and the epididymis was grossly examined for the
presence of cysts.
The study authors did not report chlordecone-induced effects in the treated dams. Female
offspring of the chlordecone-treated dams exhibited significantly decreased ovarian weight and
significantly increased adrenal weight relative to vehicle controls, as well as significantly
increased incidences of PVE (Table 4-13). In each of the control groups, all but 1 of the female
offspring were ovulatory, whereas none of the 21 of the female offspring of the chlordecone-
treated dams were ovulatory (Table 4-13). Serum estradiol levels in control female offspring
fluctuated as expected during regular 4- or 5-day estrous cycles, whereas the level in
chlordecone-treated female offspring were observed to remain at an intermediate level. The
JANUARY 2008
41
DRAFT - DO NOT CITE OR QUOTE
-------
serum estradiol levels were below 10 pg/mL in 65% of controls and 24% of the chlordecone-
treated animals. In 14% of the control animals the estradiol levels were above 47 pg/mL,
whereas none of the chlordecone treated animals had the estradiol above this level. Male
offspring of the chlordecone-treated dams exhibited no evidence of decreased fertility or altered
sex ratios in the resulting F2 generation. This study identified a LOAEL of 15 mg/kg-day for
reproductive effects in adult female offspring of rat dams administered chlordecone by gavage
during gestation days 14-20.
Table 4-13. Effects of chlordecone on adult female offspring of Sprague-
Dawley rat dams administered chlordecone by gavage on gestation
days 14-20
Treatment
Control
Untreated
Sesame oil
Chlordecone
(15 mg/kg)
Number
of rats
29
25
21
Body
weight (g)
372 ± T
338 ±6
364 ± 13
Average weight (mg)
Ovary
92 ±3
96 ±4
59±2a
Uterus
577 ± 24
621 ±26
686 ± 37
Adrenal
64 ±1
68 ±2
85±3a
Number of
rats with
PVE
2
1
12a
Number of
ovulatory
rats
1
1
21a
""Statistically significantly different from sesame-oil-treated controls (p < 0.001).
Source: Gellert and Wilson (1979).
Several groups of investigators assessed spermatogenesis in laboratory animals that had
been exposed to chlordecone. In a toxicological screen of several chemicals, chlordecone (purity
unspecified) was administered to male rats of unspecified strain at dose levels of 0.625, 1.25, 2.5,
5.0, or 10.0 mg/kg once per day for 10 days (U.S. EPA, 1986c). Untreated and vehicle controls
were included in the study. Testes and epididymides were removed for assessment of testicular
weight; sperm concentration, motility, and morphology; and histopathology. Compared with
control values, alteration of sperm concentration was noted in all chlordecone-treated groups.
There were no apparent treatment-related effects on sperm motility, testosterone level, or FSH
level and no testicular histopathologic findings. A LOAEL of 0.625 was identified for this study.
Linder et al. (1983) exposed male Sprague-Dawley rats (20/group) to dietary
concentrations of chlordecone at 0, 5, 15, or 30 ppm for 90-days. The report does not specify
how the chlordecone was added to the diet. The corresponding doses were estimated by the
researchers as 0, 0.26, 0.83, or 1.67 mg/kg-day, respectively. After 90 days of treatment, half of
the animals in each group were sacrificed for weighing and histopathological examination of the
reproductive organs and measurement of epididymal sperm characteristics. Each of the
remaining males in each group was bred to two untreated females over a 14-day unexposed
period immediately following the 90-day exposure period. The mated females were sacrificed
JANUARY 2008
42
DRAFT - DO NOT CITE OR QUOTE
-------
on day 20 of gestation, and fetal weights, fetal viability, and total implants were determined. The
mated males were maintained for a 4.5-month recovery period prior to sacrifice and examination
of sperm and reproductive organs. Some rats in the 0.83 and 1.67 mg/kg-day groups displayed
hyperexcitability and mild tremors during the treatment period. Body weight was significantly
lower than that of controls by about 7% in the 1.67 mg/kg-day group at the end of treatment, but
the lower dose groups were not affected. The decrease in final body weight was accompanied by
significant decreases in absolute prostate and seminal vesicle weight in the 1.67 mg/kg-day
group, while testis and epididymis weights were unchanged from controls. Relative weights of
all of these tissues were reported to be similar to controls, although the data were not shown. No
gross or microscopic pathology related to treatment was found.
Sperm viability, motility, and reserves in the right cauda epididymis were significantly
reduced in both the 0.83 and 1.67 mg/kg-day groups but not at 0.26 mg/kg-day (Linder et al.,
1983). The findings in the two high-dose groups were similar to each other (no clear increase in
severity with increasing dose beyond 0.83 mg/kg-day) (see Table 4-14).
Table 4-14. Sperm parameters in male Sprague-Dawley rats following
administration of chlordecone in the diet for 90 days
Endpoint
Sperm motility (% motile + SEM)
Sperm viability (% alive + SEM)
Sperm content of right cauda epididymis
(count x 106 ± SEM)
Dose (mg/kg-day)
0
37.0 ±3.9
46.0 ±4.7
308 ± 14
0.26
33.2 ±3. 8
36.2 ±3. 3
290 ± 10
0.83
19.2±4.4a
25.0 ± 3. 3a
248 ± 22a
1.67
22.6±5.5a
30.9±4.8a
249 ± 14a
aStatistically different from control groups according to ANOVA (p < 0.05).
Source: Linder etal. (1983).
Neither sperm morphology nor sperm count in the epididymal fluid was affected at any
dose. Reproductive performance (determined by number of pregnant females, number of live
litters, average live litter size, number of implants, percentage of resorptions, and fetal weight)
was similar in exposed and control groups. No effects of any type were found after the 4.5-
month recovery on control diet. In this study, subchronic dietary exposure to 0.83 mg/kg-day or
above produced significant reductions in sperm motility, viability, and reserves without affecting
sperm morphology or sperm count in the epididymal fluid or without affecting male reproductive
performance. Similar effects (oligospermia in the absence of a reduction in reproductive
performance) have also been observed in occupationally exposed humans (Guzelian, 1982a,b;
Guzelian et al., 1980; Taylor et al., 1978). Doses of 0.83 mg/kg-day and above also produced
neurological effects (hyperexcitability and tremors) in the rats, while no effects of any type were
JANUARY 2008
43
DRAFT - DO NOT CITE OR QUOTE
-------
observed at 0.26 mg/kg-day. This study identified a LOAEL of 0.83 mg/kg-day and aNOAEL
of 0.26 mg/kg-day, based on the occurrence of neurological and spermotoxic effects.
Additional reproductive studies exist that evaluate the effect of acute injected
chlordecone (at doses from 20-80 mg/kg) in experimental animals. Effects observed in these
studies were similar to studies of repeat oral administration of chlordecone and generally
included changes in estrous cyclicity and fertility (Williams and Uphouse, 1991; Johnson et al.,
1990; Pinkston and Uphouse, 1987-1988). These acute injection studies provide information to
support reproductive effects at high doses of chlordecone but do not generally contribute
additional dose-response information regarding the most sensitive effects of chlordecone
exposure.
4.3.2. Developmental Toxicity Studies
The developmental toxicity of chlordecone in humans is not known. Chlordecone
produces developmental toxicity in rats and mice at dose levels that also produced maternal
toxicity (Seidenberg et al., 1986; Chernoff and Rogers, 1976). Though the database of
developmental studies on chlordecone is small, the available animal studies indicate that
developmental or fetotoxic effects would not be expected to occur at exposures that are not also
associated with maternal toxicity.
Chernoff and Rogers (1976) administered chlordecone (purity unspecified) to groups of
pregnant CD rats at gavage doses of 0, 2, 6, or 10 mg/kg-day on gestation days 7-16. Dams
were observed for clinical signs and weight gain, and sacrificed on gestation day 21 for
assessment of liver/body weight and evaluation of fetuses. Fetal parameters evaluated include
number of implants, mortality, weight, and gross developmental abnormalities. Study results are
depicted in Table 4-15. Significant maternal toxicity was observed in high-dose dams. All
groups of dosed dams exhibited significantly depressed weight gain, and the average liver/body
weight ratio was significantly increased in the two highest dose groups (6 and 10 mg/kg-day).
Fetotoxicity was observed as significantly depressed fetal body weight and delayed ossification
in 6 and 10 mg/kg-day dose groups and significantly increased incidences of litters with fetuses
having enlarged renal pelvis, edema, undescended testes, or enlarged cerebral ventricles in the 10
mg/kg-day group relative to controls. The study identified a LOAEL of 2 mg/kg-day for
maternal toxicity, based on significantly depressed maternal body weight gain (16% lower than
controls). The study identified a NOAEL of 2 mg/kg-day and a LOAEL of 6 mg/kg-day for
fetotoxicity. The fetal effects may have been the direct result of maternal toxicity since they
occurred at doses that were clearly toxic to the dams.
JANUARY 2008 44 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-15. Maternal and fetal effects following gavage dosing of pregnant
rat dams with chlordecone on gestation days 7-16
Maternal effects3
Number inseminated
Maternal deaths
Number pregnant at sacrifice
Weight gain (g)
Liver/body weight
Fetal effects3
Implants/dam
Percent mortality
Weight at sacrifice (g)
Sternal ossification centers
Caudal ossification centers
Percent supernumerary ribs
Enlarged renal pelvis0
Edema0
Undescended testis0
Enlarged cerebral ventricles0
Dose level (mg/kg-day)
0
26
0
23
73. 5 ±3.7
5.0 ±0.1
10.2 ±0.4
9.5 ±3.0
4.1±0.1
5.6 ±0.1
4.7 ±0.1
24.4 ±6.3
1
0
0
0
2
31
1
24
62.4 ± 2.9b
5.1±0.1
10.4 ±0.6
8.1 ±2.7
4.0 ±0.1
5.5 ±0.1
4.5 ±0.1
28.1 ±5.5
2
1
0
0
6
35
0
33
33.8±2.4b
5.9±0.1b
11.0±0.4
6.5 ±1.2
3.9±0.1b
5.3 ±0.1
4.4±0.1b
24.5 ±5.4
5
0
1
0
10
42
8b
30
34.0 ± 5.6b
7.4 ± 0.2b
9.1 ±0.5
17.7 ±4.9
3.7±0.1b
5.3 ±0.1
4.0 ± 0.2b
17.4 ±3. 9
10b
10b
5b
5b
aMean ± SE.
bStatistically significantly different from controls (p < 0.05).
°Number of litters with one or more fetuses exhibiting the effect.
Source: Chernoff and Rogers (1976).
Chernoff and Rogers (1976) also administered chlordecone (purity unspecified) to groups
of pregnant CD-I mice at gavage doses of 0, 2, 4, 8, or 12 mg/kg-day on gestation days 7-16.
Dams were observed for clinical signs and weight gain, and sacrificed on gestation day 18 for
assessment of liver and body weight and evaluation of fetuses. Maternal and fetotoxicity were
assessed in the same manner as that described for the rats. In the mice, significantly depressed
maternal weight gain was noted at 8 and 12 mg/kg-day, and all dose groups exhibited
significantly increased maternal liver and body weight (Table 4-16). Signs of fetotoxicity were
observed only in the highest dose group and consisted of significantly increased fetal mortality.
The study identified a LOAEL of 2 mg/kg-day for maternal toxicity, based on a statistically
significant 10% increase in relative liver weight in the 2, 4, and 8 mg/kg-day dose groups. The
study identified a NOAEL of 8 mg/kg-day and a LOAEL of 12 mg/kg-day for fetotoxicity. The
fetal effects may have been the direct result of maternal toxicity since they occurred at doses that
were clearly toxic to the dams.
JANUARY 2008
45
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-16. Maternal and fetal effects following gavage dosing of pregnant
mouse dams with chlordecone on gestation days 7-16
Maternal effects3
Number inseminated
Maternal deaths
Number pregnant at sacrifice
Weight gain (g)
Liver/body weight
Fetal effects3
Implants/dam
Percent mortality
Weight at sacrifice (g)
Sternal ossification centers
Caudal ossification centers
Percent supernumerary ribs
Dose level (mg/kg-day)
0
26
0
16
4.3 ±0.5
6.8 ±0.3
12.8 ±0.6
15.6 ±3.3
1.0 ±0.1
5.5 ±0.1
4.0 ±0.3
33.0 ±6.8
2
16
0
14
4.1 ±0.4
7.5±0.2b
12.0 ±0.8
12.4 ±3. 5
l.OiO.l
5.3 ±0.2
3. 5 ±0.5
20.9 ±9.4
4
24
0
16
3. 3 ±0.4
7.9±0.1b
12.4 ±0.7
11.8±2.1
l.liO.l
5.6 ±0.2
4.5 ±0.4
13.8±5.1
8
25
0
19
0.7±0.9b
8.6±0.3b
11. 3 ±0.7
16.9 ±5.1
1.0 ±0.1
5.1 ±0.3
4.1 ±0.5
26.2 ±6.4
12
12
1
5
-2.8 ± 0.9b
7.6 ±0.6
11.8±1.4
53.4±19.4b
1.3 ±0.1
6.0 ±0.0
6.4 ±0.4
12.3 ±4.8
aMean±SE.
bStatistically significantly different from controls (p < 0.05).
Source: Chernoff and Rogers (1976).
Additional developmental studies exist on chlordecone administered by injection (from
5-100 mg/kg) during gestation or postnatally. Effects associated with chlordecone exposure
generally included alterations in neurological function, as well as impaired learning and
behavioral changes, alterations in sexual differentiation, and weak estrogenic effects (Laessig et
al., 2007 Sierra and Uphouse, 1986; Cooper et al., 1985; Mactutus and Tilson, 1985; Rosecrans
et al., 1985). These acute injection studies help provide information to support developmental
effects at high doses of chlordecone but do not generally contribute additional dose-response
information regarding the most sensitive effects of chlordecone exposure during development.
4.3.3. Screening Studies
In a neonatal survival screen, chlordecone (purity unspecified) was administered to
pregnant F344 rats at a gavage dose level of 0 or 10.0 mg/kg-day during gestation days 7-16
(U.S. EPA, 1986c). Neonatal survival was assessed on days 1 and 3 postpartum. Significantly
(p < 0.5) reduced survival was noted on day 3 (but not day 1) postpartum (U.S. EPA, 1986c). In
a developmental toxicity screen in the ICR/SIM mouse, chlordecone was administered by gavage
at a dose of 0 or 24 mg/kg-day during days 8 to 12 of gestation (Seidenberg et al., 1986).
Maternal toxicity was observed with decreased body weight gain and mortality in 18% of treated
dams. Decreases were also observed in neonatal body weight gain and percent survival
(Seidenberg et al., 1986).
JANUARY 2008
46
DRAFT - DO NOT CITE OR QUOTE
-------
4.4. OTHER STUDIES
4.4.1. Acute Toxicity Studies
Oral LDso values for chlordecone range from 71 mg/kg body weight for rabbits to 250
mg/kg body weight for dogs (Larson et al., 1979a). The oral LD50 value for rats is 125 mg/kg
body weight (Gaines, 1969). In experimental animals, the systemic effects of chlordecone
following short-term exposures generally include nervous system effects (tremor and
hyperexcitability), reproductive system toxicity (effects on estrous cyclicity and sperm
parameters), liver changes (hypertrophy, microsomal enzyme induction, and ultrastructural
changes), musculoskeletal effects (resulting from alterations in ATPase activity and calcium
homeostasis), and thyroid and adrenal effects (ATSDR, 1995; U.S. EPA, 1986c; WHO, 1984).
The adaptive effects observed in the liver are those generally produced by halogenated
hydrocarbons; these include increase in liver weight or size and induction of the mixed function
oxidase enzyme system (ATSDR, 1995). Chlordecone was also shown to alter lipid storage and
metabolism in mice (Carpenter et al., 1996; Chetty et al., 1993a,b), and hepatobiliary excretion
of certain chemicals was inhibited by chlordecone following acute exposure (see Section 3.4).
Other systemic effects reported following acute chlordecone exposure include decreases
in food intake and body weight gain (ATSDR, 1995; Williams et al., 1992; U.S. EPA, 1986c;
Albertson etal., 1985; Chernoff and Kavlock, 1982; Chernoff and Rogers, 1976), altered
thermoregulation resulting in a decrease in core temperature that persisted for up to 12 days
following ingestion of a single dose of 55 or 75 mg/kg in rats (Swanson and Wooley, 1982), and
slight hyperthermia in rats following 12 weeks of exposure at 7.1 mg/kg-day (Pryor et al., 1983).
The cardiovascular effects in rats after acute-duration exposure to chlordecone are limited to
biochemical changes in cardiac tissue, such as membrane enzyme inhibitions and altered protein
phosphorylation (Kodavanti et al., 1990; Desaiah et al., 1980); however, the toxicological
implications of these changes are unknown (ATSDR, 1995).
4.4.2. Potentiation of Halomethane Toxicity
Laboratory studies of chlordecone potentiation of halomethane liver toxicity provide
insight into potential mechanisms of chlordecone induced liver toxicity, though doses used in
these studies are not considered environmentally relevant doses.
Chlordecone potentiates the liver toxicity and lethality of carbon tetrachloride (CCU) and
other halomethanes (e.g., chloroform, bromotrichloromethane) in rats and mice, and this
interaction has been widely studied and reviewed (Mehendale, 1994, 1990; Faroon and
Mehendale, 1990; Mehendale et al., 1989; Plaa et al., 1987; Curtis et al., 1981). The exposure of
rats to 10 ppm chlordecone in the diet for 15 days greatly increased the liver toxicity of
halomethanes, leading to hepatic failure and death (Soni and Mehendale, 1993). Liver toxicity
was generally demonstrated by measurement of elevated serum enzyme activities and
JANUARY 2008 47 DRAFT - DO NOT CITE OR QUOTE
-------
histopathological changes, including necrosis, lipid accumulation, and hepatocyte swelling. This
effect was specific to chlordecone and was not observed following pretreatment with other
organochlorine pesticides (e.g., mirex and photomirex).
Chlordecone enhanced the oxidative metabolism of halomethanes; however, enzyme
induction was not correlated with the potentiation of liver toxicity. More efficient enzyme
inducers, such as phenobarbital, did not significantly potentiate the toxicity of CCU (Mehendale
and Klingensmith, 1988; Curtis et al., 1981). Chlordecone appears to enhance the liver toxicity
of halomethanes by suppressing the hepatocellular regeneration that is required to repair liver
injury and restore hepatolobular architecture and function (Kodavanti et al., 1992; Faroon and
Mehendale, 1990; Mehendale, 1990; Mehendale et al., 1989). Partially hepatectomized rats are
protected from chlordecone-CCU toxicity because of an increase in the rate of cell turnover as
measured by 3H-thymidine incorporation into hepatocellular DNA and an increase in the
percentage of mitotic figures (Kodavanti et al., 1989; Young and Mehendale, 1989). Protection
from liver toxicity was also provided by pretreatment with cyanidanol, which stimulated
hepatocellular regeneration evidenced by increased 3H-thymidine incorporation (Soni and
Mehendale, 1991a,b,c). Polyamine metabolism was inhibited by cotreatment with chlordecone
and bromotrichloromethane (Rao et al., 1990). Polyamines are important for the cell growth and
proliferation process that results in liver regeneration and repair.
The chlordecone suppression of liver cell regeneration and repair may be related to the
compromised energy status of hepatocytes in animals exposed to chlordecone. Treatment of rats
with chlordecone and CCU caused a decrease in liver ATP levels and an inhibition of
oligomycin-sensitive Mg2+-ATPase (Kodavanti et al., 1990). Chlordecone affects calcium
homeostasis in hepatocytes, leading to a decline in glycogen storage and a reduced energy status
(Kodavanti et al., 1993, 1990). Chlordecone-CCU administration caused an inhibition in
microsomal and mitochondrial calcium uptake and a decrease in the high affinity component of
hepatic plasma membrane Ca2+-ATPase. Administration of fructose 1,6-diphosphate to rats
resulted in protection from chlordecone-CCU hepatotoxicity due to an increase in the levels of
liver cell ATP (Rao and Mehendale, 1989). ATP administration during the early phase of liver
injury also helped to restore normal liver function through enhanced regeneration and repair
(Soni and Mehendale, 1991a,b,c).
Several studies have indicated an age-related susceptibility to the chlordecone
potentiation of CCU hepatotoxicity (Dalu et al., 1995; Cai and Mehendale, 1993). Developing
rats have been shown to be resistant to the lethal effects of the chlordecone-CCU combination
treatment. Postnatal rats recovered more quickly from CCU-induced liver injury than young
adult rats, due to the higher level of ongoing cell division and an additional stimulatory response
to liver injury (measured by 3H-thymidine incorporation into hepatocellular DNA). The
resiliency of postnatal rats was abolished by administration of the antimitotic agent colchicine,
highlighting the importance of cell turnover in liver tissue repair (Dalu et al., 1998). Aged rats
JANUARY 2008 48 DRAFT - DO NOT CITE OR QUOTE
-------
(2 years old) were also shown to be resistant to the potentiation of CCU liver toxicity by
chlordecone due to the robust and early liver tissue repair in old rats as compared with young
adult rats (3 months) (Murali et al., 2002). Gender effects were noted, with female rats being
more sensitive to chlordecone-CCU hepatotoxicity than male rats (Blain et al., 1999).
4.4.3. Neurotoxicity Studies
With tremor being the cardinal feature of chlordecone intoxication in humans, research
into the mode of action of the neurological changes has been the focus of several studies. A
number of studies have associated alterations in neurotransmitter activity (e.g., alpha-
noradrenergic, dopaminergic, and serotonergic systems) with chlordecone-induced tremor and
exaggerated startle response (Vaccari and Saba, 1995; Brown et al., 1991; Herr et al., 1987;
Tilson et al., 1986; Uphouse and Eckols, 1986; Chen et al., 1985; Desaiah, 1985; Gerhart et al.,
1985, 1983, 1982; Hong et al., 1984; Fujimori et al., 1982b; Hwang and van Woert, 1979). At
the cellular level, changes in ATPase activity and calcium homeostasis in the nervous system
have been related to chlordecone exposure across species (ATSDR, 1995). The reported effects
of chlordecone exposure on calcium balance in whole animal studies include decreased calcium
uptake in rats following a single oral dose of 40 mg/kg (End et al., 1981); decreased total
protein-bound, myelin, and synaptosomal calcium following eight consecutive daily oral doses
of 25 mg/kg-day in 4- to 6-week-old male ICR mice (Hoskins and Ho, 1982); decreased total
protein-bound and mitochondrial calcium content with increased nuclear calcium content in 24-
week-old male ICR mice following a single oral dose of 25 mg/kg (Hoskins and Ho, 1982); and
decreased brain calmodulin in rats exposed to 2.5 mg/kg-day orally for 10 consecutive days
(Desaiah et al., 1985; Desaiah, 1982). In vitro studies have supported that chlordecone may alter
calcium regulation of neuronal function (Inoue et al., 1991; Bondy and McKee, 1990; Vig et al.,
1989; End etal., 1981, 1979).
4.4.4. Endocrine Disruption Studies
Specific mechanisms of chlordecone-induced reproductive effects are not known,
although it is generally believed that an estrogenic mode of action is involved. Observed
chlordecone-induced reproductive effects include oligospermia, reduced sperm motility, and
decreased libido in occupationally exposed males (Taylor, 1985, 1982; Guzelian, 1982a; Taylor
et al., 1978) and decreased offspring production in laboratory animals (Cannon and Kimbrough,
1979; Good et al., 1965; Huber, 1965). Testicular atrophy, altered sperm characteristics,
persistent vaginal estrus, and anovulation observed in chlordecone-treated laboratory animals
mimic similar effects produced by excessive estrogen (Swartz et al., 1988; U.S. EPA, 1986c;
Uphouse, 1985; Linder et al., 1983; Larson et al., 1979a; Huber, 1965). Estrogens appear to
function by altering gene transcription in reproductive tissues via nuclear estrogen receptors.
JANUARY 2008 49 DRAFT - DO NOT CITE OR QUOTE
-------
Mechanistic studies, therefore, have been designed to assess the potential of chlordecone to
mimic the action of estrogen.
In cell-free preparations containing rat uterine estrogen receptors, 8 jiM chlordecone
inhibited the binding of [3H]estradiol by nearly 50% (Bulger et al., 1979). It was further
demonstrated that chlordecone caused the translocation of estrogen receptors from the cytosolic
to the nuclear fraction in both isolated rat uteri and ovariectomized immature rats. These results
indicate that chlordecone may act directly on the uterus. In another study, chlordecone-induced
uterine effects observed in ovariectomized immature rats were enhanced by coadministration of
estradiol, an indication that chlordecone and estradiol act at the same site in uterine tissue
(Johnson, 1996). Chlordecone demonstrated a relatively high affinity for recombinant human
estrogen receptors; 5.7 jiM (Bolger et al., 1998) and 9 jiM chlordecone (Scippo et al., 2004)
caused 50% inhibition of 17|3-estradiol binding. Chlordecone exhibits approximately equal
affinity for both subtypes of human estrogen receptors (ERa and ER|3) (Kuiper et al., 1998). In
one study, uterine levels of adenosine 3'5'-cyclic monophosphate (cAMP) decreased with
increasing uterine weight following repeated exposure to chlordecone in ovariectomized
immature rats (Johnson et al., 1995). The levels of cAMP were not decreased in similarly treated
rats that were also given the antiestrogen (ICI-182,780), indicating that the chlordecone-induced
effect on cAMP is estrogen receptor dependent.
The affinity of chlordecone for estrogen appears to be tissue dependent. Although
competition between [3H]estradiol and chlordecone was comparable in magnitude within
estrogen receptor preparations from brain or uterine tissues of rats, in vivo binding of
chlordecone in the brain of ovariectomized rats was much less than that observed in the uterus
(Williams et al., 1989). The basis for this in vivo tissue-specific difference is not clear but may
result, at least in part, from a greater time requirement for chlordecone to reach a concentration
in the brain that could result in a significant estrogenic effect. Furthermore, although
chlordecone may mimic the effect of estrogen in uterine tissue, chlordecone appears to function
as an estrogen antagonist in central nervous tissue (Huang and Nelson, 1986; Uphouse et al.,
1986).
Chlordecone interacts in vitro and in vivo with the estrogen receptor system in rat uterus.
Hammond et al. (1979) found that it competes with estradiol for binding to the cytoplasmic
receptor in vitro and also induces nuclear accumulation of estrogen receptor sites in uteri in vitro.
Chlordecone translocates estrogen receptor sites to the uterine nucleus, increases uterine weight,
and stimulates the synthesis of the progesterone receptor when it is injected into immature
female rats (Hammond et al., 1979).
Results of one recent study indicate that chlordecone-induced uterine effects may also be
induced via a pathway other than that which includes the estrogen receptor. Chlordecone up-
regulated the uterine expression of an estrogen-responsive gene, lactoferrin, in ERa knockout
mice, whereas these effects were not elicited by 17|3-estradiol (Das et al., 1997). Neither the
JANUARY 2008 50 DRAFT - DO NOT CITE OR QUOTE
-------
estrogen receptor antagonist ICI-182,780 nor 17|3-estradiol inhibited the chlordecone-induced
uterine expression of lactoferrin in these mice.
Chlordecone has been tested for its potential to bind to other receptors. The chemical
exhibited relatively high affinity for recombinant human progesterone receptors (Scippo et al.,
2004); 11 jiM chlordecone resulted in 50% inhibition of progesterone binding. Chlordecone
exhibited characteristics of a partial androgen antagonist, based on 50% reduction of inhibition
of 5a-dihydroxytestosterone-mediated activation of luciferase activity by 6.9 jiM chlordecone in
the human PC-3 prostate carcinoma cell line (Schrader and Cooke, 2000).
4.4.5. Immunological Studies
Several studies have examined the potential for general immunotoxicity associated with
chlordecone exposure, and two studies have investigated chlordecone effects on the acceleration
of an autoimmune disease. Smialowicz et al. (1985) exposed male F344 rats to technical grade
chlordecone (87% pure) in corn oil by gavage for 10 days at doses of 0.625, 1.25, 2.5, 5.0, and
10 mg/kg-day (10 rats/dose). Dose groups also included a vehicle control group (corn oil), an
untreated cage-matched control group, and cyclophosphamide (1.5-24 mg/kg-day) exposure
groups as positive controls for immunosuppression. Blood samples were taken for total and
differential white blood cell counts, and the spleen and thymus weights were recorded. Single
cell suspensions were prepared from the spleen, and the lymphoproliferative response of
splenocytes to the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (con A), the
T- and B-cell mitogen pokeweed mitogen, and the B-cell mitogen Salmonella typhimurium
mitogen (STM) were assayed. A single functional immune test, natural killer (NK) cell activity
of splenocytes, was also performed. NK activity was measured against W/Fu-Gl rat lymphoma
cells and YAC-1 mouse lymphoma cells as the target cell population. The high dose (10 mg/kg-
day) of chlordecone caused a 20% reduction in body weight as well as reduced relative spleen
and thymus weights (8 and 24% respectively). The high dose was also associated with a 69%
reduction in the concentration of circulating neutrophils, but no change was seen in the number
of lymphocytes, monocytes, or overall leukocytes. A reduced mitogenic response to PHA was
observed in the 2.5 mg/kg-day chlordecone group only. The high dose of chlordecone was
associated with a 45% reduced mitogenic response to con A, a 66% increased mitogenic
response to STM, and an almost threefold increase in background mitogenic response. In rats
exposed to the high dose of chlordecone, NK cell activity was reduced by 62 to 73% against both
target cell lines. The authors suggest that the observed effects in the high-dose animals (10
mg/kg-day) were due to overt toxicity. The authors also note that at 10 mg/kg-day rats displayed
tremors characteristic of chlordecone intoxication, and therefore the decreased body weight,
decreased spleen and thymus weight, altered lymphoproliferative response, and decreased NK
cell activity were likely effects secondary to overt toxicity.
JANUARY 2008 51 DRAFT - DO NOT CITE OR QUOTE
-------
The effects of chlordecone exposure on antibody response were examined as part of a
study of the consequences of malnutrition on antibody response in male Sprague-Dawley rats
(Chetty et al., 1993c). For the purpose of this review, only data from the control and
chlordecone-treated rats fed nutritionally sufficient diets are presented. Rats (six per group) were
exposed to 0, 10, or 100 ppm (doses calculated as 0, 0.96, or 9.6 mg/kg-day)3 chlordecone in the
diet for 2 or 4 weeks. Rats were immunized by injection of sheep red blood cells (SRBCs) 4
days before the end of chlordecone exposure. In addition to measuring body weight, the authors
measured spleen weight and antibody response to SRBCs as determined by the plaque-forming
cell (PFC) assay. Chlordecone exposure for either 2 or 4 weeks increased the PFC response.
Although the results are only presented graphically, dietary exposure of 10 ppm chlordecone
appeared to increase the PFC response about two- to threefold over controls. At this dose,
chlordecone treatment significantly reduced body weight by 15% and increased relative spleen
weight by 29%. Average body weight and spleen weight were not reported for animals exposed
to 100 ppm.
No additional studies of general immunotoxicity of chlordecone were found. As part of
an acute neurotoxicity study, however, a single dose of 75 mg/kg chlordecone to Sprague-
Dawley rats resulted in significant reductions in thymus weights (Swanson and Woolley, 1982).
As with the results from Smialowicz et al. (1985), the dose associated with thymus weight
reduction was also associated with severe generalized toxicity.
Several studies from the same laboratory have investigated the potential effects of
chlordecone treatment on autoimmune disease (Sobel et al., 2006, 2005; Wang et al., 2007).
Sobel et al. (2005) investigated the effect of chlordecone in female (NZB x NZW)Fi mice, a
murine model of systemic lupus erythematosus in which the principal clinical manifestation of
lupus is renal disease, specifically immune-mediated glomerulonephritis. In this study, female 8-
week-old (NZB x NZW)F1 control, ovariectomized, or sham-operated mice were implanted with
60-day sustained-release pellets containing doses of 0, 0.01, 0.1, 0.5, or 1 mg chlordecone
(99.2% pure). Pellets were replaced every 60 days throughout the experiment. For this phase of
the experiment, treatment groups consisted of 10 animals per group, whereas the control group
consisted of 20 animals. Urine protein, blood urea nitrogen (BUN), and body weight were
evaluated monthly for all animals. Mice were euthanized at the conclusion of the experiment if
BUN exceeded 50 mg/dL or if proteinuria exceeded 2000 mg/dL. IgG anti-double-strand DNA
liters in serum of some treatment groups were determined by indirect enzyme-linked
immunosorbent assay (ELISA). Kidneys were removed for histological examination and
glomerular damage was scored by light microscopy. Additionally, a subset of treatment groups
was examined for IgG-mediated immune complex deposition in glomeruli by using
immunohi stofluore scence.
Calculation: mg/kg-day = (ppm in feed x kg food/day)/kg body weight. Reference food consumption rates
of 0.0179 kg/day (U.S. EPA, 1988) and reported average body weight of 0.188 kg (males) were used.
JANUARY 2008 52 DRAFT - DO NOT CITE OR QUOTE
-------
Mice treated with 1.0 or 0.5 mg chlordecone pellets developed renal disease
significantly earlier than did ovariectomized controls (p < 0.05). This observation was also
correlated with proteinuria and the early appearance of immune complex glomerulonephritis.
Additionally, mice treated with chlordecone developed elevated ds-DNA autoantibody liters
earlier than ovariectomized controls. Immunohistofluorescence analysis of renal sections from a
subset of animals treated for 8 weeks with 1 mg chlordecone showed enhanced deposits of IgG
immune complexes as compared with untreated controls. The lowest dose per pellet found to
produce a significant decrease in time to onset of renal disease was found to be 0.5 mg. The
authors calculated, based on average body weight, that this would result in a dosing rate per unit
body weight of 0.2 mg/kg-day. However, blood levels of chlordecone were not examined, and
the equivalent oral dose needed to achieve this effect is uncertain.
After the demonstration that chronic chlordecone exposure accelerates the development
of autoimmunity in ovariectomized female (NZB x NZW)Fi mice (Sobel et al., 2005), additional
studies were designed to examine the effect of chlordecone on autoimmunity and renal disease in
ovary-intact female (NZB x NZW)Fi mice and female BALB/c mice, a mouse strain that is not
predisposed to the development of autoimmune-related renal disease (Sobel et al., 2006). As in
the previous study, 8-week-old female mice were implanted with 60-day sustained-release pellets
containing 0, 0.001, 0.01, 0.1, 0.5, 1, or 5 mg chlordecone subcutaneously above the shoulders.
Blood and urine were collected once per month for the assessment of renal function by BUN
analysis and urine protein content. Mice were euthanized at the conclusion of the experiment if
BUN exceeded 50 mg/dL or if proteinuria exceeded 2000 mg/dL. Blood was taken for serum
analysis and kidneys were removed for later histological analysis by light microscopy. Antigen-
specific antibody levels for anti-dsDNA and antichromatin were determined by indirect ELISA.
In the first half of the experiment, involving chlordecone treatment in ovary-intact (NZB
x NZW)Fi mice, Sobel et al. (2006 ) reported that chlordecone shortened survival, decreased the
time to onset of elevated autoantibody liters, and accelerated glomerulonephritis in a dose-
dependent manner. Median survival of control groups was 25 weeks, compared with 21 and 18
weeks in mice implanted with the 1 mg and 5 mg chlordecone pellets, respectively. Survival
curves for mice treated with chlordecone were significantly different from controls by log rank
test for trend (p = 0.01). Time to development of renal disease in mice treated with the 5 mg
pellets was significantly shorter than in controls (p < 0.05). However, histopathology associated
with renal disease was similar between the groups. Mice treated with either 1 or 5 mg
chlordecone pellets developed anti-dsDNA and antichromatin autoantibody liters significantly
earlier than controls (p < 0.005).
In the second half of the experiment, involving chlordecone treatment of BALB/c mice,
Sobel et al. (2006) performed the same assays as for the (NZB x NZW)Fi mice. No treatment-
related effects were seen in mortality, and none of the chlordecone-exposed BALB/c mice
developed renal disease. Autoantibody liters (anti-dsDNA and antichromatin) were nol differenl
JANUARY 2008 53 DRAFT - DO NOT CITE OR QUOTE
-------
from controls. Total serum IgG2a and IgGl were statistically increased in mice treated with the
1 and 5 mg chlordecone pellets (p < 0.01). The failure of chlordecone to induce renal disease or
autoantibodies in BALB/c mice (a strain not predisposed to the development of autoimmunity or
renal disease) emphasizes the importance of genetic background on the effects of chlordecone on
autoimmunity.
The mechanism by which chlordecone accelerates autoimmunity in female (NZB x
NZW)Fi mice is unknown. The (NZB x NZW)Fi mouse is a model of systemic lupus
erythematosus, an autoimmune disorder that affects women more frequently than men (Lahita,
1997). Estrogen receptor binding may play a role in some forms of autoimmune disease in
rodents and humans (Ahmed et al., 1999), and, in the (NZB x NZW)Fi mouse model of systemic
lupus erythematosus, l?p-estradiol accelerates the development of glomerulonephritis with
similar results to the effects observed following chlordecone treatment (Sobel et al., 2005).
Sobel et al. (2005) hypothesized that chlordecone's acceleration of autoimmunity may be related
to its estrogenic properties and ability of chlordecone to bind the estrogen receptor. However,
the poor correlation between autoimmune effects and estrogenic activity of chlordecone as
measured by uterine hypertrophy suggests that a non-estrogen-receptor-mediated mechanism
may be important (Sobel et al., 2005).
An additional study by the same laboratory was performed to compare the mechanism of
chlordecone-accelerated autoimmunity to that of l?p estradiol-accelerated autoimmunity in
(NZBxNZW)Fi mice by examining gene and protein expression of B cells (Wang et al., 2007).
As with the earlier experiments, 6-8-week-old ovariectomized female (NZBxNZW)Fi mice were
implanted with 60-day sustained-release pellets. In this experiment, pellets contained 1 mg
chlordecone, 5 mg chlordecone, O.OSmg estradiol, or matrix only for controls. Mice were
euthanized 5-6 weeks after implantation in order to evaluate the development of autoimmune
pathology rather than overt effects. Spleens were removed and splenic tissue and cells were
prepared for analysis. Splenocytes were analyzed for proliferation, apoptosis, and mRNA and
cDNA expression. The following markers were analyzed for expression: B220, IgM, CD 19,
CD21, CD24, CD44, CD69, CXCR4, CXCR5, ICAM-1, VCAM-F, MHC II, B7.2, and GL7.
The authors state that germinal center activity and cell surface markers of B cells in the germinal
centers were examined because of the importance of the germinal center in negative selection for
autoreactive B cells. Both chlordecone exposure and estradiol treatment activated splenic B cells
and enhanced germinal center activity as shown by upregulated protein expression of GL7,
CXCR5, and CXCR4. Both treatments also resulted in reduced B cell apoptosis and increased
patterns of protein and gene expression that may increase survival of autoreactive B cells (i.e., B
cell expression of ICAM-1 and VCAM-1 cell adhesion molecules and Bcl-2 and shp-1 gene
expression in B cells from the germinal centers). However, major differences were also
observed between the effects of chlordecone exposure and that of estradiol, particularly in the
lack of an effect of chlordecone on splenic B cell subsets such as CD138+B220" populations.
JANUARY 2008 54 DRAFT - DO NOT CITE OR QUOTE
-------
The authors conclude that differences in the effects between chlordecone and estradiol indicate
that chlordecone does not accelerate the development to systemic lupus erythematosus by
functioning strictly as an estrogen mimic.
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION
4.5.1. Genotoxicity
The weight of evidence from in vivo and in vitro studies suggests that chlordecone is not
mutagenic. The majority of studies have not shown genotoxic activity in a variety of short-term
in vitro assays. There is no evidence that chlordecone is a mutagen in S. typhimurium or
Escherichia coli (Mortelmans et al., 1986; U.S. EPA, 1986c; Probst et al., 1981; Schoeny et al.,
1979). Further, chlordecone alcohol, the major metabolite of chlordecone in humans, is not
mutagenic in S. typhimurium (Mortelmans et al., 1986). Chlordecone also gave negative results
when tested for enhancement of unscheduled DNA synthesis in primary cultures of adult rat
hepatocytes (Probst et al., 1981; Williams, 1980). The clastogenic activity of chlordecone is
unclear. Chlordecone was investigated for potential clastogenic activity in Chinese hamster
ovary (CHO) cells (Galloway et al., 1987; Bale, 1983). Bale (1983) reported that chlordecone
treatment of CHO (M3-1) cells (2, 4, or 6 |ig/mL) produced chromosome breaks, chromatid
breaks, dicentric chromosomes, and chromosome interchanges. In a later study employing
higher doses, chlordecone did not increase the frequency of CHO cells with abnormal
chromosome morphology over a nonactivated concentration range of 10-20 |ig/L or over an
Aroclor 1254-induced rat liver S9-activated concentration range of 5-15 |ig/L (Galloway et al.,
1987).
There has been limited testing of chlordecone in whole-animal genotoxicity assays. The
available data generally show that chlordecone is not genotoxic in whole-animal tests.
Chlordecone was not clastogenic in male Sprague-Dawley rat germinal cells in a dominant lethal
assay at doses of 3.6 or 11.4 mg/kg-day orally for 5 consecutive days (Simon et al., 1986, 1978).
Although chlordecone clearly increased ornithine decarboxylase activity (indicative of cellular
proliferation) in rat livers following oral exposure, it did not induce DNA damage in the target
organ (Mitra et al., 1990; Kitchin and Brown, 1989).
4.5.2. Tumor Promotion and Mechanistic Studies
Chlordecone was tested in a two-stage model of liver carcinogenesis in both male and
female Sprague-Dawley rats (Sirica et al., 1989). Male rats were subjected to two-thirds
hepatectomy and 24 hours later were administered a single gavage dose (20 mg/kg) of the
initiator chemical diethynitrosamine (DEN) in water. Ten days following initiation, rats began to
receive biweekly subcutaneous (s.c.) injections of chlordecone in corn oil at doses of 0.17, 0.34,
JANUARY 2008 55 DRAFT - DO NOT CITE OR QUOTE
-------
1.7, and 3.4 mg/kg for a total of 44 weeks. Controls for this experiment included rats given DEN
after partial hepatectomy without chlordecone administration, rats receiving biweekly
administration of chlordecone without DEN initiation, and rats receiving corn oil vehicle only.
Chlordecone (30 mg/kg) was also administered by corn oil gavage as an initiating chemical
given 24 hours after partial hepatectomy. This treatment was followed 10 days later by
administration of the tumor promoter sodium phenobarbital in the drinking water at a daily
concentration of 0.05% for 44 weeks. A second experiment was conducted that compared
promotion in the two-stage assay in male and female rats. A similar study design was used;
however, chlordecone was administered biweekly by s.c. injection at higher doses (3 or 9 mg/kg)
and the treatment was continued for only 27 weeks.
At the end of each experiment, rats were killed and their livers were evaluated
histologically for the presence of preneoplastic lesions (hyperplastic hepatocellular foci) and
tumors (hepatocellular carcinomas). Histological staining for GOT was used to identify
preneoplastic foci in nontumorous liver sections. Morphometric measurements of GGT-positive
foci were determined, and the total number of foci per cm3 of liver were quantified. The
concentration of chlordecone in the liver was measured by gas-liquid chromatography.
Body weight gain was not altered in male rats receiving chlordecone at doses between
0.17 and 3.4 mg/kg biweekly for 44 weeks (with or without DEN initiation). Higher doses did
affect body weight gain (3 and 9 mg/kg in females and 9 mg/kg only in males) when
administered biweekly for 27 weeks. The depression in body weight gain was independent of
DEN initiation. Doses greater than 3 mg/kg caused increased irritability in male and female rats,
but no obvious tremors, dermatologic changes, or liver enlargement were observed.
Nonneoplastic liver lesions were observed histologically in both male and female rats given
chlordecone doses of 3 and 9 mg/kg biweekly (s.c.) for 27 weeks. The lesions included
hypertrophy of Zone 3 hepatocytes, congestion, mild fatty change, focal necrosis, and occasional
small nests of proliferated sinusoidal cells. The severity of these lesions appeared to be dose
related, although the incidence and severity of noncancer lesions was not quantitatively
evaluated.
A dose-related increase in the number of GGT-positive foci/cm3 of liver was observed in
male rats given chlordecone at doses between 0.17 and 3.4 mg/kg biweekly (s.c) for 44 weeks
following hepatectomy and initiation with DEN (as compared with control groups that were
receiving either initiating or promoting treatment alone). Hyperplastic nodules were also
observed in 19% of male rats given the initiation and promotion treatments, while nodular liver
lesions were not observed in control rats. Chlordecone (30 mg/kg) was not effective as an
initiating chemical following partial hepatectomy and promotion with sodium phenobarbital for
44 weeks. A significant sex difference was noted in the chlordecone promotion response at
doses of 3 and 9 mg/kg. Both the median number and the size of the GGT-positive foci were
increased in female rats as compared to males following DEN initiation and 27 weeks of
JANUARY 2008 56 DRAFT - DO NOT CITE OR QUOTE
-------
chlordecone promotion. In addition, hepatocellular carcinomas were observed in female rats
(11% at 3 mg/kg and 62% at 9 mg/kg) but were not found in male rats given the same initiation-
promotion treatment. Male rats exhibited only preneoplastic foci and nodular hyperplasia under
the condition of the two-stage assay. Similar concentrations of chlordecone were measured in
the livers of male and female rats, suggesting that enhancement of the tumor promotion response
is due to increased sensitivity of females and not altered pharmacokinetics.
Chlordecone was demonstrated to be a liver tumor promoter in a two-stage assay of
hepatocarcinogenesis (Sirica et al., 1989). The mode of action for liver tumor promotion by
chlordecone is unclear; however, liver toxicity and the subsequent repair/regeneration response
may play a role at high doses. Liver toxicity (i.e., focal necrosis, hypertrophy, congestion, and
fatty change) and decreased body weight gain were evident in male and female rats at doses that
induced liver tumor promotion. The mode of action for liver tumor promotion by chlordecone is
unclear. Liver toxicity and the subsequent repair/regeneration response may play a role at high
doses. However, this study did not evaluate histological evidence of liver toxicity at lower dose
levels that were shown to cause an increase in GGT-positive foci in male rats. Therefore, the
study did not provide a clear indication of whether liver toxicity precedes liver tumor promotion
(Sirica et al., 1989).
Some in vitro evidence suggests that the promotion of liver tumors by chlordecone may
be related to suppression of proliferative control through inhibition of gap junctional cell-to-cell
communication. The metabolic cooperation between co-cultivated 6-thioguanine-sensitive and
resistant Chinese hamster V79 cells was used to evaluate intracellular communication via gap
junctions (Tsushimoto et al., 1982). 6-Thioguanine-sensitive cells are wild-type V79 cells that
are capable of metabolizing 6-thioguanine to a lethal substrate for nucleic acids that causes cell
death. Resistant cells lack the enzyme for 6-thioguanine metabolism; however, cell death can be
induced in these cells by a transfer of the lethal 6-thioguanine metabolite across gap junctions
from sensitive cells (i.e., metabolic cooperation). Chlordecone was shown to inhibit metabolic
cooperation in co-cultivated Chinese hamster V79 cells.
Chlordecone inhibition of cell-to-cell communication was also demonstrated in a dye
transfer study in embryonic palatal mesenchymal cells (Caldwell and Loch-Caruso, 1992).
Lucifer yellow was scrape-loaded into cell monolayers in the presence or absence of
chlordecone. The lucifer yellow dye is too large to cross the plasma membrane but can enter
cells through gap junctions. Junctional communication was demonstrated by the movement of
lucifer yellow fluorescence away from the scrape line. Chlordecone (20 |ig/mL) inhibited dye
transfer as demonstrated by the restriction of dye to cells near the scrape line. This effect was
reversible with a recovery of dye transfer ability 15 minutes after incubation with control culture
medium.
Chlordecone was shown to disrupt adherens junctions in human breast epithelial cells
(Starcevic et al., 2001). Human breast epithelial cells cultured on Matrigel (an extracellular
JANUARY 2008 57 DRAFT - DO NOT CITE OR QUOTE
-------
matrix) form lattice-like structures that were disrupted by incubation with 0.1 and 1.0 jiM
chlordecone (0.01 jiM chlordecone had no effect). Chlordecone was also demonstrated to
decrease the levels of the transmembrane proteins E-cadherin and p-catenin. These proteins are
components of the adherens junctions that mediate cell-to-cell interaction and may play a role in
development of neoplastic lesions.
The available data suggest that chlordecone, like many other halogenated hydrocarbons,
is not genotoxic, but may act as an epigenetic carcinogen and a tumor promoter. Chlordecone
shares similar characteristics with several other well- known tumor promoters. These features
include the following: (1) chlordecone induces hepatic enzyme induction (Trosko et al., 1983;
Williams, 1980); (2) tumors are found predominantly in rat or mouse livers (NCI, 1976a); (3)
chlordecone lacks reactive functional groups and is not genotoxic; (4) there is no evidence of
covalent binding to DNA; (5) chlordecone induces ornithine decarboxylase activity (ATSDR,
1995; Mitra et al., 1990; Kitchin and Brown, 1989); and (6) chlordecone inhibits gap-junctional-
mediated intercellular communication (Caldwell and Loch-Caruso, 1992; Tsushimoto et al.,
1982).
Most of the effects of chlordecone are thought to be produced by the parent compound,
primarily by interfering with the function of mitochondrial and cellular membranes. Disruption
of cellular homeostasis and energy production within the cell eventually leads to impaired
cellular function. In the liver, membrane perturbation and inhibition of transport proteins at the
bile canalicular membrane is thought to be related to chlordecone-induced hepatobiliary
dysfunction.
4.5.3. Structural Analog Data—Relationship to Mirex
Information on structural analogs can be instructive in predicting biological activity and
carcinogenic potential of an agent. Confidence in the conclusions of such a chemical
relationship is a function of how similar the analogs are in structure, metabolism, and biological
activity. Chlordecone is closely related to the chlorinated pesticide mirex in structure,
physiochemical properties, and biological activity.
Mirex is a fully chlorinated molecule, whereas chlordecone has a similar structure with
only the substitution of two chlorine atoms for a carbonyl group (a double-bonded oxygen atom).
This substitution impartsmore water solubility as compared to mirex. Both compounds have
very low vapor pressures and very high melting points and are crystalline solids at standard
conditions. A comparison of physiochemical properties of chlordecone and mirex is presented
below in Table 4-17.
JANUARY 2008 58 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-17. Physiochemical properties of chlordecone and mirex
Structure
Chemical formula
Molecular weight
Physical state
Octanol-water partition
coefficient
Water solubility
Vapor pressure
Chlordecone
Cl Cl Cl Cl
\ / \ /-"
\7 /
Cl^ ^Cl
\--CI
-------
observational studies of workers occupationally exposed to chlordecone found evidence of
hepatomegaly in 20 workers. Liver biopsies from 12 of these individuals showed histological
changes, including proliferation of the SER and cytoplasmic accumulation of lipofuscin
(Guzelian, 1982a; Guzelian et al., 1980; Taylor et al., 1978).
There is inadequate evidence in humans for the carcinogenicity of chlordecone and
mirex. Mirex has been shown to induce liver tumors in both sexes of rats and mice in chronic
feeding studies at similar dose levels as chlordecone. Incidence of liver tumors in chlordecone-
treated male and female rats and mice were found to be significantly elevated at 1.7, 2, 3.4, and
3.5 mg/kg-day. Increased incidence of liver tumors with chronic mirex exposure has been shown
in rats and mice at 3.8 and 7 mg/kg-day (NTP, 1990; Innes et al., 1969). In F344/N rats exposed
to 0, 0.007, 0.07, 0.7, 1.8, 3.8, and 7.7 mg/kg-day mirex in the diet, statistically significantly
increased incidences of combined liver adenomas and carcinomas were found in male and
female rats exposed to >3.8 mg/kg-day mirex (PWG, 1992; NTP, 1990). Incidences for liver
adenomas alone were statistically significantly elevated at concentrations >1.8 mg/kg-day in
male and >3.9 mg/kg-day in female F344/N rats compared with controls. In CD rats exposed
chronically in the diet to 0, 4, 7 (males), or 8 (females) mg/kg-day mirex, males showed
statistically significantly increased incidences of liver neoplastic nodules and hepatocellular
carcinomas at 7 mg/kg-day, whereas females showed increased incidences of neoplastic nodules
at 4 and 8 mg/kg-day, with no significant increases in hepatocellular carcinomas at either
exposure level (Ulland et al., 1977). In B6C3F1 and B6AKF1 mice exposed for life to 0 or 7
mg/kg-day mirex in the diet, liver tumors reported as hepatomas were found at statistically
significantly increased incidence in exposed males and females.
Liver tumors resulting from mirex and chlordecone exposure are generally described as
benign, well-differentiated masses without vascular invasion or metastases (PWG, 1992; NTP,
1990; Ulland et al., 1977; NCI, 1976a,b; Innes et al., 1969). The available studies on mirex or
chlordecone classified liver tumors as either neoplastic nodules or hepatocellular carcinoma
(Ulland et al., 1977; NCI, 1976a,b). However, classification of liver tumor types has changed
from the time chlordecone and mirex cancer bioassays were initially published in the 1970s. In
early studies, it was common for pathologists to use the term hepatocellular carcinoma for any
neoplastic lesion since it was felt that all such lesions had the capacity to become invasive and
metastasize. However, current practice is to distinguish between benign (hepatocellular
adenoma) and malignant (hepatocellular carcinoma) tumors. Both Ulland et al. (1977) and the
NCI study (1976a,b) characterized the observed hepatocellular carcinomas as well-differentiated
masses without vascular invasion or metastases. In vivo and in vitro genotoxicity studies for
mirex and chlordecone were generally negative. However, the available evidence for
chlordecone and mirex is inadequate to establish a mode of action by which these chemicals
induce liver tumors in rats and mice.
JANUARY 2008 60 DRAFT - DO NOT CITE OR QUOTE
-------
Mirex and chlordecone have exhibited similarities in reproductive effects. Decreased
sperm counts and testicular degeneration have been observed in animals (Larson et al., 1979a;
Yarbrough et al., 1981). Additionally, decreased production of litters in animals was observed
for both mirex and chlordecone (Cannon and Kimbrough, 1979; Gaines and Kimbrough, 1970).
It should be noted that although chlordecone and mirex have similar biological activity in
the liver at comperable dose levels, some of the observed noncancer effects for these structurally
related chemicals are dissimilar. For example, chlordecone exposure results in neurological
symptoms, most notably tremors, in experimental animals and in occupationally exposed humans
(Taylor, 1985, 1982; Linder et al., 1983; Guzelian, 1982a,b; Larson et al., 1979a; Taylor et al.,
1978). However, neurological effects have not been observed with mirex exposure (NTP, 1990;
Ulland et al., 1977; Innes et al., 1969). In addition, one of the most sensitive effects of mirex
exposure is the development of cataracts in offspring exposed in utero and lactationally, whereas
the development of cataracts in offspring does not occur as a result of chlordecone exposure.
Differences in distribution between chlordecone and mirex may contribute to differences in their
low-dose biological effects. For instance, it is known that mirex primarily localizes in adipose
tissue, whereas chlordecone preferentially accumulates in the liver (Hewitt et al., 1985; Morgan
et al., 1979; Cohn et al., 1978; Egle et al., 1978; Wiener et al., 1976; Kennedy et al., 1975).
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
Table 4-18 presents a summary of the noncancer results for the repeated-dose oral studies
of chlordecone toxicity in experimental animals. The primary noncancer health effects of
occupational exposure to chlordecone in humans and oral exposure in animals include liver
lesions, kidney effects (only in animals), neurotoxicity, and male reproductive toxicity. Other
reproductive effects (i.e., persistent vaginal estrus and impaired reproductive sucess) and
developmental effects also occur; however, the doses required to elicit these effects were
generally higher than those that resulted in other key effects.
JANUARY 2008 61 DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-18. Summary of noncancer results of repeat-dose studies for oral exposure of experimental animals to
chlordecone
Species
Rat
Mouse
Rat
Mouse
Rat
Rat
Dog
Mouse
Mouse
Sex
M
M
M
F
M
F
M/F
M/F
M/F
M/F
M/F
Average daily dose
(mg/kg-day)
0, 0.07
0, 10, 25, 50
0, 0.6, 1.7
0, 1.4, 2.0
0,3.4,3.9
0,3.5,7.0
0,0.3,0.5,1.6,3.9,
7.0
0, 0.06, 0.3, 0.5,
1.6,3.9,7.0
0,0.02,0.1,0.5
0, 1.9,5.6,7.0
0, 0.94
Exposure
duration
21 months
24 days
20 months
20 months
13 weeks
2 years
128 weeks
1 month prior to
mating, 100 days
after pairing
1 month prior to
mating, 5 months
after pairing
NOAEL
(mg/kg-day)
NDa
ND
ND
ND
ND
ND
0.5
0.06
0.1
1.9
ND
LOAEL
(mg/kg-day)
ND
10 (FELb)
0.6
1.4
3.4
3.5
1.6
0.3
0.5
5.6
0.94
Responses
Liver and thyroid
histopathology
Mortality and
neurotoxicity
Liver
histopathology,
neurotoxicity
Liver
histopathology,
neurotoxicity
Reproductive
toxicity
Kidney
histopathology
Decreased body
weight; organ to
body weight
changes
Reproductive
toxicity
Reproductive
toxicity
Comments
No statistically significant increase
in incidence due to small number
of animals tested and changes in
controls
Frank effect levels for
hyperexcitability, motor
incoordination, and tremor
Hyperplasia and tremors; kidney
inflammation observed at higher
doses
Hyperplasia and tremors
Testicular atrophy in a subset of
animals from the 2-year study
Glomerulosclerosis; higher doses
cause fatty changes, hyperplasia in
the liver, and tremors
Magnitude of body weight
reduction not reported; small
number of animals detract from
reliability of study
Decrease in the number of pairs
producing a second litter;
persistent vaginal estrus
Decrease in the number of pairs
producing a second litter
Reference
Chu et al.,
1981a
Huang etal.,
1980;
Fujimori et
al., 1982b
NCI, 1976a
NCI, 1976a
Larson etal.,
1979a
Larson etal.,
1979a
Larson etal.,
1979a
Huber, 1965
Good et al.,
1965
JANUARY 2008
62
DRAFT - DO NOT CITE OR QUOTE
-------
Table 4-18. Summary of noncancer results of repeat-dose studies for oral exposure of experimental animals to
chlordecone
Species
Rat
Rat
Mouse
Rat
Rat
Rat
Rat
Mouse
Sex
F
M
F
F
F
M
M
F
F
Average daily dose
(mg/kg-day)
0, 0.07, 0.4
0, 1.4
0, 1.7
0, 2, 4, 8
0,15
0,0.26,0.83, 1.67
0, 0.625, 1.25, 2.5,
5,10
0, 2, 6, and 10
0, 2, 4, 8, and 12
Exposure
duration
60 days prior to
mating and
throughout
gestation and
lactation
3 months
4 weeks
Days 14-20 of
gestation
90 days
10 days
Days 7-16 of
gestation
Days 7-16 of
gestation
NOAEL
(mg/kg-day)
ND
1.4
ND
ND
ND
0.26
ND
2
8
LOAEL
(mg/kg-day)
ND
ND
1.7
2
15
0.83
0.625
6
12
Responses
Developmental
toxicity
Reproductive
toxicity
Reproductive
toxicity
Reproductive and
developmental
toxicity
Sperm parameters,
neurotoxicity
Reproductive
toxicity
Developmental
toxicity
Developmental
toxicity
Comments
No neurobehavioral effects; no
change in pup body weight at 1, 7,
14, and 30 days of age; decreased
pup body weight at 100 days
Impaired reproductive success in
females; tremors, liver, and adrenal
lesions
Persistent vaginal estrus; higher
doses adversely affect follicle size
and condition
Persistent vaginal estrus in
offspring, decreased ovary weight,
increased adrenal weight
Decreased sperm motility and
viability, hyperexcitability, and
mild tremors
Decreased sperm concentration
Fetotoxicity (decreased fetal body
weight); maternal toxicity at lower
doses
Fetotoxicity (fetal mortality);
maternal toxicity at lower doses
Reference
Squibb and
Tilson, 1982
Cannon and
Kimbrough,
1979
Swartz et al.,
1988;
Swartz and
Mall, 1989
Gellert and
Wilson,
1979
Linderetal.,
1983
U.S. EPA,
1986c
Chernoff
and Rogers,
1976
Chernoff
and Rogers,
1976
aND = not determined.
bFEL = frank effect level.
JANUARY 2008
63
DRAFT - DO NOT CITE OR QUOTE
-------
4.6.1. Oral Exposure
Liver enlargement developed in 20 out of 32 workers exposed to high levels of
chlordecone for an intermediate to chronic exposure duration; however, evidence of significant
liver toxicity was not found (Guzelian, 1982a; Guzelian et al., 1980; Taylor et al., 1978).
Normal results were obtained for serum biochemistry, and liver biopsy samples showed
histological changes in the liver that were characterized as nonadverse in nature (see Section
4.1). Histological changes included proliferation of the SER and cytoplasmic accumulation of
lipofuscin. No evidence of fibrosis, cholestasis, or hepatocellular necrosis was found; however,
the exposure duration and latency period before examination were relatively short.
Histological changes in the liver have also been demonstrated in laboratory animals.
These effects include increased liver size and weight, hepatocellular hypertrophy, proliferation of
the SER, increased microsomal protein, CYP450 content, cytochrome c reductase activity, and
microsomal enzyme activity (see Section 3.3) (Gilroy et al., 1994; Hewitt et al., 1985;
Mehendale et al., 1978, 1977). Histopathological evidence of hepatotoxicity was also
demonstrated in animals following chronic exposure to chlordecone. The liver lesions observed
in male and female rats given chlordecone doses of 3 and 9 mg/kg biweekly (s.c.) for 27 weeks
(average daily doses of 0.86 and 2.6) included hepatocellular hypertrophy, congestion, mild fatty
change, focal necrosis, and occasional small nests of proliferated sinusoidal cells (Sirica et al.,
1989). Fatty changes and hyperplasia were also observed in rats given doses greater than 0.5
mg/kg-day for up to 2 years (Larson et al., 1979a).
Kidney toxicity was reported in laboratory animals, but was not observed in
occupationally exposed pesticide workers (Taylor, 1985, 1982; Guzelian, 1982a,b; Guzelian et
al., 1980; Sanborn et al., 1979; Cannon et al., 1978; Martinez et al., 1978; Taylor et al., 1978). It
is possible that the clinical signs of glomerulosclerosis (including proteinuria) were not observed
in occupationally exposed pesticide workers because of the relatively short exposure duration
(average exposure duration was 5-6 months), which may not be a sufficient duration for the
development of more obvious renal disease (nephropathy and frank proteinuria). It is unclear
whether clinical tests sufficient to detect glomerular damage were performed on the exposed
workers. Furthermore, a definitive diagnosis of glomerulosclerosis can only be diagnosed
through a kidney biopsy, which was not performed on any occupationally exposed worker.
Larson et al. (1979a) identified a chronic LOAEL of 0.3 mg/kg-day for proteinuria and increased
incidence of glomerulosclerosis in female Wistar rats with a corresponding NOAEL of 0.06
mg/kg-day. Renal effects were also reported in other studies at higher dose levels. NCI (1976b)
included summary tables in which chronic kidney inflammation in male Osborne-Mendel rats (at
0.6 mg/kg-day) and female Osborne-Mendel rats (at 2.0 mg/kg-day) was reported. Chu et al.
(1980) reported that 28 days of dietary exposure to chlordecone (at 0.07 mg/kg-day) produced
eosinophilic inclusions in proximal tubules in 2/10 male Sprague-Dawley rats.
JANUARY 2008 64 DRAFT - DO NOT CITE OR QUOTE
-------
Neurological symptoms, including tremor, headache, and irritability, were reported in
workers exposed to high doses of chlordecone for a period of months to years (see Section 4.1)
(ATSDR, 1995; Taylor, 1985, 1982; Guzelian, 1982a; Guzelian et al., 1980; Sanborn et al.,
1979; Cannon et al., 1978; Martinez et al., 1978; Taylor et al., 1978). Nearly half (7/16) of the
workers reported persistent symptoms (e.g., tremor, nervousness) 5 to 7 years later (Taylor,
1985). In laboratory animals, chlordecone has been shown to cause tremors, decreased motor
coordination, hyperexcitability, and an exaggerated startle response (Linder et al., 1983; Huang
et al., 1980; Larson et al., 1979a; NCI, 1976a). The hypothesized mode of action for
neurotoxicity relates to alteration in membrane transport proteins and disruption of calcium
homeostasis (see Section 4.4.3). In the chronic rat study by Larson et al. (1979a), liver lesions
were observed at slightly lower doses (>0.5 mg/kg-day) than those resulting in clinically
observable tremors (>1.6 mg/kg-day); however, hyperexcitability and mild tremors were
observed in a subchronic dietary study in rats at doses as low as 0.83 mg/kg-day (Linder et al.,
1983).
Chlordecone exposure in humans caused oligospermia, reduced sperm motility, and
decreased libido in a group of men who were occupationally exposed to chlordecone for periods
up to 1.5 years (see Section 4.1) (Taylor, 1985, 1982; Guzelian, 1982a; Taylor et al., 1978).
There was no evidence that the ability of these workers to father children was affected and male
reproductive parameters had returned to normal by 5 to 7 years following the cessation of
chlordecone exposure and treatment with cholestyramine to reduce chlordecone blood levels
(Taylor, 1982). Even though 2 of 7 workers sired children, there is no indication of the true
denominator of how many were trying to conceive and/or the fertility rate. Male reproductive
toxicity has also been observed in laboratory animal studies (Linder et al., 1983; Larson et al.,
1979a). Sperm parameters were altered by chlordecone in a subchronic dietary study (Linder et
al., 1983). Sperm viability, motility, and reserves in the right cauda epididymis were
significantly reduced at doses of 0.83 and 1.67 mg/kg-day but not at 0.26 mg/kg-day. The sperm
parameters evaluated appear to be a precursor effect in this study, because neither sperm
morphology nor sperm count in the epididymal fluid was affected at any dose. In addition,
reproductive performance (determined by number of pregnant females, number of live litters,
average live litter size, number of implants, percentage resorptions and fetal weight) was similar
across exposed and control groups. No gross or microscopic pathology of the male reproductive
system was found that could be attributed to chlordecone treatment, and recovery from the
reported sperm alterations was apparent 4.5 months following cessation of exposure. Decreased
sperm concentration was observed in rats exposed to chlordecone doses >0.625 mg/kg-day for 10
days (U.S. EPA, 1986c). Testicular atrophy was observed in rats at doses >1.6 mg/kg-day for 13
weeks (Larson et al., 1979a).
No information is available concerning chlordecone-induced reproductive effects in
women. Impaired reproductive success was, however, observed in mice and rats exposed to
JANUARY 2008 65 DRAFT - DO NOT CITE OR QUOTE
-------
chlordecone at doses of >1 mg/kg-day (see Section 4.3.1) (Cannon and Kimbrough, 1979; Good
et al., 1965; Huber, 1965). The mechanism responsible for impaired reproductive success is
unknown; however, chlordecone has been demonstrated to affect estrous cyclicity in female mice
(Swartz and Mall, 1989; Swartz et al., 1988; Huber, 1965). The doses required to induce
persistent vaginal estrus in mice were higher than the doses reported to alter sperm parameters in
male rats. Huber (1965) demonstrated that persistent vaginal estrus occurs within 8 weeks of
chlordecone treatment at doses of >5.6 mg/kg-day. Similar effects on estrous cyclicity were
noted by Swartz and Mall (1989) and Swartz et al. (1988) within 2 weeks of chlordecone
administration at dose levels of 2, 4, and 8 mg/kg-day. After 4 and 6 weeks of treatment,
ovulation was reduced in the highest chlordecone treatment group (8 mg/kg-day), which resulted
in statistically significantly lower numbers of ovulated oocytes relative to vehicle controls
(Swartz et al., 1988). Persistent vaginal estrus was also observed in offspring of female rats
given 15 mg/kg-day chlordecone by gavage on gestational days 14-20 (Gellert and Wilson,
1979). Female offspring also exhibited significantly decreased ovarian weight, significantly
increased adrenal weight (relative to vehicle controls), and a decrease in the number of animals
ovulating.
No information is available concerning developmental effects of chlordecone exposure in
humans. Laboratory animal studies demonstrated developmental toxicity in rats and mice at
dose levels that also produced maternal toxicity (Chernoff and Rogers, 1976). Chernoff and
Rogers (1976) demonstrated that chlordecone administration via gavage during days 7 to 16 of
gestation induced maternal toxicity in mice and rats at doses >2 mg/kg-day, while fetotoxicity
did not occur until doses of >6 mg/kg-day in rats and >12 mg/kg-day in mice. Maternal toxicity
was evidenced by decreased body weight and increased liver to body weight ratios. Fetotoxicity
in rats was observed as significantly depressed fetal body weight and delayed ossification in 6
and 10 mg/kg-day dose groups and significantly increased incidences of fetuses with enlarged
renal pelvis, edema, undescended testes, or enlarged cerebral ventricles in the 10 mg/kg-day
group relative to controls. Signs of fetotoxicity in mice were observed only in the highest dose
group and consisted of significantly increased fetal mortality.
The mode of action of chlordecone-induced toxicity is not completely understood.
However, limited evidence suggests that chlordecone may interact with cell membranes and
affect the membrane transport proteins (e.g., Mg2+-ATPase, Ca2+-ATPase) that are responsible
for cellular homeostasis and energetics. Disruption of cellular homeostasis and energy
production within the cell leads to impaired cellular function. In the central nervous system,
altered calcium homeostasis leads to changes in neurotransmitter activity (e.g., alpha-
noradrenergic, dopaminergic, and serotonergic systems) that may be related to chlordecone-
induced tremor and exaggerated startle response (Vaccari and Saba, 1995; Brown et al., 1991;
Herr et al., 1987; Tilson et al., 1986; Uphouse and Eckols, 1986; Chen et al., 1985; Desaiah,
1985; Gerhart et al., 1985, 1983, 1982; Hong et al., 1984; Fujimori et al., 1982b; Squibb and
JANUARY 2008 66 DRAFT - DO NOT CITE OR QUOTE
-------
Tilson, 1982; Hwang and Van Woert, 1979). In the liver, membrane perturbation and inhibition
of the active transport of glutamate at the bile canalicular membrane is thought to be related to
chlordecone-induced hepatobiliary dysfunction (Teo and Vore, 1991). Chlordecone also inhibits
oligomycin-sensitive Mg2+-ATPase activity in the rat bile canaliculi-enriched fraction of the liver
(Curtis, 1988). Treatment of rats with chlordecone and CCU caused a decrease in liver ATP
levels and an inhibition of oligomycin-sensitive Mg2+-ATPase (Kodavanti et al., 1990).
Chlordecone alters calcium homeostasis in hepatocytes, leading to a decline in glycogen storage
and a reduced energy status (Kodavanti et al., 1993, 1990). Chlordecone-CCU administration
caused an inhibition in microsomal and mitochondrial calcium uptake and a decrease in the high
affinity component of hepatic plasma membrane Ca2+-ATPase.
An estrogenic mode of action is generally considered to be involved in the reproductive
toxicity of chlordecone. Testicular atrophy, altered sperm characteristics, persistent vaginal
estrus, and anovulation observed in chlordecone-treated laboratory animals (Swartz et al., 1988;
U.S. EPA, 1986c; Linder et al., 1983; Larson et al., 1979a; Huber, 1965) mimic the effects
produced by excessive estrogen. Mechanistic studies demonstrate that chlordecone binds to the
estrogen receptor, as well as other endocrine receptors (see Section 4.4.4).
4.6.2. Mode-of-Action Information—Glomerular Lesions
The mechanism by which chronic dietary chlordecone exposure in rats results in
glomerular lesions is unclear. Larson et al., 1979a observed a significant, dose related increase
in the incidence and severity of renal lesions in female Wistar rats in the 0.3, 0.5, and 1.6 mg/kg-
day dose groups. An apparent increase in proteinuria, a clinical sign of glomerular damage, was
also observed in female rats, starting at 0.3 mg/kg-day (see also Section 4.2.2.1).
The Larson (1979a) study itself does not inform the potential mode of action of the
observed glomerular lesions; however, there are some data to suggest that the effect may be
mediated through an autoimmune mechanism. Glomerular damage is often, though not
exclusively, mediated through immune mechanisms (U.S. DHHS, 2006). Some evidence (Sobel
et al., 2006, 2005) suggests that chlordecone may accelerate glomerular lesions in susceptible
animals by way of increased deposition of immune complexes in the glomeruli (see Section
4.4.5). In similar treatment protocols Sobel et al. (2006, 2005) implanted female (NZB x
NZW)Fi mice with sustained-release pellets containing 0.001, 0.01, 0.1, 0.5, 1, or 5 mg
chlordecone subcutaneously above the shoulders. Ovary intact mice treated with either 1 mg or
5 mg chlordecone pellets developed anti-dsDNA and antichromatin autoantibody liters
significantly earlier than controls. Additionally, immunohistofluorescence analysis of renal
sections from a subset of animals treated for 8 weeks with 1 mg chlordecone showed enhanced
deposits of IgG immune complexes as compared with untreated controls. The histopathology
associated with renal disease was similar between chlordecone-treated mice and controls.
JANUARY 2008 67 DRAFT - DO NOT CITE OR QUOTE
-------
An alternate theory holds that chlordecone damages the glomeruli directly. Chlordecone
predominantly binds plasma proteins and lipoproteins (especially albumin and HDL); this
binding has been demonstrated in exposed workers and in animal models (Soine et al., 1982;
Skalsky et al., 1979). The glomeruli are the functional units of the kidney that are predominantly
responsible for filtering high molecular weight proteins, including albumin, from the blood (Hart
and Kinter, 2005). Therefore, this region of the kidney may be subjected to relatively high
concentrations of chlordecone that could potentially result in direct chemical insult. Distribution
studies of chlordecone in experimental animals (see Section 3.2) have indicated that chlordecone,
by various routes of exposure, predominantly localizes in the liver but is also distributed to the
kidneys (Belfiore et al., 2007; Heatherington et al., 1998; Hewitt et al., 1985; Kavlock et al.,
1980). A dermal study of chlordecone organ distribution found that kidney concentration was
second only to liver concentration (Heatherington et al., 1998).
Uncertainty surrounds the two proposed mechanisms for the observed glomerular damage
following chlordecone exposure. It is conceivable that chlordecone may not cause glomerular
damage per se but may accelerate or increase the severity of the disease in animals with
preexisting susceptibility to glomerular damage. For example, though a significant dose-
response relationship was seen in the principal study between glomerulosclerosis and increasing
doses of chlordecone, the control animals also exhibited a background incidence of glomerular
lesions, which was particularly high in the male rats (12% incidence in females and 55% in
males). In addition, Sobel et al. (2006, 2005) indicated that chlordecone exposure increased the
severity and accelerated the development of renal damage and autoantibodies in a susceptible
mouse strain, (NZB x NZW)Fi. However, a follow-up experiment by Sobel et al. (2006) treated
BALB/c mice, a strain in which spontaneous development of glomerular damage is rare, and
found that treatment of these mice with chlordecone for up to 1 year did not produce elevated
autoantibody liters or renal disease. Arguably, the two strains tested by Sobel et al. (2006)
represent two extremes in genetic propensity to autoimmunity in rodents and are not
representative of the genetic heterogeneity of human populations regarding autoimmune
susceptibility.
4.7. EVALUATION OF CARCINOGENICITY
4.7.1. Summary of Overall Weight of Evidence
Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a),
there is suggestive evidence of carcinogenic potential in humans based on data from an oral
cancer bioassay in rats and mice demonstrating an increase in the incidence of hepatocellular
carcinomas in both sexes of both species (NCI, 1976 a,b). This characterization lies at the high
end of the continuum for this weight of evidence descriptor. NCI (1976 a,b) demonstrated a
statistically significant increase in hepatocellular carcinomas in both sexes of mice. Male and
JANUARY 2008 68 DRAFT - DO NOT CITE OR QUOTE
-------
female rats exhibited increased incidences of hepatocellular carcinomas at high doses that were
statistically significant when compared with pooled controls. The incidence of hepatocellular
carcinomas was not statistically significant in comparison with matched controls for rats of either
sex. The tumor response was particularly robust in male and female mice at the highest doses
(Table 4-1). NCI (1976 a,b) also demonstrated a decrease in the time to tumor in both sexes of
both species. There are some limitations associated with the design and conduct of the only
cancer bioassay for chlordecone (NCI, 1976 a,b) which create uncertainty in the characterization
of the overall weight of the evidence for the carcinogenic potential of chlordecone in humans.
The major limitations in the NCI (1976 a,b) study are described below and in Section 4.2.2.1.
Issues related to the study design and conduct make it difficult to draw a confident conclusion
regarding human carcinogenic potential. It should be noted that mirex, a structurally similar
chemical, also induces hepatocellular adenomas or carcinomas in both sexes of rats and mice.
This weight of evidence conclusion collectively takes into consideration the NCI (1976a,b)
cancer bioassay and its limitations, the available human studies, and other chronic animal
bioassays.
There are no studies in humans that adequately assess the carcinogenic potential of
chlordecone. NCI (1976a,b) reported a statistically significant increased incidence of liver
tumors (hepatocellular carcinomas) in rats and mice, following dietary exposure to chlordecone
for 20 months. A strong liver tumor response was seen in both sexes of mice and female rats,
with a weak response noted among male rats. No other tumor types were significantly increased
in either rats or mice in this study. The NCI (1976a,b) study had several limitations in study
design, conduct, and reporting. One major issue included early mortality and toxicity, indicating
initial use of excessively high doses. Due to the initial toxicity and mortality, the study authors
adjusted dose levels to one-half to one-sixth of the initial dose levels. The adjustment in dosing
during the study led to inconsistent exposure concentrations and difficulty in determining time-
weighted-average daily dosage. In addition, some of the surviving male rats in the discontinued
high-dose group were moved into the next lower dose group at 6 weeks, resulting in a lack of
homogeneity in this dose group. Decreased survival was also seen in treated groups when
compared with matched controls for all dose groups except female mice (see Table 4.4). The
authors did not explain whether decreased survival was secondary to toxic or carcinogenic
effects. Survival to study termination for male rats in the low- and high-dose groups was
decreased 33 and 53% as compared with matched controls. However, liver tumor incidence was
low in these groups (1/50 and 3/44, respectively), indicating that decreased survival in male rats
was not due to carcinogenic effects. Further limitations of the NCI (1976a,b) study included
incomplete reporting in the published study. For example, about 10% of rats in the high-dose
groups of both sexes are unaccounted for by the authors. An additional study limitation includes
the use of only two dose groups, making characterization of a dose-response relationship
difficult.
JANUARY 2008 69 DRAFT - DO NOT CITE OR QUOTE
-------
Similarities in the tumor profile of chlordecone and mirex, a structurally related
chemical, have been observed in animals. A statistically significantly increased incidence of
liver tumors in F344/N and CD rats and B6C3F1 and B6AKF1 mice has been observed
following chronic oral exposure to mirex at similar dose levels as chlordecone. The liver tumors
resulting from exposure to mirex, similar to exposure to chlordecone, are described as
predominantly benign, well-differentiated masses without vascular invasion or metastases
(PWG, 1992; NTP, 1990; Ulland et al., 1977; NCI, 1976a,b; Innes et al., 1969). Mirex and
chlordecone also produce noncancer effects in the liver at similar doses. It should be noted that,
though chlordecone and mirex appear to have closely related biological activity and
carcinogenicity in the liver at similar dose levels (though the mode of action for each is
unknown), several noncancer effects reported following exposure to mirex and chlordecone are
dissimilar. For example, the characteristic neurotoxicity observed following exposure to
chlordecone has not been described for mirex.
The mode of carcinogenic action of chlordecone in the livers of rats and mice is
unknown. Most genotoxicity tests for chlordecone are negative. For the liver tumors in rats and
mice, some data suggest that chlordecone may induce cell proliferation and lead to a promotion
in the growth of preinitiated cells. However, key precursor events linked to observed cell
proliferation have not been identified. The liver tumors observed in the NCI cancer bioassay in
rats and mice were considered relevant to the assessment of the carcinogenic potential of
chlordecone.
No animal cancer bioassay data following inhalation exposure to chlordecone are
available. However, EPA's Guidelines for Carcinogen Risk Assessment (2005a) indicate that,
for tumors occurring at a site other than the initial point of contact, the carcinogenic potential
may apply to all routes of exposure that have not been adequately tested at sufficient doses.
Thus, for chlordecone, there is suggestive evidence of carcinogenic potential by any route of
exposure.
This is the first IRIS assessment for chlordecone. Therefore, no previous
characterization of cancer potential or quantitative cancer evaluation exists.
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
Few studies are available that directly assess the carcinogenic potential of chlordecone.
Limited data on the carcinogenic potential in humans can be garnered from observational studies
of a single group of 133 workers occupationally exposed to chlordecone at a chlordecone
manufacturing plant in Hopewell, Virginia, in the late 1970s (Taylor, 1985, 1982; Guzelian,
1982a; Guzelian et al., 1980; Sanborn et al., 1979; Cannon et al., 1978; Martinez et al., 1978;
Taylor et al., 1978). A subset of 32 of these workers with clinical signs or symptoms of
chlordecone toxicity and high chlordecone blood levels (>0.6 ug/mL at the time of diagnosis)
were examined specifically for hepatotoxicity (Guzelian et al., 1980). Hepatomegaly was
JANUARY 2008 70 DRAFT - DO NOT CITE OR QUOTE
-------
observed in 20 of 30 of these workers. However, liver biopsy samples taken from 12 of these
workers showed no evidence of liver neoplasia (Guzelian, 1982a; Guzelian et al., 1980). The
average exposure duration of these subjects was 5-6 months, and they were physically examined
for this study within 10 months of exposure cessation. Upon follow-up of the exposed workers
2-3 years after exposure cessation, hepatomegaly had resolved in all workers and biopsies were
negative for abnormal histopathological findings (Guzelian et al., 1980). Conclusions regarding
cancer from this study are limited by the small number of workers examined, uncertainties
concerning exposure dose and route, the relatively brief duration of exposures, and the absence
of a sufficient latency period for tumor development.
Occupational exposures to chlordecone also provide evidence for the preferential
accumulation of chlordecone in the liver. For example, in 32 workers exposed to chlordecone
for a period that ranged from 3 to 16 months, high concentrations of chlordecone were found in
blood, liver, and subcutaneous fat (Cohn et al., 1978). The ratio of the chlordecone
concentration in fat as compared to the chlordecone concentration in the blood was 7:1, which is
relatively low for a lipophilic organochlorine pesticide. However, the liver to blood
concentration ratio in exposed workers was reported to be 15:1 (Table 3-2). Chlordecone has
also been shown to bind plasma proteins and lipoproteins and preferentially accumulate in the
liver, where it is slowly eliminated, in experimental animals and exposed workers (Cohn et al.,
1978; Egle et al., 1978). Thus, due to the preferential accumulation of chlordecone in the liver,
humans may be susceptible to chlordecone-induced liver toxicity.
The human case reports and clinical observations of occupational chlordecone exposure
lack sufficient design, power, and follow-up to determine carcinogenic potential of chlordecone
in humans; however, the observations from these studies provide valuable information on human
susceptibility to chlordecone. A review of biological and epidemiological evidence of cancer
found no population-based studies on cancer in humans related to chlordecone exposure
(Ahlborg et al., 1995).
Animal studies provide suggestive evidence for the carcinogenic potential of
chlordecone. Chlordecone has been shown to induce liver tumors in Osborne-Mendel rats and
B6C3F1 mice in a single study performed by the National Cancer Institute (NCI, 1976a,b).
B6C3F1 mice (50/sex/group) and Osborne-Mendel rats (50/sex/group) were exposed to
chlordecone in the diet for 20 months. Dietary concentrations of chlordecone began at 0, 15, 30,
or 60 ppm for male rats and 0, 30, or 60 ppm for female rats. In mice, dietary concentrations of
chlordecone began at 0 or 40 ppm (two groups at this concentration) for males and 0, 40 or 80
ppm for females. During the course of the study, concentrations were reduced at least once in
each treatment group due to toxicity (see Figures 4-1 to 4-4). Time-weighted-average dietary
concentrations were 0, 8, or 24 ppm (0, 0.6, or 1.7 mg/kg-day) for male rats and 0, 18, or 26 ppm
(0, 1.4, or 2.0 mg/kg-day) for female rats. In mice, time-weighted-average dietary
concentrations were 0, 20, or 23 ppm (0, 3.4, or 3.9 mg/kg-day) for male mice and 0, 20, or 40
JANUARY 2008 71 DRAFT - DO NOT CITE OR QUOTE
-------
ppm (0, 3.5, or 7.0 mg/kg-day) for female mice. Liver tumors described as hepatocellular
carcinomas were observed in high-dose female rats at an incidence that was significantly
elevated compared with the pooled control incidence (0/100, 0/10, 1/49, and 10/45 in the pooled
control, matched control, and low-dose and high-dose groups, respectively). Incidences of male
rats with hepatocellular carcinomas were lower at 0/105, 0/10, 1/50, and 3/44, respectively. The
incidence of carcinomas in high-dose males was significant (p = 0.049) in comparison with
pooled controls. The incidence of hepatocellular carcinomas was not statistically significant in
comparison with matched controls for rats of either sex. A significant dose-response trend was
observed for the incidence of hepatocellular carcinoma in both male and female rats (Cochran-
Armitage test conducted for this review). In mice, statistically significant elevated incidences of
hepatocellular carcinomas were found in both exposed groups compared with matched and
pooled control incidences (NCI, 1976a). Incidences for matched control, low-, and high-dose
groups were 6/19, 39/48, and 43/49 for male mice and 0/10, 26/50, and 23/49 for female mice.
No other tumor types in rats or mice were found to be significantly elevated in this study.
Decreases in survival rates and decreased body weight gain were observed in all animal
groups except the low- and high-dose female mice (see Table 4.4). A robust liver tumor
incidence of 26/50 (52%) was observed in the low-dose group (3.5 mg/kg-day) of female mice, a
group that had survival rates and body weight gains that were comparable with controls. While
is it is true that high toxicity was observed in the high-dose groups (specifically of male rats and
mice), the conclusion that high toxicity is required for tumor induction may not be warranted.
Significant limitations in the study design and outcome with respect to toxicity and
mortality exist for the NCI (1976a,b) cancer bioassay. The primary limitation of the NCI
(1976a,b) bioassay relates to the dose selection. The initial dietary concentrations in the high-
dose groups were excessively high and induced high mortality, tremors, anemia, and dermatitis
in both sexes of both species. During the course of the study, concentrations were reduced at
least once in each treatment group due to toxicity (see Figures 4-1 to 4-4). In both male rats and
mice, the initial high-dose group was discontinued due to excessive toxicity and mortality
(animals were sacrificed). Issues related to the dosing regimen of this study make it unsuitable
for quantification of cancer risk. Because of changes in chlordecone dietary exposure levels, the
dose metric related to the development of liver tumors cannot be determined. The study reports
time-weighted-average dietary concentrations for chlordecone in rats and mice; however, the
tumorigenic effects observed may not occur following chronic exposure to these lower average
concentrations. For example, it is not known whether the high initial dietary concentrations
caused significant early liver injury resulting in the subsequent development of the observed
liver tumors.
Conclusions from cancer bioassays utilizing potentially excessive doses are regarded with
caution for several reasons. Doses of an agent that cause high toxicity to the animals may result
in early deaths directly resulting from toxicity, which could decrease the ability of the assay to
JANUARY 2008 72 DRAFT - DO NOT CITE OR QUOTE
-------
detect tumor effects. Animal mortality in the NCI (1976a,b) study was high in comparison to
controls; however, this did not prevent the detection of elevated rates of hepatocellular
carcinoma in the high-dose groups. Alternately, there is concern that high doses may result in
tumor effects that are secondary to toxic effects (e.g., cytotoxicity) or altered toxicokinetics (U.S.
EPA, 2005a). It is possible that high doses of chlordecone used in the NCI study resulted in
tumor effects that were secondary to liver cytotoxicity and thus would not be likely at low doses.
However, there is not sufficient data to support this mode of action. In the absence of data that
indicate that direct liver cytotoxicity at high doses precedes tumor development, the increased
incidence of liver tumors observed in the NCI cancer bioassay cannot be completely discounted.
There are no data to support the concern that elevated levels of hepatocellular carcinoma
detected by the NCI study in rats and mice are the direct result of altered toxicokinetics from
excessive chlordecone levels. In fact, animal data support the conclusion that the liver is
especially sensitive to chlordecone-induced lesions even at very low doses that do not result in
overt toxicity to the animal or decreased survival (Chu et al., 1981a; Larson et al., 1979a).
Additionally, chlordecone has been demonstrated in humans and animals to preferentially
accumulate in the liver (Cohen et al., 1987; Hewitt et al., 1985; Egle et al., 1978). Therefore it is
not likely that liver tumors arising after high exposures to chlordecone are due to altered
toxicokinetics.
Besides the NCI (1976a,b) cancer bioassay, Larson et al. (1979a) and Chu et al. (198la)
are the only additional chronic dietary studies of chlordecone exposure in animals. Larson et al.
(1979a) fed groups of Wistar rats (40/sex/group) diets estimated to result in dose levels of 0,
0.06, 0.3, 0.5, 1.6, 3.9, or 7.0 mg/kg-day for up to 2 years. Increased incidence of liver lesions
(characterized as fatty changes and hyperplasia) were seen in females at 0.5 mg/kg-day and in
males at 1.6 mg/kg-day. Liver lesions in three females in the 0.5 mg/kg-day group and one
female and two males in the 1.6 mg/kg-day group were described by the authors as being
possibly carcinomatous in nature, though the authors reported that an independent review by four
pathologists was equivocal. Therefore, this study can only be considered to provide a lack of
positive evidence for chlordecone carcinogenicity in Wistar rats. However, it should be noted
that very few animals were available for pathological examination at the end of the study,
limiting the study's power to detect carcinogenic effects.
A chronic dietary exposure study by Chu et al. (1981a) detected an apparent increase in
liver lesions in rats in the single chlordecone exposure group (5/6 compared to 3/7) of
0.07 mg/kg-day but did not report tumors. However, the very small number of animals and use
of only a single low-dose group severely limit this study's power to assess carcinogenic
potential. Additionally, neither toxicity nor changes in body weight gain were observed in the
dose tested. Therefore, the dose utilized cannot be considered adequately high to detect
carcinogenic potential for chlordecone.
JANUARY 2008 73 DRAFT - DO NOT CITE OR QUOTE
-------
The structurally related chemical mirex has been shown to induce liver tumors in both
sexes of rats and mice in chronic feeding studies at similar dose levels as chlordecone.
Incidences of liver tumors in chlordecone-treated male and female rats and mice were found to
be significantly elevated at 1.7, 2, 3.4, and 3.5 mg/kg-day. Increased incidence of liver tumors
(adenomas or carcinomas) with chronic mirex exposure has been shown in rats and mice at 3.8
and 7 mg/kg-day (PWG, 1992; NTP, 1990; Ulland et al., 1977; Innes et al., 1969). Liver tumors
resulting from mirex and chlordecone exposure are generally described as benign, well-
differentiated masses without vascular invasion or metastases (PWG, 1992; NTP, 1990; Ulland
et al., 1977; NCI, 1976a,b; Innes et al., 1969). The available studies on mirex or chlordecone
classified liver tumors as either neoplastic nodules or hepatocellular carcinoma (Ulland et al.,
1977; NCI, 1976a,b). In vivo and in vitro genotoxicity studies for mirex and chlordecone were
generally negative. However, the available evidence for chlordecone and mirex is inadequate to
clearly establish a mode of action by which these chemicals induce liver tumors in rats and mice.
Chlordecone and mirex exposure in experimental animals results in similar noncancerous liver
lesions that may be precursor lesions to the development of liver tumors. Liver lesions common
to mirex and chlordecone include hypertrophy, hyperplasia, fatty changes, cytoplasmic
vacuolation, and anisokaryosis (NTP, 1990; Chu et al., 1981b,c; Larson et al., 1979a,b; NCI,
1976a,b). However, though chlordecone and mirex appear to have related biological activity and
carcinogenicity in the liver, this evidence is limited by the observation of several dissimilar
noncancer effects.
In summary, under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA,
2005a), there is suggestive evidence of carcinogenic potential for chlordecone in humans. This is
based primarily on a statistically significant increase in liver tumors in both sexes of mice in
response to oral chlordecone exposure in a cancer bioassay by the National Cancer Institute
(NCI, 1976a,b). Male and female rats exhibited increased incidences of hepatocellular
carcinomas at high doses that were statistically significant when compared with pooled controls.
The incidence of hepatocellular carcinomas was not statistically significant in comparison with
matched controls for rats of either sex. Uncertainty exists due to limitations in the NCI study
related to design and conduct (as reviewed above and in Section 4.2.2.1.).
4.7.3. Mode-of-Action Information
The majority of studies on chlordecone were negative for genotoxic activity in a variety
of short-term in vitro and in vivo assays (see Section 4.4.5). One hypothesis for the mode of
action of chlordecone induced tumorigenicity is sustained proliferation of spontaneously
transformed liver cells, resulting in the eventual formation of liver tumors. Proliferative liver
lesions (hyperplasia) were found in a chronic dietary study in Wistar rats at doses greater than
0.5 mg/kg-day in females and 1.6 mg/kg-day in males (Larson et al., 1979). Additionally, the
NCI (1976a,b) chronic dietary cancer bioassay that reported increased incidences of liver tumors
JANUARY 2008 74 DRAFT - DO NOT CITE OR QUOTE
-------
in both sexes of rats and mice also noted extensive liver hyperplasia in both sexes of both
species. Though the incidence of hyperplasia was not noted in the study, the authors reported
that the incidence of hyperplasia in the matched control mice was low as compared to the treated
groups. In rats, the authors reported that no liver hyperplasia was seen in the matched controls.
Chlordecone was demonstrated to be a liver tumor promoter, rather than an initiator or a
complete hepatic carcinogen, in a two-stage tumor promotion assay in male and female Sprague-
Dawley rats (Sirica et al., 1989). This study also demonstrated a greater tumor response in
female rats, suggesting that hormonal involvement may be important in the promotion of
chlordecone-induced liver tumors. The NCI (1976a,b) study provides further support for this
potential mode of action for chlordecone. Specifically, the authors reported an increased
incidence of liver tumors and shorter time to tumor formation in female rats exposed to the high
dose compared to male rats exposed to the high dose (NCI, 1976a).
Chlordecone is one of a large number of organochlorine chemicals that produce liver
tumors in rodents and do not exhibit genotoxicity in short-term tests. Many of these pesticides
(including chlordane, heptachlor, and hexachlorocyclohexane) have been shown to promote liver
tumors in rodent livers when administered after an initiating dose of a known carcinogen (Demi
and Oesterle, 1987; Williams, 1983; Williams and Numoto, 1984). However, the mode of action
by which chlordecone produces liver tumors is unknown.. Precursor events in which
chlordecone may promote proliferation of transformed liver cells are uncertain, and data
regarding a plausible temporal progression from chlordecone-induced liver lesions to eventual
liver tumor formation are not available. Therefore, the available evidence is inadequate to
clearly establish a mode of action by which chlordecone induces liver tumors in rats and mice.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.8.1. Possible Childhood Susceptibility
Neurological studies suggest that the immature brain may be sensitive to subtle effects
from chlordecone exposure. As reported in Section 4.3, exposure of female rats to chlordecone
for 60 days prior to mating through lactation day 12 produced subtle neurological changes in
male but not female offspring later in life that suggested an alteration in dopamine sensitivity
(Squibb and Tilson, 1982). An abstract by Rosenstein et al. (1977) suggested altered brain
activity from gestational and lactational exposure of rats to chlordecone. Brain electrical activity
(recorded by EEG) was significantly different in the pups, but not the dams, exposed to
chlordecone (maternal dose was 1, 2, or 4 mg/kg-day orally in corn oil) at 24 days postpartum.
No additional study details concerning this effect are provided. In a lactation exposure study,
Sprague-Dawley rat pups were exposed to chlordecone in milk by treating lactating dams
immediately after birth with 0 (corn oil vehicle) or 2.5 mg/kg-day chlordecone by gavage (Jinna
et al., 1989). In vitro assays of brain P2 fractions showed that the exposed pups (through day 20)
JANUARY 2008 75 DRAFT - DO NOT CITE OR QUOTE
-------
exhibited increased activity of Na+, K+, and Ca++-ATPase activity. As compared to effective
doses in adult rats (8.3 or 10 mg/kg-day orally for 3 days [Kodavanti et al., 1990, and Desaiah et
al., 1980, respectively]), the exposure doses expected via lactation are lower, suggesting that the
maturing ATPases of neonatal rats may be more sensitive to chlordecone exposure. At the
cellular level, Hoskins and Ho (1982) also reported significant differences in calcium content and
subcellular distribution in brain in adult (24 weeks old) as compared to young (4-6 weeks old)
male ICR mice following acute oral chlordecone exposure (25 mg/kg-day in corn oil).
In summary, some studies have indicated that developing animals may be more
susceptible to subtle neurological effects of chlordecone including alterations in dopamine
sensitivity, ATP-ase activity, calcium concentration and subcelluar distribution, and EEG
activity.
4.8.2. Possible Gender Differences
The extent to which men and women differ in susceptibility to chlordecone toxicity is not
known. No human data are available to suggest that there are gender differences in the toxicity
or carcinogenicity of chlordecone.
In the NCI (1976a) bioassay of chlordecone carcinogenicity, a strong liver tumor
response was seen in female rats, and only a weak response was noted among male rats. Tumors
were seen in both genders of mice; however, female mice were more resistant to the lethal
effects of chlordecone at high doses. A significant sex difference was noted in the liver tumor
promotion response in a two-stage assay of hepatocarcinogenesis (Sirica et al., 1989). Both the
median number and the size of the GGT-positive foci were increased in female rats as compared
to males following DEN initiation and 27 weeks of chlordecone promotion. In addition,
hepatocellular carcinomas were observed in female rats but were not found in male rats given the
same initiation-promotion treatment. Similar concentrations of chlordecone were measured in
the livers of male and female rats, suggesting that enhancement of the tumor promotion response
is due to increased sensitivity of females and not altered pharmacokinetics. It is possible that the
estrogenic properties of chlordecone may play a role in the sensitivity of female rats to tumor
promotion. Female rats in this study were also more susceptible to decreases in body weight
gain, suggesting that enhanced toxicity may play a role in tumor promotion; however,
histological examination of noncancerous portions of the liver did not indicate significant gender
differences in liver toxicity.
Chlordecone induces reproductive effects in both male and female laboratory
animals.However, some evidence exists to suggest that female reproductive toxicity has a larger
effect on reproductive success at the same chlordecone dose level. Reproductive toxicity has
been demonstrated by altered sperm parameters, testicular atrophy, altered estrous cyclicity, and
impaired reproductive success in animals. Although the most sensitive endpoint evaluated
appeared to be alterations in sperm parameters induced by subchronic chlordecone exposure in
JANUARY 2008 76 DRAFT - DO NOT CITE OR QUOTE
-------
male rats (Linder et al., 1983), these decreases were observed at doses where reproductive
success was unaffected. Effects on the estrous cycle and ovulation are observed at higher doses
as compared to sperm effects (Swartz and Mall, 1989; Swartz et al., 1988; Huber, 1965);
however, a crossover study in rats that paired control males with treated females and control
females with treated males suggests that female reproductive toxicity had a larger effect on
reproductive success at the same chlordecone dose level (Cannon and Kimbrough, 1979). In
male and female rats fed diets containing 25 ppm chlordecone (1.4 or 1.7mg/kg-day) for 3
months, 12 of 20 pairs of treated males and control females produced offspring, while none of
the 20 pairs of treated females and control males produced offspring.
JANUARY 2008 77 DRAFT - DO NOT CITE OR QUOTE
-------
5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
The only available data concerning health effects of chlordecone in humans are derived
from studies of a single group of 133 men exposed occupationally to chlordecone in the late
1970s at a chlordecone manufacturing facility in Hopewell, Virginia (Taylor, 1985, 1982;
Guzelian, 1982a; Guzelian et al., 1980; Sanborn et al., 1979; Cannon et al., 1978; Martinez et al.,
1978; Taylor et al., 1978). Due to inadequate industrial safety measures at the factory,
substantial inhalation, dermal, and oral exposures likely occurred (Cannon et al., 1978). Toxicity
observed in the exposed workers included effects on the nervous system, liver, and reproductive
system. Of the 133 men, 76 experienced neurological symptoms, especially tremors,
nervousness, and headaches, sometimes persisting for as long as 9-10 months after cessation of
exposure and the start of treatment (Cannon et al., 1978). In addition, a subset of the men
experienced reproductive effects, including oligospermia, reduced sperm motility, and decreased
libido (Taylor, 1982). A subset of 32 of the occupationally exposed workers with clinical signs
or symptoms of chlordecone toxicity and high chlordecone blood levels (>0.6 ug/mL at the time
of diagnosis) were examined specifically for hepatotoxicity (Guzelian et al., 1980).
Hepatomegaly was observed in 20 of 32 workers. Minimal elevation (less than two fold) of
serum alkaline phosphatase (SALP) was noted in seven patients; however, other liver enzymes
were normal including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and
y-glutamyl transpeptidase (GGT) (Guzelian et al., 1980). Sulfobromophthalein retention, a
measure of liver clearance, was normal in a subset of 18 workers tested (Guzelian et al., 1980).
Upon biopsy of 12 workers with hepatomegaly, histological changes included proliferation of the
smooth endoplasmic reticulum (SER) and cytoplasmic accumulation of lipofuscin. These
changes in the liver were characterized by the authors as nonadverse in nature and were
suggested to be adaptive changes rather than a reflection of hepatotoxicity (Guzelian, 1982a,b;
Guzelian et al., 1980; Taylor et al., 1978). Upon follow-up of the exposed workers 2-3 years
after exposure cessation, hepatomegaly had resolved in all workers and biopsies were negative
for abnormal histopathological findings (Guzelian et al., 1980).
Because of uncertainties regarding exposure routes and exposure levels at the facility,
NOAELs or LOAELs could not be established for the observed neurological, liver, and
reproductive effects in the occupationally exposed workers. Additionally, workers may have had
concomitant exposure to the chemical precursors used to manufacture chlordecone. Because of
these major uncertainties, health effects data in these workers are unsuitable for derivation of an
RfD.
JANUARY 2008 78 DRAFT - DO NOT CITE OR QUOTE
-------
The toxicity database for oral exposure in laboratory animals includes a few chronic
duration studies (Chu et al., 198la; Larson et al., 1979a, NCI, 1976a) and several subchronic
studies (see Section 4.5 and Table 4-18).
Chu et al. (1981a) fed rats (10/group) chlordecone at 0.07 mg/kg-day for 21 months. The
authors reported an increase in liver lesions (described as pericentral cytoplasmic vacuolation
with mild anisokaryosis) compared to the control group (5/6 compared to 3/7). Chu et al.
(1981a) also reported an apparent increase in thyroid lesions (described as mild degenerative and
proliferative changes in the epithelium). However, because of small study size and high
incidence of effects in the controls, these increases were not statistically significant (Chu et al.
198 la). Thus, due to limited study size, dosing regimen, and high incidence of effects in the
control group, this study was not selected as the principal study.
NCI (1976a,b) conducted a 20-month feeding study in B6C3F1 mice and Osborne-
Mendel rats. Though treatment groups consisted of 50/sex/group for both rats and mice, only 10
(19 for male mice) matched controls per sex were used. Pooled control groups (from the same
laboratory with birth dates within 3-4 months of the treatment groups) contained about
100/sex/group. During the course of the study, toxicity and mortality in the high-dose groups
prompted the investigators to reduce dietary chlordecone concentrations to one-half to one-sixth
of the previous levels. The resulting time-weighted-average dietary concentrations were 0, 8, or
24 ppm (0, 0.6, or 1.7 mg/kg-day) for male rats and 0, 18, or 26 ppm (0, 1.4, or 2.0 mg/kg-day)
for female rats. In mice, time-weighted-average dietary concentrations were 0, 20, or 23 ppm (0,
3.4, or 3.9 mg/kg-day) for male mice and 0, 20, or 40 ppm (0, 3.5, or 7.0 mg/kg-day) for female
mice. Noncancer effects reported in response to chlordecone treatment included tremors,
dermatologic changes, and liver lesions, though incidences for these effects were not reported.
The observed liver lesions were characterized as extensive hyperplasia and atypia in both male
and female mice in both dose groups.
This study exhibits several design and conduct issues which limit its interpretation (as
reviewed in Section 4.2.2.1). These issues include incomplete reporting (lack of incidence data
on observed liver effects in treated animals and controls), inconsistent dose levels (making it
difficult to define a dose-response relationship for liver lesions), use of only two dose groups,
high early toxicity and mortality in high-dose male animals, decreased 2-year survival in most
dose groups, and low numbers of matched controls (10/sex/group). Though the evidence of liver
hyperplasia in rats and mice in the NCI (1976a) study provide qualitative information indicating
that the liver is a target organ of chlordecone toxicity, for the reasons discussed above, this study
is not adequate to support quantitative risk assessment.
Larson et al. (1979a) fed groups of Wistar rats (40/sex/group) diets estimated (based on
graphically depicted food consumption and body weight data) to result in dose levels of 0, 0.06,
0.3, 0.5, 1.6, 3.9, or 7.0 mg/kg-day for up to two years. All rats in the highest two dose groups
died within the first 6 months. Though the two highest dose groups were uninformative because
JANUARY 2008 79 DRAFT - DO NOT CITE OR QUOTE
-------
of high mortality, four acceptable low-dose exposure groups exist (with adequate numbers of
animals). The most sensitive effects observed in this study include kidney lesions in female rats,
testicular atrophy in males, and liver lesions in both sexes. The authors reported increased
incidence of liver lesions and an increase in relative liver weights in female rats at 0.5 mg/kg-day
and male rats at 1.6 mg/kg-day. The liver lesions observed were characterized primarily as fatty
changes and hyperplasia.
In addition to liver lesions and testicular effects, Larson et al. (1979a) also observed a
significant, dose-related increase in the incidence and severity of renal lesions in female Wistar
rats in the 0.3, 0.5, and 1.6 mg/kg-day dose groups. The background incidence of renal lesions
in male rats was high (56% as compared to 12% in female rats) and, as such, effects in dosed
animals did not achieve statistical significance. An apparent increase in proteinuria, a clinical
sign of glomerular damage, was observed in female rats, starting at 0.3 mg/kg-day, though data
from individual animals were not reported, precluding statistical analysis for this endpoint.
Larson et al. (1979a) identified a LOAEL of 0.3 mg/kg-day for proteinuria and increased
incidence of glomerulosclerosis in female Wistar rats with a corresponding NOAEL of 0.06
mg/kg-day.
A supporting study by Sobel et al. (2005) found that chlordecone, at doses estimated to
be >0.2 mg/kg-day, increased the severity and decreased the latency of glomerular disease in
subcutaneously treated mice of a strain known to be susceptible to autoimmunity mediated
glomerulonephritis, (NZB x NZW)Fi. Female ovariectomized mice were exposed
subcutaneously to sustained-release pellets containing 0.01, 0.1, 0.5, or 1.0 mg chlordecone for
up to 30 weeks. Mice treated with 0.5 mg chlordecone pellets (calculated by the authors as an
average exposure level of 0.20 mg/kg-day) developed renal impairment (proteinuria and
glomerulonephritis) significantly earlier than did ovariectomized controls (p < 0.05). Renal
sections from the chlordecone-treated mice demonstrated severe proliferative glomerulonephritis
with the deposition of immune complexes. A follow-up study by the same group (Sobel et al.,
2006), utilizing the same doses and protocol, found that chlordecone treatment of BALB/c mice
(a strain not prone to autoimmune disease or glomerular lesions) for up to a year did not produce
elevated autoantibody liters or renal disease.
A study by Chetty et al. (1993c) indicates that chlordecone treatment significantly
elevates serum indicators of kidney (specifically glomerular) and liver damage in rats treated for
15 days. Male Sprague-Dawley rats (six/group) were treated with 0, 1, 10, 50, or 100 ppm
chlordecone in the diet (purity unspecified) for 15 days. Based on the reported average animal
weight (175 g) and food intake values (U.S. EPA, 1988) the average doses were calculated as
0.1, 1.0, 4.9, and 9.7 mg/kg-day. After 15 days of chlordecone exposure, serum levels of total
protein, urea nitrogen, uric acid, creatinine, serum glutamic oxaloacetic transaminase (SGOT),
serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase (SALP), and serum
creatine kinase (SCK) were measured. SGPT was elevated at doses starting at 1.0 mg/kg-day,
JANUARY 2008 80 DRAFT - DO NOT CITE OR QUOTE
-------
additionally all other serum enzymes tested were statistically significantly elevated at the highest
dose tested (9.7 mg/kg-day). The alterations of serum enzyme levels of SGOT, SGPT, SCK, and
SALP suggest chlordecone-induced liver damage. Urea nitrogen was statistically significantly
elevated over controls at doses >4.9 mg/kg-day. At 9.7 mg/kg-day, urea nitrogen, uric acid and
creatinine were statistically significantly elevated. Urea nitrogen is the end product of protein
catabolism in most species; >90% is excreted via the kidney, being freely filtered at the
glomerulus but also diffusible out of the tubule (Hart and Kinter, 2005). Creatinine is also a
compound whose excretion is almost exclusively renal as the consequence of glomerular
filtration (Hart and Kinter, 2005). The increased concentrations of these compounds in the
serum of chlordecone-treated animals suggest kidney dysfunction. Furthermore, the impaired
excretion of creatinine indicates that this dysfunction is most likely glomerular in nature.
Most serum indications of kidney function are insensitive. For example, in order for
serum creatinine or blood urea nitrogen concentrations to be detectable, approximately 50% of
renal function must be lost (Hart and Kinter, 2005). However, it is unclear whether these
elevations in serum proteins and enzymes as seen in Chetty et al. (1993c) were accompanied by
pathological lesions, as the scope of the study did not include a histological examination of the
liver or kidney.
Renal effects with chlordecone exposure were also reported in other studies. NCI
(1976b) reported chronic kidney inflammation in male Osborne-Mendel rats (at 0.6 mg/kg-day)
and female Osborne-Mendel rats (at 2.0 mg/kg-day). Chu et al. (1980) reported that 28 days of
dietary exposure to chlordecone (at 0.07 mg/kg-day) produced eosinophilic inclusions in
proximal tubules in 2/10 male Sprague-Dawley rats. A 32-month oral exposure study in beagles
(Larson et al., 1979a) reported increased relative kidney weights in the 0.5 mg/kg-day
chlordecone exposure group, though renal histology findings were negative. A 3-month oral
study observed increased relative kidney weight in female rats exposed to 1.6-1.7 mg/kg-day,
though no histological findings were noted (Cannon and Kimbrough, 1978). Furthermore,
Chernoff and Rodgers (1976) reported that gestational exposure to chlordecone (gestation days
7-16) in mice resulted in a statistically significant increase in the incidence of fetuses with
enlarged renal pelvis, though this dose was much higher than kidney effects reported in the
aforementioned studies (10 mg/kg-day).
Support in the chlordecone database exists for a variety of reproductive effects with
chlordecone exposure. Larson et al. (1979a) observed testicular atrophy in male rats treated with
chlordecone for 13 weeks at dose levels of >1.6 mg/kg-day. The incidence of testicular atrophy
at 13 weeks was reported as 1/10, 0/5, 1/5, 4/5, 4/5, and 5/5 at 0, 0.3, 0.5, 1.6, 3.9, and 7.0
mg/kg-day. Other animal studies have also shown male reproductive effects, such as decreased
sperm viability, motility, and concentration, following exposure to chlordecone (EPA, 1986c;
Linder et al., 1983). However, it is unclear whether these effects on sperm, though statistically
significant, can be considered biologically significant. EPA (1986c) reported decreased sperm
JANUARY 2008 81 DRAFT - DO NOT CITE OR QUOTE
-------
concentration in male rats treated orally for 10 days with 0.625 mg/kg-day chlordecone. Linder
et al. (1983) saw sperm effects (decreased viability, motility, and concentration) in rats at 0.83
and 1.67 mg/kg-day (90 days of treatment); however, the authors did not see any treatment-
related histological lesions or an effect on reproductive performance (number of pregnant
females, number of live litters, average live litter size, number of implants, percentage of
resorptions, and fetal weight) when treated males were mated to untreated females. This study
and a study by Cannon and Kimbrough (1979) indicate that decreased reproductive success in
experimental animals may not be solely attributable to male reproductive effects. Cannon and
Kimbrough (1979) reported that treated female rats (1.6-1.7 mg/kg-day for 3 months) mated to
control rats failed to produce litters, whereas treated males (1.2-1.6 mg/kg-day for 3 months)
mated to control females had reproductive success similar to controls. Good et al. (1965)
reported in a continuous breeding study that male and female mice treated with 0.94 mg/kg-day
for 1 month prior to mating and 5 months during mating had impaired reproductive success;
specifically, reduced production of litters was seen in treated mice and the mated offspring of
treated mice. However, the general confidence in this study is limited by incomplete reporting of
the variance of reproductive parameters and decreased fertility of the control mice one
generation apart. Two other reproductive studies (Good et al., 1965; Huber et al., 1965) treated
outbred mice in the diet for 1 month prior to mating and 3-5 months during the mating period
with doses of chlordecone starting at 1.9 mg/kg-day and did not see a depression of reproductive
parameters until doses of 3.3 or 5.6 mg/kg-day (Good et al., 1965, and Huber et al., 1965,
respectively). Additional studies have reported reproductive toxicity but at higher doses (Swartz
and Mall, 1989; Swartz et al., 1988; Gellert and Wilson, 1979; Huber, 1965). Taken together,
the available studies showing reproductive effects following chlordecone exposure suggest that
functional reproductive deficits are seen at levels higher than the level reported to cause renal
lesions in chronically treated rats (Linder et al., 1983; Cannon and Kimbrough, 1979; Larson et
al., 1979a; Good et al., 1965; Huber et al., 1965). Therefore, reproductive effects were not
selected as the critical effect of chlordecone exposure. Nevertheless, potential points of
departure (PODs) for reproductive endpoints from Larson et al. (1979a) and Good et al. (1965)
were considered in the derivation of a RfD (see Section 5.1.2 and Appendix B).
In consideration of the available studies reporting effects of chronic and subchronic
chlordecone exposure in humans and animals, Larson et al. (1979a) was chosen as the principal
study. This study was adequately designed with several acceptable dose groups and adequate
numbers of animals. Results were sufficiently reported for most endpoints. Sensitive endpoints
identified in this study include glomerulosclerosis, liver lesions, and testicular atrophy. Though
testicular atrophy was observed at 13 weeks, the only lesions observed chronically that were
reported to be treatment related were in the liver and kidney. This observation coupled with the
lack of support for testicular lesions in other studies in rats of similar dose and duration (Linder
et al., 1983; Cannon and Kimbrough, 1978) decreases confidence in this endpoint. Additionally,
JANUARY 2008 82 DRAFT - DO NOT CITE OR QUOTE
-------
the liver lesions observed in the principal study (characterized as fatty changes and hyperplasia)
occurred at higher doses as compared with the observed kidney lesions. After consideration of
all endpoints, the increased incidence of glomerulosclerosis in female rats was determined to be
the most sensitive and biologically significant effect detected in this study. Furthermore, the
chlordecone database contains additional support for the specific endpoint of glomerular damage
(Sobel et al., 2006, 2005; Chetty et al., 1993c) and general support for the kidney as a target
organ as determined by increased kidney weights seen in studies in addition to the principal
study (Cannon and Kimbrough, 1978; NCI, 1976a).
Glomerulosclerosis is believed to be an irreversible effect that can result in renal
impairment. The mechanism by which chlordecone causes kidney lesions is not known;
however, there is no indication that kidney lesions would not occur in humans chronically
exposed to chlordecone. Though clinical indications of kidney dysfunction were not detected in
workers occupationally exposed to chlordecone, this may be because the relatively short average
exposure duration of workers (5-6 months) was not sufficient for the development of detectable
kidney impairment. Therefore, for the above reasons, Larson et al. (1979a) was chosen as the
principal study and renal lesions as the critical effect.
5.1.2. Methods of Analysis
All available models in the EPA Benchmark Dose Software (BMDS) version 1.3.2 were
fit to quantal incidence data for histopathologic renal lesions in female Wistar rats from a 2-year
dietary study (Larson et al., 1979a). The data modeled are shown below in Table 5-1.
Table 5-1. Incidence of histopathologic renal lesions (glomerulosclerosis
grades 1, 2, or 3 combined) in male or female Wistar rats following
administration of chlordecone in the diet for 1-2 years
Gender
Male
Female3
Dose (mg/kg-day)
0
12/22 (55%)
4/34 (12%)
0.06
3/11(27%)
2/13 (15%)
0.3
4/6 (67%)
8/17 (47%)b
0.5
6/9 (67%)
8/12 (67%)b
1.6
3/4 (75%)
3/4 (75%)b
aStatistically significant trend for increased incidence by Cochran-Armitage test (p < 0.01).
bStatistically significantly different from controls according to Fisher's exact test (p < 0.05) performed for this
review.
Source: Larson et al. (1979a).
Biological and statistical considerations were taken into account in the selection of a
benchmark response level for this data set. Statistically, a 10% level of response is intended to
select a response level near the lower range of detectable observations in typical studies
conducted with 50 animals per dose group (U.S. EPA, 2000c). The data set for the critical effect
JANUARY 2008
83
DRAFT - DO NOT CITE OR QUOTE
-------
from Larson et al. (1979a) relies on notably smaller groups of animals (4-22 animals per group),
therefore, use of a benchmark response (BMR) below 10% would result in a POD further outside
of the observable range and would involve greater uncertainty. Biologically speaking, a BMR of
a 10% increase in glomerulosclerosis was selected under an assumption that it represents a
minimal biologically significant change. Therefore, for this dataset, a response level of 10%
was used. The results of benchmark dose (BMD) modeling of the data are discussed below.
Statistical analysis of the incidence of glomerulosclerosis (grades 1, 2, or 3 combined) in
each dose by sex revealed that the incidence of glomerulosclerosis in female rats exhibited a
significant dose response trend (according to the Cochran-Armitage test). Therefore, the
incidence data for renal lesions in female rats in the 0, 0.06, 0.3, 0.5, and 1.6 mg/kg-day dose
groups were used to fit models to derive BMDs. It should be noted that all animals from the two
highest dose groups (3.9 and 7.0 mg/kg-day) died within the first 6 months of the study, and thus
data from these animals were not available for use in the dose-response assessment.
As shown in Appendix B, most models provided adequate fits to the data for
histopathologic renal lesions (glomerulosclerosis) in female rats from the Larson et al. (1979a)
study (Table 5-1), as assessed by a chi-square goodness-of-fit test and visual inspection of the
respective plots of observed and predicted values from the various models. The log-probit model
provided the best fit to the female rat data as assessed by Akaike's Information Criterion (AIC).
Additionally, this model exhibits the best fit to the incidence data at low doses (i.e., in the
vicinity of the BMR) as evidenced by examining the chi-square scaled residuals and the visual fit
of the model to the data in the plot from the BMDS output. Thus, the log-probit model was
selected to estimate the BMD for glomerulosclerosis data in female rats from Larson et al.
(1979a). The BMDio associated with a 10% extra risk for glomerulosclerosis in female rats was
0.12 mg/kg-day, and its lower 95% confidence limit (BMDLio) was 0.08 mg/kg-day.
Reproductive effects observed following oral exposure to chlordecone were also
evaluated as potential PODs. Reproductive endpoints, such as testicular atrophy (Larson et al.,
1979a), and functional reproductive outcomes, such as decreases in first and second litters (Good
et al., 1965; Huber et al., 1965), were investigated. The most sensitive functional reproductive
endpoint identified in the chlordecone database of dietary repeat exposure studies is a
freestanding LOAEL of 0.94 mg/kg-day identified in Good et al. (1965) for the reduced
production of second litters in chlordecone treated BALB/c mice and reduced reproduction in
offspring of treated mice (reduced production of first litters). Upon examination of the data set,
it was determined that these data were not amenable to BMD modeling; specifically, the
continuous endpoints reported (percent of pairs producing first and second litters, pair days per
litter) were averages and did not include any measure of the variability, such as standard
deviation.
The incidence of testicular atrophy in male Wistar rats, following 3 months of dietary
chlordecone exposure (Larson et al., 1979a), was determined to be the only biologically
JANUARY 2008 84 DRAFT - DO NOT CITE OR QUOTE
-------
significant reproductive endpoint with a data set amenable to BMD modeling, though uncertainty
surrounds this endpoint since it was not detected in the same study at the chronic time point.
Regardless, the BMD modeling results for testicular lesions in rats are included as part of
Appendix B. The multistage and quantal linear models provided the best fit for this dataset as
assessed by a chi-square goodness-of-fit test, an AIC, and a visual inspection of the respective
plots of observed and predicted values from the various models. The BMDio associated with a
10% extra risk for testicular atrophy in rats was 0.206 mg/kg-day, and its lower 95% confidence
limit (BMDLio) was 0.119 mg/kg-day.
5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)
Of the endpoints shown in Table 4-18, the increased incidence of histopathological renal
lesions (glomerulosclerosis) among female Wistar rats receiving chlordecone in the diet
continuously for 2 years (Larson et al., 1979a) is the most sensitive endpoint. BMD modeling
revealed that the BMDLio associated with this effect is 0.08 mg/kg-day. The BMDLio provides
the POD for the RfD.
A total UF of 300 was applied to the POD of 0.08 mg/kg-day: 10 for interspecies
extrapolation from animals to humans (OTA); 10 for human intraspecies variability (UFn); and 3
to account for database deficiencies (UFD).
A 10-fold UF was used to account for uncertainties in extrapolating from laboratory rats
to humans. Aside from a difference in metabolism (humans produce chlordecone alcohol,
whereas rats do not), the available data do not suggest differential toxicity of these forms, nor do
the toxicity data from various animal species provide marked evidence that rats or any other
species are more sensitive to chlordecone than humans. Consequently, the default UF of 10 for
extrapolating from laboratory animals to humans was applied.
A 10-fold UF was used to account for variation in susceptibility among members of the
human population (i.e., interindividual variability). Insufficient information is available to
predict potential variability in human susceptibility.
An UF of 3 was applied to account for deficiencies in the chlordecone toxicity database.
The database includes limited human data from observational studies of occupationally exposed
workers. The database also includes several studies in laboratory animals, including chronic and
subchronic dietary exposure studies and several subchronic reproductive and developmental
studies, as well as one specifically assessing developmental neurotoxicity. The chlordecone
database does not have an appropriately designed multigenerational reproductive study, but
includes approximately 10 oral repeat-exposure studies assessing reproductive and
developmental toxicity, including several single-generation reproductive toxicity studies and
three developmental studies in rats and mice (Linder et al., 1983; Squibb and Tilson, 1982;
Cannon and Kimbrough, 1979; Chernoff and Rogers, 1976; Good et al., 1965; Huber et al.,
1965). Several of these reproductive studies have indicated decreased reproductive success in
JANUARY 2008 85 DRAFT - DO NOT CITE OR QUOTE
-------
chlordecone-treated animals (Cannon and Kimbrough, 1979; Good et al., 1965; Huber et al.,
1965). The database also includes two nonstandard multigenerational studies that evaluate
reproductive success of chlordecone-treated animals (Gellert and Wilson, 1979; Good et al.,
1965). Due to limited scope and design, these studies are not considered adequate for the
assessment of potential multigenerational reproductive toxicity.
In addition, some limited evidence exists to suggest that the critical effect (glomerular
lesions) may be mediated through an autoimmune mechanism. Therefore, in consideration of the
entire database for chlordecone, a partial database UF of 3 is considered appropriate to account
for the lack of an adequately designed two-generational reproductive study and for the lack of
immunotoxicity studies where some data indicate chlordecone may exhibit immunological
effects. There are no available data to indicate whether these effects would be expected to occur
at doses lower than those observed for the critical effect.
Because the POD was selected from a dose associated with an endpoint identified by a
chronic dietary study (Larson et al., 1979a), no uncertainty factor is needed for exposure duration
(subchronic to chronic). A UF for LOAEL-to-NOAEL extrapolation was not used because the
current approach is to address this factor as one of the considerations in selecting a BMR for
BMD modeling. In this case, a BMR of a 10% increase in glomerulosclerosis was selected under
an assumption that it represents a minimal biologically significant change.
The oral RfD for chlordecone was calculated as follows:
RfD = BMDLio - UF
= 0.08 mg/kg-day - 300
= 0.0003 or 3E-4 mg/kg-day
5.1.4. RfD Comparison Information
Kidney (glomerular) lesions, liver lesions, testicular atrophy, and decreased fertility are
observed low-level effects, following subchronic or chronic oral exposure to chlordecone
(Larson et al., 1979; Good et al., 1965). Table 5-2 provides a tabular summary of potential
PODs and resulting RfDs for these endpoints. Additionally, Figure 5-1 provides a graphical
representation of this information. This figure should be interpreted with caution since the PODs
across studies are not necessarily comparable, nor is the confidence the same in the data sets
from which the PODs were derived. The PODs presented in this figure are based on either a
BMDLio (for kidney, testicular, or liver lesions) or a LOAEL (in the case of Good et al., 1965).
Some indication of the confidence associated with the resulting RfD is reflected in the magnitude
of the total UF applied to the POD (i.e., the size of the bar); however, the text of Sections 5.1.1
and 5.1.2 should be consulted for a more complete understanding of the issues associated with
each dataset and the rationale for the selection of the principal study and the critical effect used
JANUARY 2008 86 DRAFT - DO NOT CITE OR QUOTE
-------
to derive the RfD. As discussed in Section 5.1.1., among the studies considered, the chronic
study by Larson et al. (1979a) provided the data set most appropriate for the derivation of the
RfD.
JANUARY 2008 87 DRAFT - DO NOT CITE OR QUOTE
-------
Table 5-2. Possible PODs with applied uncertainty factors and resulting
RfDs
Effect
Kidney lesions
Testicular atrophy
Liver lesions
Decreased production of
litters
POD
0.08b
0.12b
0.14b
0.94C
Species
Rat
Rat
Rat
Mouse
Uncertainty factors3
Total
300
3000
300
3000
A
10
10
10
10
H
10
10
10
10
L
10
s
10
D
3
3
3
3
RfD
3 xlO^1
4 x 10~5
5 x IQ-4
3 x 10^
Uncertainty factors: A = animal to human (interspecies); H = interindividual (intraspecies); L = LOAEL
to NOAEL; S = subchronic-to-chronic duration; D = database deficiency.
bPOD based on BMDL determined through BMD modeling of a 10% response. Source: Larson et al.
(1979a).
°POD based on a freestanding LOAEL for a 65% decrease in second-generation animals producing litters.
Source: Good et al. (1965).
00
10 T
1
0.1 --
0.01 _. '
§ 0.001
Q
0.0001
0.00001
/
/
Point of Departure
LTF, animal to human
LTF, human variability
LTF, database
LTF, subchronic to chronic
LTF, LOAEL to NOAEL
RfD
Kidney
lesions;
rats
1'3'6
Reduced
reproductive
success; mice
2' 4
Liver
lesions;
rats
1'3
Testicular
atrophy;
rats1'3'5
Figure 5-1. RfD comparison array for alternate points of departure.
1 Larson etal.(1979a).
2Goodetal. (1965).
3BMDL10 used as the POD.
4POD based on a freestanding LOAEL for a 65% decrease in second-generation animals producing litters.
5Subchronic endpoint (13 weeks) not observed at chronic durations (1-2 years).
6Selected critical effect for the derivation of the RfD.
JANUARY 2008
88
DRAFT - DO NOT CITE OR QUOTE
-------
The PODs presented for kidney, liver, and testicular lesions were derived through BMD
modeling of the dichotomous data by using a 10% response level. BMD modeling outputs for
these three endpoints are included in Appendix B. The PODs based on BMD methods have an
inherent advantage over the use of a NOAEL or LOAEL by making greater use of all the dose-
response data from a given data set. The POD for reduced reproductive success in mice was
based on a freestanding LOAEL.
Although the RfD based on kidney lesions has the lowest POD, the RfD based on
testicular atrophy results in a lower RfD due to an added magnitude of uncertainty applied for the
use of a subchronic endpoint. The POD for testicular atrophy was derived from a 3-month
exposure duration (within the chronic study by Larson et al. [1979a]); however, testicular effects
were not noted for the longer exposure durations (1-2 years) in the same study, nor were
testicular lesions detected in other studies in rats treated with similar doses for the same duration
(Linder et al., 1983; Cannon and Kimbrough, 1979). Therefore, because of lower confidence in
this endpoint and the evidence in the database for more sensitive effects in the kidney, testicular
lesions were not selected as the critical effect for the derivation of the chlordecone RfD.
5.1.5. Previous RfD Assessment
An oral assessment for chlordecone was not previously available on IRIS.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
Although adverse health effects from an occupational exposure incident may have
resulted from inhalation exposure (in combination with oral and dermal exposures), the data do
not identify doses at which effects occur (Taylor, 1985, 1982; Guzelian, 1982a; Guzelian et al.,
1980; Sanborn et al., 1979; Cannon et al., 1978; Martinez et al., 1978; Taylor et al., 1978).
Consequently, the human data cannot be used to define a dose-response relationship for
inhalation exposure to chlordecone. No studies on the toxicity of chlordecone following
inhalation exposure in laboratory animals were located. This lack of data precludes the
derivation of an RfC.
Consideration was given to route-to-route extrapolation to derive inhalation doses from
existing oral dose-response data for development of an RfC. Route-to-route extrapolation from
the oral database, however, is precluded by deficiencies in the database. The available rat PBTK
models for chlordecone do not include the inhalation route of exposure (see Section 3.5), and
human PBTK models with both oral and inhalation portals of entry have not yet been developed.
In the absence of PBTK models that include oral and inhalation routes of exposure, and lacking
inhalation absorption efficiency data in humans and rats, a route-to-route extrapolation from oral
to inhalation for chlordecone would be highly uncertain. As discussed in Chapter 2, only very
JANUARY 2008 89 DRAFT - DO NOT CITE OR QUOTE
-------
small amounts of chlordecone will evaporate from soil or water surfaces, and any chlordecone in
the air is likely to be removed by deposition of particles.
5.3. CANCER ASSESSMENT
Utilizing the EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there
is suggestive evidence of carcinogenic potential for chlordecone. This characterization lies at the
high end of the continuum for this weight of evidence descriptor. An animal cancer bioassay
(NCI, 1976a,b) provides evidence of carcinogenic potential of chlordecone, following high-dose
oral exposure in Osborne-Mendel rats and B6C3F1 mice. In this study the incidence of
hepatocellular tumors was statistically significantly increased in both sexes of B6C3F1 mice.
Male and female rats exhibited increased incidences of hepatocellular carcinomas at high doses
that were statistically significant when compared with pooled controls. The incidence of
hepatocellular carcinomas was not statistically significant in comparison with matched controls
for rats of either sex. In addition, a decrease in latency for time to tumor appearance was
observed in dosed animals compared with controls. Review of the NCI (1976a,b) bioassay for
chlordecone raises concerns regarding the study design and conduct (see Sections 4.2.2.1 and
4.7.1). The study was limited in scope (only two dose groups) and utilized high-dose levels
designed to elicit a maximal carcinogenic response. Following the observation of marked
toxicity in the high-dose groups of both species, dosing levels were lowered to one-half to one-
sixth of the initial dose levels. Due to this change in chlordecone exposure levels, the dose
metric related to the development of liver tumors cannot be determined. Because of these
limitations, the NCI (1976a,b) study is not suitable for low-dose extrapolation for human cancer
risk assessment. Therefore, in the absence of adequate cancer bioassay data, no quantitative
dose-response cancer assessment can be performed to derive an oral slope factor for chlordecone.
JANUARY 2008 90 DRAFT - DO NOT CITE OR QUOTE
-------
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
Chlordecone was previously used as an insecticide to control agricultural pests, including
slugs, snails, and fire ants. Chlordecone was first produced in the United States in the early
1950s; however, production in the United States ended in 1975 due to intoxication from severe
industrial exposure in employees who worked at the only chlordecone manufacturing plant in the
country. Its registration was cancelled in 1976. Chlordecone is very resistant to degradation in
the environment. It is expected to adsorb to soil and to stick to suspended solids and sediments
in water. Very small amounts of chlordecone will evaporate from soil or water surfaces, and any
chlordecone in the air is likely to be removed by deposition of particles. Chlordecone has a very
high potential for bioaccumulation in fish and other aquatic organisms.
Chlordecone is well absorbed following oral exposure. Once absorbed, it is widely
distributed and eventually concentrates in the liver. It is metabolized by humans and some
animal species to chlordecone alcohol. Glucuronide conjugates of chlordecone and chlordecone
alcohol, as well as unconjugated chlordecone, are slowly excreted in the bile and eliminated in
the feces. Fecal excretion is delayed by enterohepatic recirculation.
The primary noncancer health effects of oral exposure to chlordecone in humans and
animals include liver effects, kidney lesions (only in animals), neurotoxicity, and male
reproductive toxicity. Other reproductive effects (i.e., persistent vaginal estrus, impaired
reproductive success) and developmental effects have also been observed in laboratory animals;
however, the doses required to elicit these effects were generally higher than those that resulted
in liver and kidney effects, neurotoxicity, and/or male reproductive toxicity.
Liver enlargement developed in workers exposed to high levels of chlordecone for an
intermediate exposure duration; however, evidence of significant liver toxicity was not found.
Histological changes were observed in liver biopsy samples; however, these were characterized
as nonadverse in nature. Similar changes in the liver were also demonstrated in laboratory
animals, including increased liver size and weight, hepatocellular hypertrophy, proliferation of
the SER, increased microsomal protein, CYP450 content, cytochrome c reductase activity, and
microsomal enzyme activity. Chronic animal studies also demonstrated evidence of
hepatotoxicity, including hepatocellular hypertrophy, hyperplasia, congestion, mild fatty change,
focal necrosis, and occasional small nests of proliferated sinusoidal cells.
Neurological symptoms were also reported in workers exposed to high doses of
chlordecone, including tremor, headache, irritability, poor recent memory, rapid random eye
movements, muscle weakness, gait ataxia, incoordination, and slurred speech. The effects
persisted for as long as 9-10 months after cessation of exposure and the start of treatment.
JANUARY 2008 91 DRAFT - DO NOT CITE OR QUOTE
-------
Chlordecone also causes tremors, decreased motor coordination, hyperexcitability, and an
exaggerated startle response in laboratory animals.
Chlordecone exposure in humans caused oligospermia, reduced sperm motility, and
decreased libido in a group of men who were occupationally exposed to chlordecone for periods
up to 1.5 years. There was no evidence that the ability of these workers to father children was
affected, and male reproductive parameters had returned to normal by 5 to 7 years following the
cessation of chlordecone exposure and treatment with cholestyramine to reduce chlordecone
blood levels. Chlordecone also induces reproductive toxicity in male and female laboratory
animals, as demonstrated by altered sperm parameters, testicular atrophy, altered estrous
cyclicity, and impaired reproductive success. Chlordecone induced developmental toxicity in
rats and mice at dose levels that also produced significant maternal toxicity.
Kidney toxicity was reported in laboratory animals but was not observed in
occupationally exposed pesticide workers. However, it is unclear if clinical indicators of renal
damage were specifically examined in occupationally exposed workers. Several animal studies
reported kidney effects from chlordecone exposure. Proteinuria and increased incidence of
kidney lesions were observed in female Wistar rats and in (NZB x NZW)Fi mice. Chronic
kidney inflammation was observed in male and female Osborne-Mendel rats. Twenty-eight days
of dietary exposure to chlordecone produced eosinophilic inclusions in proximal tubules in male
Sprague-Dawley rats. Gestational exposure to chlordecone resulted in a statistically significant
increase in the incidences of fetuses with enlarged renal pelvis.
Most of the effects of chlordecone are thought to be produced by the parent compound,
primarily by interfering with the function of mitochondrial and cellular membranes. Disruption
of cellular homeostasis and energy production within the cell eventually leads to impaired
cellular function. In the central nervous system, altered calcium homeostasis leads to changes in
neurotransmitter activity. In the liver, membrane perturbation and inhibition of transport
proteins at the bile canalicular membrane is thought to be related to chlordecone-induced
hepatobiliary dysfunction. The reproductive and developmental effects of chlordecone are most
likely related to endocrine disruption. Chlordecone exhibits estrogenic properties that may be
related to impaired reproductive success and adverse effects on sperm.
6.2. DOSE RESPONSE
6.2.1. Noncancer
No studies on the toxicity of chlordecone following inhalation exposure in humans or
laboratory animals were located. This lack of data precludes the derivation of the RfC.
The database for chlordecone includes limited human data from observational studies of
occupationally exposed workers. The database also includes several studies in laboratory
animals, including chronic and subchronic dietary exposure studies, and several subchronic
studies with a wide variety of tissues and endpoints assessed. The database also includes several
JANUARY 2008 92 DRAFT - DO NOT CITE OR QUOTE
-------
reproductive and developmental studies, including one specifically assessing developmental
neurotoxicity. Endpoints associated with oral exposure to chlordecone include lesions in the
liver, kidney, and testis; neurological effects (specifically tremors); and reduced fertility.
Support for these endpoints exists across a range of diverse studies; nevertheless, data gaps have
been identified and uncertainties associated with data are discussed below.
The observation of kidney, liver, and testicular effects in the principal study at similar
dose levels creates some uncertainty in the selection of a critical effect that would be most
appropriate in a chronic low-dose human exposure paradigm. The most sensitive effect observed
from chronic dietary exposure to chlordecone is the increased incidence of kidney lesions in
female Wistar rats (Larson et al., 1979a). Furthermore, several additional animal studies, in both
rats and mice, support findings of kidney effects with chlordecone exposure (Sobel et al., 2006,
2005; Chetty et al., 1993c; Chu et al., 1981a; Chernoff and Rodgers, 1976; NCI, 1976b). In
light of the weight of evidence for kidney, testicular, and liver lesions seen in the chlordecone
animal literature (see Section 5.1.1), kidney lesions were deemed to be the most supported,
biologically significant effect on which to base the RfD. Some uncertainty exists regarding the
lack of observable effects on the kidney in humans. However, it is unknown whether the
relatively short average exposure duration of workers (5-6 months) was sufficient for the
development of detectable kidney impairment. Additionally, it is unclear from the literature
whether clinical tests sensitive to early kidney impairment were administered to exposed
workers.
After consideration of all potential PODs, the RfD of 3 x 10^ mg/kg-day was based on
the increased incidence of kidney lesions in female Wistar rats, following chronic dietary
administration of chlordecone (Larson et al., 1979a). To derive the RfD, the uncertainty factor
approach, following EPA practices (U.S. EPA, 2002), was applied to the POD determined
through BMD modeling of the critical effect of kidney lesions in female rats. Factors to account
for uncertainties associated with the extrapolation from the POD derived from an animal study to
a diverse human population of varying susceptibilities were applied. This extrapolation was
accomplished through the application of default UFs due to limitations in the chlordecone
database that precluded the derivation of chemical specific adjustment factors.
The choice of BMD model is not expected to introduce a considerable amount of
uncertainty in the risk assessment since the chosen response rate of 10% additional risk is within
the observable range of the data. Furthermore, the ratio of the BMD to the BMDL for the model
that best describes the incidence data for the critical effect is less than a factor of two, indicating
a relatively precise BMD estimate.
Additional BMD modeling for other amenable data sets, including liver lesions and
testicular atrophy, was also conducted to provide other PODs for comparison purposes (see
Appendix B). A graphical representation of these potential PODs and resulting reference values
is shown below in Figure 6-1.
JANUARY 2008 93 DRAFT - DO NOT CITE OR QUOTE
-------
*
00
O
Q
10 -
1 -
0.1 -
0.01 _
0.001 -
Itn
1
* Point of Departure
§ UF, animal to human
g] UF, human variability
£3 UF, database
[]J| UF, subchronic to chronic
g UF, LOAEL to NOAEL
• RfD
0.0001 _.
\ /
0.00001
Kidney
lesions;
rats1'3'6
Reduced
reproductive
success; mice2' 4
Liver
lesions;
rats1'3
Testicular
atrophy;
rats1'3'5
Figure 6-1. RfD comparison array for alternate points of departure.
1 Larson etal.(1979a)
2Goodetal. (1965).
3BMDL10 used as the POD.
4POD based on a freestanding LOAEL for a 65% decrease in second-generation animals producing litters.
5Subchronic endpoint (13 weeks) not observed at chronic durations (1-2 years).
6Selected critical effect for the derivation of the RfD.
The default UF of 10 for the extrapolation from animals and humans is a composite of
uncertainty to account for toxicokinetic differences and toxicodynamic differences between the
animal species in which the POD was derived and humans. PBTK models can be useful for the
evaluation of interspecies toxicokinetics; however, the chlordecone database lacks an adequate
model that would inform potential differences. Data from workers occupationally exposed to
chlordecone provide some information on the absorption, distribution, metabolism, and
elimination of chlordecone in humans and indicate qualitatively that the toxicokinetics of
chlordecone are similar between humans and animals. Additionally, biological effects, including
neurological, hepatic, and reproductive effects, observed in animals and humans are similar in
nature, indicating similar toxicodynamics. However, the magnitude of the similarities or
differences in toxicokinetic and toxicodynamic parameters cannot be calculated due to
uncertainties regarding routes of exposure and doses for the occupationally exposed workers.
Therefore, an UF of 10 to account for interspecies differences was used.
JANUARY 2008 94 DRAFT - DO NOT CITE OR QUOTE
-------
Limited data exist on effects of chlordecone in a small population of occupationally
exposed workers. However, since potential variability in responses to chlordecone in the greater
human population is unknown, the default uncertainty factor of 10 for intrahuman variability was
not reduced. Human variation may be larger or smaller; however, chlordecone-specific data to
examine the potential magnitude of human variability of response are unknown.
Uncertainties associated with data gaps in the chlordecone database have been identified.
Specifically, data more fully characterizing potential multigenerational reproductive and
immunological effects are lacking. Some data suggest that the selected critical effect of kidney
lesions may be an immune-mediated effect.However, additional data to evaluate this potential
effect is lacking (Sobel et al., 2006, 2005). Additionally, uncertainty exists in the database
concerning the dose-response characterization of potential multigenerational reproductive
effects. Several one-generational reproductive studies have indicated decreased reproductive
success in chlordecone-treated animals (Cannon and Kimbrough, 1979; Good et al., 1965; Huber
et al., 1965). In addition, two nonstandard multigenerational studies exist that evaluate
reproductive success of chlordecone-treated animals (Gellert and Wilson, 1979; Good et al.,
1965). However, due to limited scope and design, these studies are not considered adequate for
the assessment of multigenerational reproductive toxicity. Therefore, for the above data gaps in
the chlordecone database, an UF of 3 was applied to the POD in the derivation of the RfD.
The overall confidence in the RfD and the principal study (Larson et al., 1979a) is
medium. The principal study involves a sufficient number of animals per group, several
acceptable dose levels, and a wide range of tissues and endpoints assessed. Confidence in the
database is medium. The chlordecone database includes case studies of occupationally exposed
workers, chronic and subchronic dietary exposure studies in laboratory animals, and several
subchronic reproductive and developmental studies, including one developmental neurotoxicity
study. However, the database is lacking a multigenerational reproductive toxicity study.
Therefore, reflecting medium confidence in both the database and the principal study, confidence
in the RfD is medium.
6.2.2. Cancer
Though uncertainty exists regarding the classification of the carcinogenic
potential of chlordecone due to limitations in the design and conduct of the primary cancer
bioassay, under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the database
for chlordecone provides suggestive evidence of carcinogenic potential. This characterization
lies at the high end of the continuum for this weight of evidence descriptor. This determination
is primarily based on the NCI (1976a,b) study, which found positive evidence of liver tumors in
both sexes of rats and mice after chronic chlordecone dietary exposure. Additionally, data on
mirex, a structurally similar chemical also demonstrates an increase in hepatocellular adenomas
or carcinomas in both sexes of rats and mice. However, unlike the observed cancer effects, some
JANUARY 2008 95 DRAFT - DO NOT CITE OR QUOTE
-------
but not all of the noncancer effects noted for these two chemicals are similar as described in
Section 4.5.3. This weight of evidence conclusion collectively takes into consideration the NCI
(1976a,b) cancer bioassay and its limitations, the available human studies, and other chronic
animal bioassays. Due to design and conduct issues in the primary study (NCI, 1976a,b),
including inconsistent dosing levels and the use of doses that may have been excessively high,
the data available are not sufficient for a stronger conclusion and are not suitable for
quantification of cancer risk.
JANUARY 2008 96 DRAFT - DO NOT CITE OR QUOTE
-------
7. REFERENCES
Adir, J; Caplan, YH; Thompson, BC. (1978) Kepone serum half-life in humans. Life Sci 22(8):699-702.
Ahlborg, UG; Lipworth, L; Titus-Ernstoff, L; et al. (1995) Organochlorine compounds in relation to breast cancer,
endometrial cancer, and endometriosis: an assessment of the biological and epidemiological evidence. CRC Crit
Rev Toxicol 25(6):463-531.
Ahmed, SA; Hissong, BD; Verthelyi, D; et al. (1999) Gender and risk of autoimmune diseases: possible role of
estrogenic compounds. Environ Health Perspect 107(Suppl. 5):681-686.
Albertson, TE; Joy, RM; Stark, LF. (1985) Chlorinated hydrocarbon pesticides and amygdaloid kindling. Neurobeh
Toxicol Teratol 7(3):233-237. (as cited in ATSDR, 1995)
Aldous, CN; Chetty, CS; Desaiah, D. (1983) Alterations in tissue distribution of chlordecone (Kepone) in the rat
following phenobarbital or SKF-525A administration. J Toxicol Environ Health 11(3):365-372.
ATSDR (Agency for Toxic Substances and Disease Registry). (1995) Toxicological profile for mirex and
chlordecone. Public Health Service, U.S. Department of Health and Human Services, Atlanta, GA. Available online
at http://www.atsdr.cdc.gov/toxpro2.html.
Bale, SS. (1983) Cytological effects of Kepone on Chinese hamster cells. J Hered 74(2): 123-124.
Belfiore, CJ; Yang, RS; Chubb, LS; et al. (2007) Hepatic sequestration of chlordecone and hexafluoroacetone
evaluated by pharmacokinetic modeling. Toxicology 234(l-2):59-72.
Blain, RB; Reeves, R; Ewald, KA; et al. (1999) Susceptibility to chlordecone-carbon tetrachloride induced
hepatotoxicity and lethality is both age and sex dependent. Toxicol Sci 50(2):280-286.
Blanke, RV; Fariss, MW; Guzelian, PS; et al. (1978) Identification of a reduced form of chlordecone (Kepone) in
human stool. Bull Environ Contam Toxicol 20:782-785.
Bolger, R; Wiese, TE; Ervin, K; et al. (1998) Rapid screening of environmental chemicals for estrogen receptor
binding capacity. Environ Health Perspect 106(9):551-557.
Bondy, SC; McKee, M. (1990) Prevention of chemically induced synaptosomal changes. J Neurosci Res 25:229-
235.
Boylan, JJ; Cohn, WJ; Egle, JL, Jr; et al. (1979) Excretion of chlordecone by the gastrointestinal tract: evidence for
a nonbiliary mechanism. ClinPharmacol Ther 25:579-585.
Brown, HE; Salamance, S; Stewart, G; et al. (1991) Chlordecone (Kepone) on the night of proestrus inhibits female
sexual behavior in CDF-344 rats. Toxicol Appl Pharmacol 110(1):97-106.
Bulger, WH; Muccitelli, RM; Kupfer, D. (1979) Studies on the estrogenic activity of chlordecone (Kepone) in the
rat: effects on uterine estrogen receptor. Mol Pharmacol 15:515-524.
Bungay, PM; Dedrick, RL; Matthews, HB. (1979) Pharmacokinetics of halogenated hydrocarbons. Ann NY Acad
Sci 320:257-270.
Bus, JS; Leber, AP. (2001) Miscellaneous chlorinated hydrocarbon pesticides. In: Bingham, E; Cohrssen, B;
Powell, CH; eds. Patty's toxicology. Electronic version available through subscription to Wiley Interscience.
Accessed March 24, 2004 at http://www.mrw.interscience.wiley.com/pattys/pattys_search_fs.html.
Cai, Z; Mehendale, HM. (1993) Resiliency to amplification of carbon tetrachloride hepatotoxicity by chlordecone
during postnatal development in rats. Pediatr Res 33(3):225-232.
JANUARY 2008 97 DRAFT - DO NOT CITE OR QUOTE
-------
Caldwell, V; Loch-Caruso, R. (1992) Chlordecone rapidly and reversibly inhibits gap junctional communication in
human embryonic palatal mesenchyme cells. In Vitro Toxicol 5(2): 113-122.
Cannon, SB; Kimbrough, RD. (1979) Short-term chlordecone toxicity in rats including effects on reproduction,
pathological organ changes, and their reversibility. Toxicol Pharmacol 47:469-476.
Cannon, SB; Veazey, JM; Jackson, RS; et al. (1978) Epidemic Kepone poisoning in chemical workers. Am J
Epidemiol 107:529-537.
Carpenter, HM; Curtis, LR. (1989) A characterization of chlordecone pretreatment-altered pharmacokinetics in
mice. Drug Metab Dispos 17(2): 131-138.
Carpenter, HM; Curtis, LR. (1991) Low dose chlordecone pretreatment altered cholesterol disposition without
induction of cytochrome P-450. Drug Metab Dispos 19(3):673-678.
Carpenter, HM; Hedstrom, OR; Siddens, LK; et al. (1996) Ultrastructural, protein, and lipid changes in liver
associated with chlordecone treatment of mice. Fundam Appl Toxicol 34(1): 157-164.
Chadwick, RW; Copeland, MF; Rosenstein, L. (1979) The effect of Kepone exposure during gestation and lactation
on the metabolism of lindane by weanling rats. Toxicol Lett 4:247-252.
Chen, PH; Tilson, HA; Marbury, GD; et al. (1985) Effect of chlordecone (Kepone) on the rat brain concentrations of
3-methoxy-4-hydroxyphenylglycol: evidence for a possible involvement of the norepinephrine system in
chlordecone-induced tremor. Toxicol Appl Pharmacol 77:158-164. (as cited in ATSDR, 1995)
Chernoff, N; Rogers, EH. (1976) Fetal toxicity of Kepone in rats and mice. Toxicol Appl Pharmacol 38:189-194.
Chernoff, N; Kavlock, RJ. (1982) An in vivo teratology screen utilizing pregnant mice. J Toxicol Environ Health
10:541-550.
Chetty, KN; Walker, J; Brown, K; et al. (1993a) The effects of dietary calcium and chlordecone on cholinsterase,
triglyderides, low density lipoproteins, and cholesterol in serum of rat. Arch Environ Contam Toxicol 24(3):365-
367.
Chetty KN, Brown K, Walker J; et al. (1993b) Effects of chlordecone and malnutrition on immune response in rats.
LifeSci52(18):PL175-180.
Chetty, KN; Walker, J; Brown, K; et al. (1993c) Influence of dietary calcium on chlordecone-induced biochemical
changes in serum of rat. Ecotoxicol Environ Saf 26(2):248-252.
Chu, I; Villeneuve, DC; Becking, GC; et al. (1980) Short-term study of the combined effects of mirex, photomirex,
and Kepone with halogenated biphenyls in rats. J Toxicol Environ Health 6:421-432.
Chu, I; Villeneuve, DC; Valli, VE; et al. (1981a) Chronic toxicity of photomirex in the rat. Toxicol Appl Pharmacol
59:268-278.
Chu, I; Villeneuve, DC; MacDonald, BL; et al. (1981b) Reversibility of the lexicological changes induced by
photomirex and mirex. Toxicology 21(3):235-250.
Chu, I; Villeneuve, DC; Secours, VE; et al. (1981c) Effects of photomirex and mirex on reproduction in the rat.
Toxicol Appl Pharmacol 60(3):549-556.
Cohn, WJ; Boylan, JJ; Blanke, RV; et al. (1978) Treatment of chlordecone (Kepone) toxicity with cholestyramine:
results of a controlled trial. N Engl J Med 298:243-248.
Cooper, JR; Vodicnik, MJ; Gordon JH. (1985) Effects of perinatal Kepone exposure on sexual differentiation of the
rat brain. Neurotoxicology 6(1): 183-90.
JANUARY 2008 98 DRAFT - DO NOT CITE OR QUOTE
-------
Curtis, LR. (1988) Chlordecone is a potent in vitro inhibitor of oligomycin-insensitive Mg +-ATPase of rat bile
canaliculi-enriched fraction. J Biochem Toxicol 3:321-328. (as cited in ATSDR, 1995)
Curtis, LR; Mehendale, HM. (1979) The effects of Kepone pretreatment on biliary excretion of xenobiotics in the
male rat. Toxicol Appl Pharmacol 47:295-303. (as cited in ATSDR, 1995)
Curtis, LR; Thureson-Klein, AK; Mehendale, HM. (1981) Ultrastructural and biochemical correlates of the
specificity of chlordecone-potentiated carbon tetrachloride hepatotoxicity. J Toxicol Environ Health 7(3-4):499-
517.
Dalu, A; Warbritton, A; Bucci, TJ; et al. (1995) Age-related susceptibility to chlordecone-potentiated carbon
tetrachloride hepatotoxicity and lethality is due to hepatic quiescence. PediatrRes 38(2): 140-148.
Dalu, A; Rao, PS; Mehendale, HM. (1998) Colchicine antimitosis abolishes resiliency of postnatally developing
rats to chlordecone-amplified carbon tetrachloride hepatotoxicity and lethality. Environ Health Perspect
106(9):597-606.
Das, SK; Taylor, JA; Korach, KS; et al. (1997) Estrogenic responses in estrogen receptor-alpha deficient mice reveal
a distinct estrogen signaling pathway. Proc Natl Acad Sci 94:12786-12791.
Demi E, Oesterle D. (1987) Dose-response of promotion by polychlorinated biphenyls and chloroform in rat liver
foci bioassay. Arch Toxicol 60:209-1 l.Desaiah, D; Gilliland, T; Ho, IK; et al. (1980) Inhibition of mouse
synaptosomal ATPases and ouabain binding by chlordecone. Toxicol Lett 6:275-285. (as cited in ATSDR, 1995)
Desaiah, D. (1982) Biochemical mechanisms of chlordecone neurotoxicity: a review. Neurotoxicology 3(2): 103-
110.
Desaiah, D. (1985) Chlordecone interaction with catecholamine binding and uptake in rat brain synaptosomes.
Neurotoxicology 6(1): 159-165.
Desaiah, D; Chetty, CS; Rao, KS. (1985) Chlordecone inhibition of calmodulin activated cacium ATPase in rat
brain synaptosomes. J Toxicol Environ Health 16:189-195. (as cited in ATSDR, 1995)
Egle, JL, Jr.; Fernandez, JB; Guzelian, PS; et al. (1978) Distribution and excretion of chlordecone (Kepone) in the
rat. Drug Metab Dispos 6(l):91-95.
el-Masri, HA; Thomas, RS; Benjamin, SA; et al. (1995) Physiologically based pharmacokinetic/pharmacodynamic
modeling of chemical mixtures and possible applications in risk assessment. Toxicology 105(2-3):275-282.
End, DW; Carchman, RA; Ameen, R; et al. (1979) Inhibition of rat brain mitochondrial calcium transport by
chlordecone. Toxicol Appl Pharmacol 51:189-196. (as cited in ATSDR, 1995)
End, DW; Carchman, RA; Dewey, WL. (1981) Neurochemical correlates of chlordecone neurotoxicity. J Toxicol
Environ Health 8(5-6):707-718.
Fariss, MW; Blanke, RV; Saady, V; et al. (1980) Demonstration of major metabolic pathways for chlordecone
(Kepone) in humans. Drug Metab Dispos 8:434-438.
Faroon, OM; Mehendale, HM. (1990) Bromotrichlormethane hepatotoxicity. The role of stimulated hepatocellular
regeneration in recovery: biochemical and histopathological studies in control and chlordecone pretreated male rats.
Toxicol Pathol 18(4 Pt. 2):667-677. (as cited in ATSDR, 1995)
Fujimori, K; Benet, H; Mehendale, HM; et al. (1982a) Comparison of brain discrete area distributions of
chlordecone and mirex in the mouse. Neurotoxicology 3(2): 125-129.
Fujimori, K; Nabeshima, T; Ho, IK; et al. (1982b) Effects of oral administration of chlordecone and mirex on brain
biogenic amines in mice. Neurotoxicology 3(2): 143-148.
JANUARY 2008 99 DRAFT - DO NOT CITE OR QUOTE
-------
Galloway, SM; Armstrong, MJ; Reuben, C; et al. (1987) Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen 10(Suppl. 10): 1-175.
Gaines TB. (1969). Acute toxicity of pesticides. Toxicol Appl Pharmacol 14:515-534.
Gaines, TB; Kimbrough, RD. (1970) Oral toxicity of mirex in adult and suckling rats. With notes on the
ultrastructure of liver changes. Arch Environ Health 21:7-14.
Gerhart, JM; Hong, JL; Uphouse, LL; et al. (1982) Chlordecone-induced tremor: quantification and
pharmacological analysis. Toxicol Appl Pharmacol 66:234-243. (as cited in ATSDR, 1995)
Gerhart, JM; Hong, JS; Tilson, HA. (1983) Studies on the possible sites of chlordecone-induced tremor in rats.
Toxicol Appl Pharmacol 70:382-389. (as cited in ATSDR, 1995)
Gerhart, JM; Hong, JS; Tilson, HA. (1985) Studies on the mechanism of chlordecone-induced tremor in rats.
Neurotoxicology 61:211-229. (as cited in ATSDR, 1995)
Gellert, RJ; Wilson, C. (1979) Reproductive function in rats exposed prenatally to pesticides and poly chlorinated
biphenyls (PCB). Environ Res 18:437-443.
Gibson, JR; Ivie, GW; Dorough, HW. (1972) Fate of mirex and its major photodecomposition product in rats. J
Agric Food Chem 20(6): 1246-1248.
Gilroy, DJ; Carpenter, HM; Curtis, LR. (1994) Chlordecone pretreatment alters [14C]chlordecone and
[14C]cholesterol transport kinetics in the perfused rat liver. Fundam Appl Toxicol 22(2):286-292.
Good, EE; Ware, GW; Miller, DF. (1965) Effects of insecticides on reproduction in the laboratory mouse: I.
Kepone. J Econ Entomol 58(4):754-757.
Guzelian, PS. (1982a) Chlordecone poisoning: a case study in approaches for the detoxification of humans exposed
to environmental chemicals. Drug Metab Rev 13:663-679.
Guzelian, PS. (1982b) Comparative toxicology of Chlordecone (Kepone) in humans and experimental animals. Ann
Rev Pharmacol Toxicol 22:89-113.
Guzelian, PS; Vranian, G; Boylan, JJ; et al. (1980) Liver structure and function in patients poisoned with
Chlordecone (Kepone). Gastroenterology 78(2):206-213.
Hammond B; Katyzenellenbogen BS; Krauthammer N; et al. (1979). Estrogenic activity of the insecticide
Chlordecone (Kepone) and interaction with uterine estrogen receptors. Proc Natl Acad Sci 76:6641-6645.Hansch, C;
Leo, A; Hoekman, D. (1995) Exploring QSAR: hydrophobic, electronic, and steric constants. ACS Professional
Reference Book. Washington, DC: American Chemical Society; p. 65.
Hart, SE; Kinter, LB. (2005). Assessing renal effects of toxicants in vivo. In: Tarloff, JB; Lawrence, LH; eds.
Toxicology of the kidney. New York, NY: CRC Press; pp. 81-147.
Heatheringon, AC; Fisher, HL; Sumler, MR; et al. (1998) Percutaneous absorption and disposition of
[14C]chlordecone in young and adult female rats. Environ Res 79(2): 138-155.
Herr, DW; Gallus, JA; Tilson, HA. (1987) Pharmacological modification of tremor and enhanced acoustic startle by
Chlordecone and p,p'-DDT. Psychopharmacology 91:320-325. (as cited in ATSDR, 1995)
Hewitt, LA; Caille, G; Plaa, GL; et al. (1985) Temporal relationships between biotransformation, detoxication, and
Chlordecone potentiation of chloroform-induced hepatotoxicity. Can J Physiol Pharmacol 64:477-482.
JANUARY 2008 100 DRAFT - DO NOT CITE OR QUOTE
-------
Hong, JS; Tilson, HA; Uphouse, LL; et al. (1984) Effects of chlordecone exposure on brain neurotransmitters:
possible involvement of the serotonin system in chlordecone-elicited tremor. Toxicol Appl Pharmacol 73:336-344.
(as cited in ATSDR, 1995)
Hoskins, B; Ho, IK. (1982) Chlordecone-induced alterations in content and subcellular distribution of calcium in
mouse brain. J Toxicol Environ Health 9:535-544. (as cited in ATSDR, 1995)
Houston, TE; Mutter, LC; Blanke, RV; et al. (1981) Chlordecone alcohol formation in the Mongolian gerbil
(Meriones unguiculatus): A model for human metabolism of chlordecone (Kepone). Fundam Appl Toxicol
l(3):293-298.
Huang, ESR; Nelson, FR. (1986) Anti-estrogenic action of chlordecone in rat pituitary gonadotrophs in vitro.
Toxicol Appl Pharmacol 82:62-69.
Huang, TP; Ho, IK; Mehendale, HM. (1980) Assessment of neurotoxicity induced by oral administration of
chlordecone (Kepone) in the mouse. Neurotoxicology 2:113-124.
Huber, JJ. (1965) Some physiological effects of the insecticide Kepone in the laboratory mouse. Toxicol Appl
Pharmacol 7:516-524.
Hwang, EC; Van Woert, MH. (1979) Serotonin-norepinephrine interactions in the tremorolytic actions of
phenoxybenzamine and trazodone. Pharmacol Biochem Behav 10(l):27-29. (as cited in ATSDR, 1995)
IARC (International Agency for Research on Cancer). (1979) Chlordecone. IARC monographs on the evaluation of
carcinogenic risk of chemicals to humans. Vol. 20. Some halogenated hydrocarbons. Lyon, France: International
Agency for Research on Cancer; p. 67.
Innes, JRM; Ulland, BM; Valerio, MG; et al. (1969) Bioassay of pesticides and industrial chemicals for
tumorigenicity in mice: apreliminary note. JNatl Cancer Inst42(6):1101-1114.
Inoue, K; Nakazawa, K; Obama, T; et al. (1991) Chlordecone inhibits three types of ion channels in a neural cell
line. Pharmacol Toxicol 68:444-446. (as cited in ATSDR, 1995)
Ivie, GW; Gibson, JR; Bryant, HE; et al. (1974) Accumulation, distribution, and excretion of mirex-14C in animals
exposed for long periods to the insecticide in the diet. J Agric Food Chem 22(4):646-653.
Jinna, RR; Uzodinma, JE; Desaiah, D. (1989) Age-related changes in rat brain ATPases during treatment with
chlordecone. J Toxicol Environ Health 27(2): 199-208.
Johnson, DC. (1996) Estradiol-chlordecone (Kepone) interactions: additive effect of combinations for uterotropic
and embryo implantation functions. Toxicol Lett 89:57-64.
Johnson, DC; Sen, M; Kogo, H; et al. (1990) Initiation of embryo implantation and maintenance of early pregnancy
in the rat by chlordecone (Kepone). Proc Soc Exp Biol Med 195(1):44-50.
Johnson, DC; Banerjee, S; Chatterjee, S. (1995) Estradiol and chlordecone (Kepone) decrease adenosine 3'5'-cyclic
monophosphate concentrations in the ovariectomized immature rat uterus. Proc Soc Exp Biol Med 210(l):33-38.
Kavlock, RJ; Chemoff, N; Rogers, E; et al. (1980) Comparative tissue distribution of mirex and chlordecone in fetal
and neonatal rats. Pestic Biochem Physiol 14(3):227-235.
Kennedy, MW; Pittman, KA; Stein, VM. (1975) Fate of 14C mirex in the female rhesus monkey. Toxicol Appl
Pharmacol 33:161-162.
Kilzer, L; Scheunert, I; Geyer, H; et al. (1979) Laboratory screening of the volatilization rates of organic chemicals
from water and soil. Chemosphere 10:751-761.
JANUARY 2008 101 DRAFT - DO NOT CITE OR QUOTE
-------
Kitchin, KT; Brown, JL. (1989) Biochemical studies of promoters of carcinogenesis in rat liver. Teratogen
Carcinogen Mutagen 9:273-285.
Kocarek, TA; Schuetz, EG; Guzelian, PS. (1991) Selective induction of cytochrome P450eby Kepone
(chlordecone) in primary cultures of adult rat hepatocytes. Mol Pharmacol 40:203-210.
Kocarek, TA; Schuetz, EG; Guzelian, PS. (1994) Regulation of cytochrome P450 2B1/2 mRNAs by Kepone
(chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel. Toxicol Lett
71(2):183-196.
Kodavanti, PR; Joshi, UM; Mehendale, HM; et al. (1989) Chlordecone (Kepone)-potentiated carbon tetrachloride
hepatotoxicity in partially hepatectomized rats: a histomorphometric study. J Appl Toxicol 9(6):367-375.
Kodavanti, PR; Kodavanti, UP; Mehendale, HM. (1990) Altered hepatic energy status in chlordecone (Kepone)-
potentiated CC14 hepatotoxicity. Biochem Pharmacol 40(4):859-866.
Kodavanti, PRA; Kodavanti, UP; Faroon, OM; et al. (1992) Pivitol role of hepatocellular regeneration in the
ultimate hepatotoxicity of CC14 in chlordecone-, mirex-, or phenobarbital-pretreated rats. Toxicol Pathol
20(4):556-569.
Kodavanti, PRA; Rao, VC; Mehendale, HM. (1993) Loss of calcium homeostasis leads to progressive phase of
chlordecone-potentiated carbon tetrachloride hepatotoxicity. Toxicol Appl Pharmacol 122:77-87.
Kuiper, GGJM; Lemmen, JG; Carlsson, B; et al. (1998) Interaction of estrogenic chemicals and phytoestrogens with
estrogen receptor beta. Endocrinology 139(10):4252-4263.
Laessig, SA; Auger, AP; McCarthy, MM; et al. (2007) Effects of prenatal chlordecone on sexually differentiated
behavior in adult rats. Neurotoxicol Teratol 29(2):255-263.
Lahita, RG. (1997) Predisposing factors to autoimmune disease. Int J Fertil Womens Med 42(2): 115-119.
Larson, PS; Egle, JL, Jr; Hennigar, GR. (1979a) Acute, subchronic, and chronic toxicity of chlordecone. Toxicol
Appl Pharmacol 48:29-41.
Larson, PS; Egle, JL, Jr; Hennigar, GR; et al. (1979b) Acute and subchronic toxicity of mirex in the rat, dog and
rabbit. Toxicol Appl Pharmacol 49(2):271-277.
Lide, DR; ed. (2000) CRC handbook of chemistry and physics. 81st edition. Boca Raton, FL: CRC Press; p. 3-204.
Linder, RE; Scotti, TM; McElroy, WK; et al. (1983) Spermotoxicity and tissue accumulation of chlordecone
(Kepone) in male rats. J Toxicol Environ Health 12:183-192.
Mactutus, CF; Tilson, HA. (1984) Neonatal chlordecone exposure impairs early learning and retention of active
avoidance in the rat. Neurobehav Toxicol Teratol 6(l):75-83.
Mactutus, CF; Tilson, HA. (1985) Evaluation of long-term consequences in behavioral and/or neural function
following neonatal chlordecone exposure. Teratology 31(2): 177-186.
Martinez, AJ; Taylor, JR; Dyck, PJ; et al. (1978) Chlordecone intoxication in man: II. Ultrastructure of peripheral
nerves and skeletal muscle. Neurology 28:631-635.
Mehendale, HM. (1990) Potentiation of halomethane hepatotoxicity by chlordecone: a hypothesis for the
mechanism. Med Hypotheses 33(4):289-299.
Mehendale, HM. (1994) Amplified interactive toxicity of chemicals at nontoxic levels: mechanistic considerations
and implications to public health. Environ Health Perspect 102(Suppl. 9): 139-149.
JANUARY 2008 102 DRAFT - DO NOT CITE OR QUOTE
-------
Mehendale, HM; Klingensmith, JS. (1988) In vivo metabolism of CC14 by rats pretreated with chlordecone, mirex,
or phenobarbitol. Toxicol Appl Pharmacol 93(2):247-256.
Mehendale, HM; Takanaka, A; Desaiah, D; et al. (1977) Kepone induction of hepatic mixed function oxidases. Life
Sci20(6):991-997.
Mehendale, HM; Takanaka, A; Desaiah, D; et al. (1978) Effect of preexposure to Kepone on hepatic mixed function
oxidases in the female rat. Toxicol Appl Pharmacol 44:171-180.
Mehendale, HM; Purushotham, KR; Lockard, VG. (1989) The time course of liver injury and [3H]thymidine
incorporation in chlordecone-potentiated CHC13 hepatotoxicity. ExpMolPathol 51:31-47.
Metcalf, RL. (2002) Insect control. In: Ullmann's encyclopedia of industrial chemistry. Wiley Interscience.
Accessed March 22, 2004. Available online at http://www.mrw.interscience.wiley.com/ueic/ueic_search_fs.html.
Mitra, A; Richards, I; Kitchin, K; et al. (1990) Mirex induces ornitine decarboxylase in female rat liver. J Biochem
Toxicol 5(2): 119-124.
Molowa, DT; Wrighton, SA; Blanke, RV; et al. (1986) Characterization of a unique aldo-keto reductase responsible
for the reduction of chlordecone in the liver of the gerbil and man. J Toxicol Environ Health 17:375-384.
Morgan, DP; Sandifier, SH; Hetzler, HL; et al. (1979) Test for in vivo conversion of mirex to Kepone. Bull Environ
Contam Toxicol 22( 1-2):238-244 (as cited in ATSDR, 1995).
Mortelmans, K; Haworth, S; Lawlor, T; et al. (1986) Salmonella mutagenicity tests: II. Results from the testing of
270 chemicals. Environ Mutagen 8(Suppl. 7): 1-119.
Murali, B; Korrapati, MC; Anand, SS; et al. (2002) Age-dependent susceptibility of F344 rats to chlordecone
potentiated CC14 hepatotoxicity and lethality. Int J Toxicol 21(6):524.
NCI (National Cancer Institute). (1976a) Report on carcinogenesis bioassay of technical grade chlordecone
(Kepone) (CAS No. 143-50-0). Carcinogenesis Program, Division of Cancer Cause and Prevention, National
Cancer Institute, Public Health Service, U.S. Department of Health and Human Services; NTP TR-00. Available
online at http://ntp.niehs.nih.gov/.
NCI. (1976b) Microfiche for NCI 1976a study. Project # 455-5224.
NIOSH (National Institute for Occupational Safety and Health). (2004) Pocket guide to chemical hazards. Centers
for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services,
Cincinnatti, Ohio. Accessed March 23, 2004 at http://www.cdc.gov/niosh/npg/.
NLM (National Library of Medicine). (2004a) Chlordecone. HSDB (Hazardous Substances Data Bank). National
Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland. Last review dated August
25, 1989. Available online at http://toxnet.nlm.nih.gov/cgi-bin/sis/htmlgen7HSDB.
NLM (National Library of Medicine). (2004b) Chlordecone. ChemlDplus. National Institutes of Health, U.S.
Department of Health and Human Services, Bethesda, Maryland. Accessed February 6, 2007 at
http://chem.sis.nlm.nih.gov/chemidplus/isp/common/ChemFull.jsp?MW=490.639.
NLM. (2004c) Mirex. ChemlDplus. National Institutes of Health, U.S. Department of Health and Human Services,
Bethesda, Maryland. Accessed February 6, 2007 at
http://chem.sis.nlm.nih.gov/chemidplus/isp/common/ChemFull.jsp?MW=545.546.
NRC (National Research Council). (1983) Risk assessment in the federal government: managing the process.
Washington, DC: National Academy Press.
JANUARY 2008 103 DRAFT - DO NOT CITE OR QUOTE
-------
NRC (National Research Council). (1994) Science and judgment in risk assessment. Washington, DC: NationalAcademy Press.
NTP (National Toxicology Program). (1990) Toxicology and carcinogenesis studies of mirex (CAS No. 2385-85-5)
in F344/N rats (feed studies). Public Health Service, U.S. Department of Health and Human Services; NTP TR-313;
NIH Publ. No. 90-2569. Available from the National Institute of Environmental Health Sciences, Research Triangle
Park, NC, and online at http://ntp.niehs.nih.gov/ntpweb/index.cfrn?objectid=08480D3B-9D26-6528-
FDD1F546F54EE341.
O'Neil, MJ; ed. (2001) The Merck index: an encyclopedia of chemicals, drugs, and biologicals. 13th edition.
Whitehouse Station, NJ: Merck and Co., Inc.; p. 359.
PWG (Pathology Working Group). (1992) Pathology Working Group report on mirex chronic
toxicity/carcinogenicity study in F344 rats. Prepared by R.M. Sauer, PATHCO, Inc. Ijamsville, MD. (unpublished
report)
Pinkston, G; Uphouse, L. (1987-1988) Postovulatory reduction of fertility in chlordecone treated female rats.
Reprod Toxicol 1(2):105-109.
Pittman, KA; Wiener, W; Treble, DH. (1976) Mirex kinetics in the rhesus monkey. II. Pharmacokinetic model.
Drug Metab Dispos 4(3):288-295.
Plaa, GL; Caille, G; Vezina, M; et al. (1987) Chloroform interaction with chlordecone and mirex: correlation
between biochemical and histological indexes of toxicity and quantitative tissue levels. Fundam Appl Toxicol
9:198-207.
Probst, GS; McMahont, RE; Hill, LE; et al. (1981) Chemically-induced unscheduled DNA synthesis in primary rat
hepatocyte cultures: a comparison with bacterial mutagenicity using 218 compounds. Environ Mutagen 3:11-32.
Pryor, GT; Uyeno, ET; Tilson, HA; et al. (1983) Assessment of chemicals using a battery of neurobehavioral tests: a
comparative study. Neurobehav Toxicol Teratol 5(1):91-117. (as cited in ATSDR, 1995)
Rao, SB; Mehendale, HM. (1989) Protection from chlordecone (Kepone)-potentiated CC14 hepatotoxicity in rats by
fructose 1,6-diphosphate. Int JBiochem21(9):949-954.
Rao, SB; Young, RA; Mehendale, HM. (1990) Perturbations inpolyamines and related enzymes following
chlordecone-potentiated bromotrichloromethane hepatotoxicity. J Biochem Toxicol 5(l):23-32.
Rochelle, LG; Curtis LR. (1994) Distribution of chlordecone to liver plasma membranes and recovery from
hepatobiliary dysfunction in rats. Toxicology 86(1-2): 123-134.
Rochelle, LG; Miller, TL; Curtis, LR. (1990) Chlordecone impairs Na(+)-stimulated L-[3H]glutamate transport and
mobility of 16-doxyl stearate in rat liver plasma membrane vesicles. Toxicol Appl Pharmacol 105(2):234-242.
Rosecrans, JA; Squibb, RE, Jr; Johnson, JH; et al. (1985) Effects of neonatal chlordecone exposure on pituitary-
adrenal function in adult Fischer-344 rats. Neurobehav Toxicol Teratol 7(l):33-37.
Rosenstein, L; Brice, A; Rogers, N; et al. (1977) Neurotoxicity of Kepone in perinatal rats following inutero
exposure. Toxicol Appl Pharmacol 41:142-143.
Sanborn, GE; Selhorst, JB; Calabrese, VP; et al. (1979) Pseudotumor cerebri and insecticide intoxication.
Neurology 29(9 Pt 1): 1222-1227.
Schoeny, RS; Smith, CS; Loper, JC. (1979) Non-mutagenicity for salmonella of the chlorinated hydrocarbons
aroclor 1254, 1,2,4-trichlorobenzene, mirex and Kepone. MutatRes 68:125-132.
Schrader, TJ; Cooke, GM. (2000) Examination of selected food additives and organochlorine food contaminants for
androgenic activity in vitro. Toxicol Sci 53:278-288.
JANUARY 2008 104 DRAFT - DO NOT CITE OR QUOTE
-------
Scippo, ML; Argiris, C; Van De Weerdt, C; et al. (2004) Recombinant human estrogen, androgen and progesterone
receptors for detection of potential endocrine disrupters. Anal Bioanal Chem 378:664-669.
Seidenberg, JM; Anderson, DG; Becker, RA. (1986) Validation of an in vivo developmental toxicity screen in the
mouse. Teratogen Carcinogen Mutagen 6:361-374.
Shah, PV; Fisher, HL; Sumler, MR; et al. (1987) Comparison of the penetration of 14 pesticides through the skin of
young and adult rats. J Toxicol Environ Health 21(3):353-366.
Sierra, V; Uphouse, L. (1986) Long-term consequences of neonatal exposure to chlordecone. Neurotoxicology
7(2):609-621.
Simon, GS; Kipps, BR; Tardiff, RG; et al. (1978) Failure of Kepone and hexachlorobenzene to induce dominant
lethal mutations in the rat. Toxicol Appl Pharmacol 45(l):330-331.
Simon, GS; Egle, JL, Jr; Dougherty, RW; et al. (1986) Dominant lethal assay of chlordecone and its distribution in
the male reproductive tissues of the rat. Toxicol Lett 30:237-245.
Sirica, AE; Wilkerson, CS; Wu, LL; et al. (1989) Evaluation of chlordecone in a two-stage model of
hepatocarcinogenesis: a significant sex difference in the hepatocellular carcinoma incidence. Carcinogenesis
10(6): 1047-1054.
Skalsky, HL; Farris, MW; Blanke, RV; et al. (1979) The role of plasma proteins in the transport and distribution of
chlordecone (Kepone ) and other polyhalogenated hydrocarbons. Ann NYT Acad Sci 320:231-237.
Smialowicz, RJ; Luebke, RW; Riddle, MM; et al. (1985) Evaluation of the immunotoxic potential of chlordecone
with comparison to cyclophosphamide. J Toxicol Environ Health 15(5):561-574.
Sobel, ES; Gianini, J; Butfiloski, EJ; et al. (2005) Acceleration of autoimmunity by organochlorine pesticides in
(NZB x NZW)Fj mice. Environ Health Perspect 113(3):323-328.
Sobel, ES; Wang, F; Butfiloski, EJ; et al. (2006) Comparison of chlordecone effects on autoimmunity in (NZB *
NZW) F(l) and BALB/c mice. Toxicology 218(2-3):81-89.
Soine, PJ; Blanke, RV; Guzelian, PS; et al. (1982) Preferential binding of chlordecone to the protein and high
density lipoprotein fractions of plasma from humans and other species. J Toxicol Environ Health 9:107-118.
Soine, PJ; Blanke, RV; Schwartz, CC. (1983) Chlordecone metabolism in the pig. Toxicol Lett 17(l-2):35-41.
Soine, PJ; Blanke, RV; Chinchilli, VM; et al. (1984) High-density lipoproteins decrease the biliary concentration of
chlordecone in isolated perfused pig liver. J Toxicol Environ Health 14(2-3):319-335.
Soni, MG; Mehendale, HM. (1991a) ATP protection of chlordecone-amplified carbon tetrachloride hepatotoxicity
and lethality. Fed Am Soc Exp Biol J 5(6):A1604.
Soni, MG; Mehendale, HM. (1991b) Protection from chlordecone-amplified carbon tetrachloride toxicity by
cyanidanol: regeneration studies. Toxicol Appl Pharmacol 108(l):58-66.
Soni, MG; Mehendale, HM. (1991c) Protection from chlordecone-amplifed carbon tetrachloride toxicity by
cyanidanol: biochemical and histological studies. Toxicol Appl Pharmacol 108(l):46-57.
Soni, MG; Mehendale, HM. (1993) Hepatic failure leads to lethality of chlordecone-amplified hepatotoxicity of
carbon tetrachloride. Fundam Appl Toxicol 21(4):442-450.
Squibb, RE; Tilson, HA. (1982) Effects of gestational and perinatal exposure to chlordecone (Kepone) on the
neurobehavioral development of Fischer-344 rats. Neurotoxicology 3(2): 17-26.
JANUARY 2008 105 DRAFT - DO NOT CITE OR QUOTE
-------
Starcevic, SL; Bortolin, S; Woodcraft, KJ; et al. (2001) Kepone (chlordecone) disrupts adherens junctions in human
breast epithelial cells cultured on Matrigel. In Vivo 15:289-294.
Swanson, KL; Woolley, DE. (1982) Comparison of the neurotoxic effects of chlordecone and dieldrin in the rat.
Neurotoxicology 3(2):81-102. (as cited in ATSDR, 1995)
Swartz, WJ; Mall, GM. (1989) Chlordecone-induced follicular toxicity in mouse ovaries. Reprod Toxicol 3:203-
206.
Swartz, WJ; Eroschenko, VP; Schutzmann, RL. (1988) Ovulatory response of chlordecone (Kepone)-exposed mice
to exogenous gonadotropins. Toxicology 51(2-3):147-153.
Taylor, JR. (1982) Neurological manifestations in humans exposed to chlordecone and follow-up results.
Neurotoxicology 3(2):9-16.
Taylor, JR. (1985) Neurological manifestations in humans exposed to chlordecone: follow-up results.
Neurotoxicology 6(1):231-236.
Taylor, JR; Selhorst, JB; Houff, SA; et al. (1978) Chlordecone intoxication in man: I. Clinical observations.
Neurology 28:626-630.
Teo, S; Vore, M. (1991) Mirex inhibits bile acid secretory function in vivo and in the isolated perfused rat liver.
Toxicol Appl Pharmacol 109(1): 161-170.
Tilson, HA; Hudson, PM, Hong, JS. (1986) 5,5-Diphenylhydantoin antagonizes neurochemical and behavioral
effects of p,p'-DDT but not of chlordecone. J Neurochem 47(6): 1870-1878. (as cited in ATSDR, 1995)
Trosko, JE; Jone, C; Chang, CC. (1983) The role of tumor promoters onphenotypic alterations affecting
intercellular communication and tumorigenesis. Ann NY Acad Sci. 407:316-327.
Tsushimoto, G; Trosko, JE; Chang, CC; etal. (1982) Inhibition of intercellular communication by chlordecone
(Kepone) and mirex in Chinese hamster v79 cells in vitro. Toxicol Appl Pharmacol 64:550-556.
Ulland, BM; Page, NP; Squire, RL; et al. (1977) A carcinogenicity assay of mirex in Charles River CD rats. J Natl
Cancer Inst 58:133-140.
Uphouse, L. (1985) Effects of chlordecone on neuroendocrine function of female rats. Neurotoxicology 6(1):191-
210.
Uphouse, L; Eckols, K. (1986) Serotonin receptors in striatum after chlordecone treatment of adult female rats.
Neurotoxicology 7(l):25-32. (as cited in ATSDR, 1995)
Uphouse, L; Eckols, K; Sierra, V; et al. (1986) Failure of chlordecone (Kepone) to induce behavioral estrus in adult
ovariectomized rats. Neurotoxicology 7(1): 127-142.
U.S. DHHS (Department of Health and Human Services). (2006) Glomerular diseases. National Kidney and
Urologic Diseases Information Clearinghouse, Bethesda, MD; NIH Publication No. 07-4358. Available online at
http://kidney.niddk.nih.gov/kudiseases/pubs/glomerular/.
U.S. EPA (Environmental Protection Agency). (1986a) Guidelines for the health risk assessment of chemical
mixtures. Federal Register 51(185):34014-34025. Available online at http://www.epa.gov/ncea/raf/rafguid.htm.
U.S. EPA. (1986b) Guidelines for mutagenicity risk assessment. Federal Register 51(185):34006-34012. Available
online at http://www.epa.gov/ncea/raf/rafguid.htm.
U.S. EPA. (1986c) Final report on the evaluation of four toxic chemicals in an in vivo/in vitro toxicological screen:
Acrylamide, chlordecone, cyclophosphamide, and diethylstilbestrol. EPA/600/1-86/002.
JANUARY 2008 106 DRAFT - DO NOT CITE OR QUOTE
-------
U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk assessment.
Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Cincinnati, OH;
EPA/600/6-87/008. Available from the National Technical Information Service, Springfield, VA; PB88-179874/AS,
and online at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=34855.
U.S. EPA. (1991) Guidelines for developmental toxicity risk assessment. Federal Register 56(234):63798-63826.
Available online at http://www.epa.gov/ncea/raf/rafguid.htm.
U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity: notice of
availability. Federal Register 59(206):53799. Available online at http://www.epa.gov/EPA-
PEST/1994/October/Day-26/pr-11 .html.
U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation
dosimetry. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH; EPA/600/8-90/066F. Available from the National Technical Information Service, Springfield, VA,
PB2000-500023, and online at http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=71993.
U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/R-94/007. Available from the National Technical Information Service, Springfield, VA,
PB95-213765, and online at http://cfpub.epa.gov/ncea/raf/raf_pubtitles.cfm?detype=document&excCol=archive.
U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessment. Federal Register 61(212):56274-56322.
Available online at http://www.epa.gov/ncea/raf/rafguid.htm.
U.S. EPA. (1998a) Guidelines for neurotoxicity risk assessment. Federal Register 63(93):26926-26954. Available
online at http://www.epa.gov/ncea/raf/rafguid.htm.
U.S. EPA. (1998b) Science policy council handbook: peer review. Office of Science Policy, Office of Research and
Development, Washington, DC; EPA/100-B-98-001. Available from the National Technical Information Service,
Springfield, VA, PB98-140726, and online at http://www.epa.gov/waterscience/WET/pdf/prhandbk.pdf.
U.S. EPA. (2000a) Science policy council handbook: peer review. 2nd edition. Office of Science Policy, Office of
Research and Development, Washington, DC. EPA/100-B-OO-OOl. Available online at
http://www.epa.gov/OSA/spc/2peerrev.htm.
U.S. EPA. (2000b) Science policy council handbook: risk characterization. Office of Science Policy, Office of
Research and Development, Washington, DC. EPA/100-B-00-002. Available online at
http://www.epa.gov/OSA/spc/pdfs/prhandbk.pdf.
U.S. EPA. (2000c) Benchmark dose technical guidance document [external review draft]. Risk Assessment Forum,
Washington, DC; EPA/630/R-00/001. Available online at http://cfpub.epa.gov/ncea/cfm/
nceapublication.cfm?ActType=PublicationTopics&detype=DOCUMENT&subject=BENCHMARK+DOSE&subjty
pe=TITLE&excCol=Archive.
U.S. EPA. (2000d) Supplementary guidance for conducting health risk assessment of chemical mixtures. Risk
Assessment Forum, Washington, DC; EPA/630/R-00/002. Available online at
http://cfpub.epa.gov/ncea/raf/chem_mix.cfm.
U.S. EPA. (2002) A review of the reference dose concentration and reference concentration processess. Risk
Assessment Forum, Washington, DC; EPA/630/P-02/002F. Available online at
http://cfpub.epa. gov/ncea/raf/raf_pubtitles.cfm?detype=document&excCol=archive.
U.S. EPA. (2005a) Guidelines for carcinogen risk assessment. Federal Register 70(66): 17765-18717. Available
online at http://www.epa.gov/cancerguidelines.
U.S. EPA. (2005b) Supplimental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens.
EPA/630/R-03/003F Available online athttp://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=160003.U.S. EPA.
JANUARY 2008 107 DRAFT - DO NOT CITE OR QUOTE
-------
(2005c) Peer review handbook. 3rd edition. Review draft. Science Policy Council, Washington, DC. Available
online at http://intranet.epa.gov/ospintra/scipol/prhndbk05.doc.
Vaccari, A; Saba, P. (1995) The tyramine-labelled vesicular transporter for dopamine: a putative target of pesticides
and neurotoxins. Euro J Pharmacol 292:309-314.
Vig, PJS; Mehrotra, BD; Pennington, A; et al. (1989) Chlordecone interaction of calmodulin binding with
phosphodiesterase. FASEB J 3(4):A1037.
Wang, TP; Ho, IK; Mehendale HM. (1981) Correlation between neurotoxicity and chlordecone (Kepone) levels in
brain and plasma in the mouse. Neurotoxicology 2(2):373-381.
Wang, F; Roberts, S. M; Butfiloski, E. J.; et al. (2007). Acceleration of autoimmunity by organochlorine pesticides:
A comparison of splenic B-cell effects of chlordecone and estradiol in (NZBxNZW)Fl mice. Toxicol. Sci. 99:141-
152.
WHO (World Health Organization). (1984) Chlordecone. Environmental health criteria. Vol. 43. International
Programme on Chemical Safety, Geneva, Switzerland. Available online at
http://www.inchem.org/documents/ehc/ehc/EHC43.htm.
Wiener, M; Pittman, KA; Stein, V. (1976) Mirex kinetics in the rhesus monkey. I. Disposition and excretion. Drug
Metab Dispos 4(3):281-287.
Williams, GM. (1980) Classification of genotoxic and epigenetic hepatocarcinogens using liver culture assays. Ann
NY Acad Sci 349:273-282.
Williams GM. (1983) Epigenetic Effects of Liver Tumor Promoters and Implications for Health Effects. Environ
Health Perspect 50:177-183.
Williams GM, Numoto S. (1984) Promotion of mouse liver neoplasms by the organochlorine pesticides chlordane
and heptachlor in comparison to dichlorodiphenyltrichloroethane. Carcinogenesis 5:1689-96.
Williams, J; Uphouse, L. (1991) Vaginal cyclicity, sexual receptivity, and eating behavior of the female rat
following treatment with chlordecone. Reprod Toxicol 5(1):65-71.
Williams, J; Eckols, K; Uphouse, L. (1989) Estradiol and chlordecone interactions with the estradiol receptor.
Toxicol Appl Pharmacol 98:413-421.
Williams, J; Montanez, S; Uphouse, L. (1992) Effects of chlordecone on food intake and body weight in the male
rat. Neurotoxicology 13(2):453-462.
Young, RA; Mehendale, HM. (1989) Carbon tetrachloride metabloism in partially hepatectomized and sham-
operated rats pre-exposed to chlordecone (Kepone). J Biochem Toxicol 4(4):211-219.
JANUARY 2008 108 DRAFT - DO NOT CITE OR QUOTE
-------
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND
PUBLIC COMMENTS AND DISPOSITION
[Place holder]
JANUARY 2008 A-1 DRAFT - DO NOT CITE OR QUOTE
-------
APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE RfD
Kidney Lesions (Glomerulosclerosis) in Female Rats Exposed to Chlordecone in the Diet for
1-2 years
The Larson et al. (1979a) study did not include statistics for renal lesions as described
in Section 4.2.2. Statistical analysis (performed for this review) of the frequency of renal lesions
in each dose by sex (Fisher's exact test) revealed that the incidence of glomerulosclerosis (grades
1, 2, or 3 combined) in some of the exposure groups of female rats was statistically different
from control. Additionally, a significant dose-response trend was seen by the Cochran-Armitage
test. All available models in the EPA Benchmark Dose Software (BMDS) version 1.3.2 were fit
to quantal incidence data (Table B-l) for histopathologic glomerulosclerosis in female Wistar
rats from a 2-year dietary study (Larson et al., 1979a). To provide potential points of departure
for RfD derivation, benchmark response (BMR) levels were selected as 10% extra risk for
quantal incidence data. The results of statistical analysis and BMD modeling for each sex are
described below.
Table B-l. Incidence of histopathologic renal lesions (glomerulosclerosis
grades 1, 2, or 3 combined) in female Wistar rats following administration of
chlordecone in the diet for 2 years
Gender
Male
Female3
Dose (mg/kg-day)
0
12/22
4/34
0.06
3/11
2/13
0.3
4/6
8/17b
0.5
6/9
8/12b
1.6
3/4
3/4b
""Statistically significant trend for increased incidence by Cochran-Armitage test (p < 0.01).
bStatistically significantly different from controls according to Fisher's exact test (p < 0.05) performed for this
review.
Source: Larson et al. (1979a).
As shown in Table B-l, the frequency of renal lesions (glomerulosclerosis) in female rats
was statistically different from the incidence among control rats at doses of 0.3 mg/kg-day and
higher. Most dichotomous models provided adequate fit to the female rat incidence data, based
on the summary results reported in the BMDS output and a more detailed examination of the
graphs and goodness-of-fit statistics (summarized in Table B-2 and Figure B-l).
As shown in Table B-2, the log-probit model had the best fit as indicated by the lowest
Akaike's Information Criterion (AIC) and visual inspection (Figure B-l). Additionally, this
model also exhibits the best fit to the incidence data at low doses (i.e., in the vicinity of the
BMR) as evidenced by examining the chi-square scaled residuals and the visual fit of the model
to the data in the plot from the BMDS output. Thus, it was selected to calculate a potential point
JANUARY 2008 B-l DRAFT - DO NOT CITE OR QUOTE
-------
of departure for the RfD based on the incidence data for renal lesions (glomerulosclerosis)
among female rats. The model-predicted benchmark dose (BMD) associated with a 10% extra
risk for glomerulosclerosis was 0.12 mg/kg-day (Table B-2). The lower 95% confidence limit on
the benchmark dose (BMDLio), a potential point of departure for the RfD, was 0.08 mg/kg-day
(Table B-2).
Table B-2. BMD modeling results for the incidence of histopathologic renal
lesions (glomerulosclerosis) in female Wistar rats, following administration
of chlordecone in the diet for 2 years
Model
Log-probita
Quantal linear
Multistage
Weibull
Gamma
Log-logistic
Quantal quadratic
BMD10
0.116
0.071
0.071
0.071
0.071
0.067
0.264
BMDL10
0.076
0.045
0.045
0.045
0.045
0.026
0.188
yfp-value
0.62
0.56
0.56
0.56
0.56
0.72
0.0002b
AIC
84.3
84.7
84.7
84.7
84.7
85.7
93.0
aForm of the probit model:
P(response) = background + [1 -background] x CumNorm[intercept + slope x log(dose)]
Where: CumNorm is the cumulative normal distribution function; background = 0.117647; intercept = 0.723913;
slope = 1.
bQuantal quadratic model provided inadequate fit to the data.
Probit Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
Probit
BMDLJ jBMD
0.2
0.4
0.6
0.8
dose
1.2
1.4
1.6
Figure B-l. Observed and predicted incidence of histopathologic renal lesions
(glomerulosclerosis grades 1, 2, or 3 combined) in female Wistar rats following
administration of chlordecone in the diet for 1-2 years.
JANUARY 2008
B-2
DRAFT - DO NOT CITE OR QUOTE
-------
Log-Probit Model of U.S. EPA Benchmark Dose Software (Version 1.3.2).
The computer output from the log-Probit model of the glomerulosclerosis data follows:
Probit Model SRevision: 2.1 $ $Date: 2000/02/26 03:38:53 $
Input Data File: C:\BMDS\KIDNEY_LESIONS.(d)
Gnuplot Plotting File: C:\BMDS\KIDNEY_LESIONS.plt
Wed May 09 15:06:562007
BMDS MODEL RUN
The form of the probability function is:
P [response] = Background
+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose)),
where CumNormQ is the cumulative normal distribution function
Dependent variable = COLUMN 1
Independent variable = COLUMN3
Slope parameter is restricted as slope >= 1
Total number of observations = 5
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
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0.117647
intercept = 0.723913
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -slope
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix)
background intercept
background 1 -0.36
intercept -0.36 1
JANUARY 2008 B-3 DRAFT - DO NOT CITE OR QUOTE
-------
Parameter Estimates
Variable Estimate Std. Err.
background 0.123642 0.0510126
intercept 0.869701 0.276028
slope 1 NA
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model Log(likelihood) Deviance Test DF P-value
Full model -39.5379
Fitted model -40.1501 1.22434 3 0.7472
Reduced model -49.6869 20.2979 4 0.0004361
AIC:
84.3002
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000
0.0600
0.3000
0.5000
1.6000
0.1236
0.1464
0.4471
0.6232
0.9210
4.204
1.903
7.601
7.479
3.684
4
2
8
8
3
34
13
17
12
4
-0.1062
0.07598
0.1948
0.3105
-1.268
Chi-square= 1.76 DF = 3 P-value = 0.6241
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD= 0.116338
BMDL = 0.0756267
JANUARY 2008
B-4
DRAFT - DO NOT CITE OR QUOTE
-------
Testicular Atrophy in Male Rats Receiving Chlordecone in the Diet for 3 Months
The Larson et al. (1979a) study did not include statistics for the testicular atrophy
observed in male rats (see Section 4.2.2). Statistical analysis (performed for this review) of the
frequency of renal lesions in each dose by sex (Fisher's exact test) revealed that the incidence of
testicular atrophy in male rats in some of the exposure groups of male rats was statistically
different from control. Additionally, a significant dose response trend was seen by the Cochran-
Armitage test. All available models in the EPA BMDS version 1.3.2 were fit to quantal
incidence data (Table B-3) for testicular atrophy in male Wistar rats, following 3 months of
dietary exposure (Larson et al., 1979a). To provide potential points of departure for RfD
derivation, benchmark response levels were selected as 10% extra risk for quantal incidence
data. The results of statistical analysis and BMD modeling for each sex are described below.
As shown in Table B-3, the frequency of testicular atrophy in male rats was statistically
different from the incidence among control rats at doses of 1.6 mg/kg-day and higher. However,
the highest dose groups of 3.9 and 7 mg/kg-day were not included in the dose response modeling
as animals in these dose groups suffered from overt toxicity, leading to death of all animals in
these groups by 6 months into the study. Testicular atrophy in the highest exposed rats may have
resulted from frank toxic effects including decreased body weight gain.
Most of the dichotomous models provided adequate fit to the testicular atrophy incidence
data based on the summary results reported in the BMDS output and a more detailed
examination of the graphs and goodness-of-fit statistics (summarized in Table B-4 and Figure B-
2). As shown in Table B-4, the multistage and quantal linear models provided the best fit as
indicated by the lowest AIC values and visual inspection (Figure B-2). Both models predicted
the BMD associated with a 10% extra risk for testicular atrophy as 0.21 mg/kg-day (Table B-4).
The lower 95% confidence limit on the benchmark dose (BMDLio), a potential point of
departure for the reference dose (RfD), was 0.12 mg/kg-day (Table B-4).
Table B-3. Incidence of testicular atrophy in male rats receiving chlordecone
in the diet for 3 months
Dietary level (ppm)
Average dose3 (mg/kg-day)
Incidence of testicular atrophyb
0
0
1/10
5
0.3
0/5
10
0.5
1/5
25
1.6
4/5c
50
3.9
4/5c
80
7.0
5/5c
"Average doses to male rats, based on graphically depicted food consumption data presented by the authors.
bStatistically significant trend for increased incidence by Cochran-Armitage test (p < 0.01).
Statistically significantly different from controls according to Fisher's exact test (p < 0.05) performed for this
review.
Source: Larson et al. (1979a).
JANUARY 2008
B-5
DRAFT - DO NOT CITE OR QUOTE
-------
Table B-4. BMD modeling results for the incidence of testicular atrophy in
male Wistar rats, following administration of chlordecone in the diet for 3
months
Model
Gamma
Logistic
Log-logistic
Multistage (l°)a
Probit
Log-probit
Quantal linear
Quantal quadratic
Weibull
BMD10
0.393
0.560
0.436
0.206
0.563
0.444
0.206
0.776
0.338
BMDL10
0.126
0.323
0.125
0.119
0.350
0.203
0.119
0.541
0.123
X2 p-value
0.42
0.35
0.49
0.58
0.36
0.51
0.58
0.26
0.41
AIC
30.97
30.52
30.37
29.54
30.58
30.28
29.54
30.87
31.15
Torm of the multistage model:
P[response] = background + (1-background) x (l-EXP(-beta
Where: background = 0.0672234; beta(l)= 0.510742.
dose"1)]
Multistage Model with 0.95 Confidence Level
T3
D
0.8
0.6
o
'•3 0.4
CD
0.2
Multistage
3 4
dose
11:3905/092007
Figure B-2. Observed and predicted incidence of testicular atrophy in male Wistar
rats, following administration of chlordecone in the diet for 3 months.
JANUARY 2008
B-6
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Model of U.S. EPA Benchmark Dose Software (Version 1.3.2).
The computer output from the Multistage model of the male testicular atrophy follows:
Multistage Model. SRevision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: G:\KEPONE DOSE-RESPONSE
MODELING\MALE_RAT_TESTES_LARSON_1979.(d)
Gnuplot Plotting File: G:\KEPONE DOSE-RESPONSE
MODELING\MALE_RAT_TESTES_LARSON_1979.plt
Wed May 09 11:39:012007
BMDS MODEL RUN
The form of the probability function is:
Pfresponse] = background + (1-background)* [1-EXP(
-beta 1* dose Al)]
The parameter betas are restricted to be positive
Dependent variable = Response
Independent variable = Dose
Total number of observations = 6
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 1.27121e+019
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
JANUARY 2008 B-7 DRAFT - DO NOT CITE OR QUOTE
-------
Background 1 -0.41
Beta(l) -0.41 1
Parameter Estimates
Variable Estimate Std. Err.
Background 0.0672234 0.22791
Beta(l) 0.510742 0.227823
Analysis of Deviance Table
Model Log(likelihood) Deviance Test DF P-value
Full model -10.7569
Fitted model -12.7712 4.02865 4 0.4021
Reduced model -23.9018 26.2898 5 <.0001
AIC:
29.5424
Goodness of Fit
Dose Est._Prob. Expected Observed Size ChiA2 Res.
1
0
0
0
1
3
7
.0000
.3000
.5000
.6000
.9000
.0000
0
0
0
0
0
0
.0672
.1997
.2774
.5880
.8727
.9739
0
0
1
2
4
4
.672
.999
.387
.940
.364
.869
1
0
1
4
4
5
10
5
5
5
5
5
0
-1.
-0.
0.
-0.
1.
.523
250
386
875
655
027
Chi-square= 2.87 DF = 4 P-value = 0.5800
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.206289
BMDL= 0.118596
JANUARY 2008 B-8
DRAFT - DO NOT CITE OR QUOTE
-------
Liver Lesions (Fatty Changes and Hyperplasia) in Male and Female Rats Exposed to
Chlordecone in the Diet for 1-2 years
The Larson et al. (1979a) study did not include statistics for liver lesions. Statistical
analysis by Syracuse Research Corporation of the frequency of liver lesions in each dose by sex
(Fisher's exact test and Cochran-Armitage trend test) revealed that the incidence of liver lesions
in some of the exposure groups was statistically different from controls. An examination liver
lesion incidence based on sex indicated (by Fisher's exact test) no significant differences; the
incidence data for males and females was combined. The incidence data were used to fit various
dichotomous models available in the EPA BMDS version 1.3.2. The frequency of liver lesions
(fatty changes and hyperplasia) in both sexes combined was statistically different from control at
0.5 and 1.6 mg/kg-day (see Table B-5). In addition, the Cochran-Armitage trend test showed a
statistically significant dose-response trend in the frequency of liver lesions (fatty changes and
hyperplasia) for both sexes combined.
Table B-5. Incidence of histopathologic liver lesions (fatty changes and
hyperplasia) in Wistar rats, following administration of chlordecone in the
diet for 1-2 years
Endpoint
Liver lesions3
Male rats
Female rats
Both
Dose (mg/kg-day)
0
1/22
2/34
3/56
0.06
1/11
1/13
2/24
0.3
2/6
2/17
4/23
0.5
2/9
4/12b
6/2 lb
1.6
3/4b
1/4
4/8b
""Statistically significant trend for increased incidence by Cochran-Armitage test.
bStatistically significantly different from controls according to Fisher's exact test performed for this review.
All models for dichotomous variables available in the EPA BMDS version 1.3.2 were fit
to the data in Table B-5. All dichotomous models provided adequate fit to the data based on the
summary results reported in the BMDS output and a more detailed examination of the graphs
and goodness-of-fit statistics (summarized in Table B-6).
The gamma, multistage, quantal linear, and Weibull models had equally good fit as
indicated by equally low AIC values for these models. Thus, these were selected to calculate a
potential point of departure for the RfD, based on the incidence data for liver lesions (fatty
changes and hyperplasia) among rats. The model-predicted BMDs associated with a 10% extra
risk for liver lesions (fatty changes and hyperplasia) were all equal to 0.23 mg/kg-day. The
lower 95% confidence limit on the BMDL, a potential point of departure for the RfD, was equal
to 0.14 mg/kg-day.
JANUARY 2008
B-9
DRAFT - DO NOT CITE OR QUOTE
-------
Table B-6. BMD modeling results for the increased incidence of liver lesions
in rats (both sexes combined), following administration of chlordecone in the
diet for 1-2 years
Model
Gamma
Log-logistic
Multistage (l°)a
Probit
Quantal linear
Quantal quadratic
Weibull
BMD10
0.225
0.200
0.225
0.327
0.225
0.534
0.225
BMDL10
0.136
0.106
0.136
0.217
0.136
0.380
0.136
X2p-value
0.97
0.95
0.97
0.74
0.97
0.23
0.97
AIC
98.9
100.7
98.9
99.9
98.9
102.7
98.9
Multistage model was run as 3rd degree polynomial with betas > 0.
Gamma Multi-Hit Model with 0.95 Confidence Level
0.9
0.8
0.7
0.6
1 0.5
o
'•5
2
0.4
0.3
0.2
0.1
0
Gamma Multi-Hit
BMDL BMD
0.2
0.4
0.6
0.8
dose
1.2
1.4
1.6
16:2204/202004
Figure B-3. Observed and predicted incidence of liver lesions in male and female
Wistar rats following administration of chlordecone in the diet
for 1-2 years.
Source: Larson et al. (1979a).
JANUARY 2008
B-10
DRAFT - DO NOT CITE OR QUOTE
-------
Gamma Model of U.S. EPA Benchmark Dose Software (Version 1.3.2).
The computer output from the Gamma model of the incidence of liver lesions follows:
SRevision: 2.2 $ $Date: 2001/03/14 01:17:00 $
Input Data File: C:\BMDS\LARSON_BOTHSEXES_DATA.(d)
Gnuplot Plotting File: C:\BMDS\LARSON_BOTHSEXES_DATA.plt
TueApr20 16:22:312004
BMDS MODEL RUN
The form of the probability function is:
P [re sponse] = background+( 1 -background) * CumGamma[slope * dose,power],
where CumGammaQ is the cummulative Gamma distribution function
Dependent variable = Frequency
Independent variable = Dose
Power parameter is restricted as power >=1
Total number of observations = 5
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0614035
Slope = 0.901339
Power = 1.3
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Power
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background Slope
Background 1 -0.38
Slope -0.38 1
JANUARY 2008 B-l 1 DRAFT - DO NOT CITE OR QUOTE
-------
Parameter Estimates
Variable Estimate Std. Err.
Background 0.0554334 0.0274998
Slope 0.467464 0.165121
Power 1 NA
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model Log(likelihood) Deviance Test DF P-value
Full model -47.3181
Fitted model -47.428 0.219805 3 0.9743
Reduced model -54.3907 14.1451 4 0.006846
AIC:
98.8561
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000
0.0600
0.3000
0.5000
1.6000
0.0554
0.0816
0.1790
0.2523
0.5529
3.104
1.957
4.118
5.298
4.423
3
2
4
6
4
56
24
23
21
8
-0.06089
0.03177
-0.06401
0.3525
-0.3009
Chi-square= 0.22 DF = 3 P-value = 0.9737
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.225388
BMDL= 0.136075
JANUARY 2008
B-12
DRAFT - DO NOT CITE OR QUOTE
------- |