£% niA United States
Environmental Protection
mmAgency
EPA/690/R-13/011F
Final
8-14-2013
Provisional Peer-Reviewed Toxicity Values for
Endosulfan Sulfate
(CASRN 1031-07-8)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER
Jon Reid, PhD, DABT
National Center for Environmental Assessment, Cincinnati, OH
DRAFT DOCUMENT PREPARED BY
ICF International
9300 Lee Highway
Fairfax, VA 22031
PRIMARY INTERNAL REVIEWERS
Ghazi Dannan, PhD
National Center for Environmental Assessment, Washington, DC
Anuradha Mudipalli, MSc, PhD
National Center for Environmental Assessment, Research Triangle Park, NC
This document was externally peer reviewed under contract to
Eastern Research Group, Inc.
110 Hartwell Avenue
Lexington, MA 02421-3136
Questions regarding the contents of this document may be directed to the U.S. EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center (513-569-7300).
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TABLE OF CONTENTS
COMMONLY USED ABBREVIATIONS	iii
BACKGROUND	1
DISCLAIMERS	1
QUESTIONS REGARDING PPRTVs	1
INTRODUCTION	2
BASIS FOR USE OF ENDOSULFAN TOXICITY DATA AS AN ESTIMATE OF
TOXICITY I OR ENDOSULFAN SULFATE	6
BIODEGRADATION OF ENDOSULFAN	7
REVIEW OF POTENTIALLY RELEVANT DATA (CANCER AND NONCANCER)	7
HUMAN STUDIES	15
Oral Exposures	15
Short-term Studies	15
Long-term Studies	15
Chronic-duration Studies	15
Inhalation Exposures	15
ANIMAL STUDIES	15
Oral Exposures	15
Sub chronic-duration Studies	15
Chronic-duration Studies	25
Developmental and Reproductive Studies	25
Inhalation Exposures	37
Short-term Studies	37
Sub chronic-duration Studies	37
Chronic-duration Studies	37
Developmental Studies	37
Reproductive Studies	37
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)	37
DERIVATION OF PROVISIONAL VALUES	38
DERIVATION OF ORAL REFERENCE DOSES	40
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)	40
Derivation of Chronic RfD (Chronic RfD)	40
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS	40
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)	40
Derivation of Chronic Provisional RfC (Chronic p-RfC)	40
CANCER WOE DESCRIPTOR	40
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES	41
Derivation of Provisional Oral Slope Factor (p-OSF)	41
Derivation of Provisional Inhalation Unit Risk (p-IUR)	41
APPENDIX A. PROVISIONAL SCREENING VALUES	42
APPENDIX B. ADDITIONAL INFORMATION	47
APPENDIX C. DATA TABLES	78
APPENDIX D. BMD OUTPUTS	87
APPENDIX E. REFERENCES	92
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COMMONLY USED ABBREVIATIONS
BMC
benchmark concentration
BMCL
benchmark concentration lower confidence limit
BMD
benchmark dose
BMDL
benchmark dose lower confidence limit
HEC
human equivalent concentration
HED
human equivalent dose
IUR
inhalation unit risk
LOAEL
lowest-observed-adverse-effect level
LOAELadj
LOAEL adjusted to continuous exposure duration
LOAELhec
LOAEL adjusted for dosimetric differences across species to a human
NOAEL
no-ob served-adverse-effect level
NOAELadj
NOAEL adjusted to continuous exposure duration
NOAELhec
NOAEL adjusted for dosimetric differences across species to a human
NOEL
no-ob served-effect level
OSF
oral slope factor
p-IUR
provisional inhalation unit risk
POD
point of departure
p-OSF
provisional oral slope factor
p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
RfC
inhalation reference concentration
RfD
oral reference dose
UF
uncertainty factor
UFa
interspecies uncertainty factor
UFC
composite uncertainty factor
UFd
database uncertainty factor
UFh
intraspecies uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFS
subchronic-to-chronic uncertainty factor
WOE
weight of evidence
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PROVISIONAL PEER-REVIEWED TOXICITY VALUES FOR
ENDOSULFAN SULFATE (CASRN 1031-07-8)
BACKGROUND
A Provisional Peer-Reviewed Toxicity Value (PPRTV) is defined as a toxicity value
derived for use in the Superfund Program. PPRTVs are derived after a review of the relevant
scientific literature using established Agency guidance on human health toxicity value
derivations. All PPRTV assessments receive internal review by a standing panel of National
Center for Environment Assessment (NCEA) scientists and an independent external peer review
by three scientific experts.
The purpose of this document is to provide support for the hazard and dose-response
assessment pertaining to chronic and subchronic exposures to substances of concern, to present
the major conclusions reached in the hazard identification and derivation of the PPRTVs, and to
characterize the overall confidence in these conclusions and toxicity values. It is not intended to
be a comprehensive treatise on the chemical or toxicological nature of this substance.
The PPRTV review process provides needed toxicity values in a quick turnaround
timeframe while maintaining scientific quality. PPRTV assessments are updated approximately
on a 5-year cycle for new data or methodologies that might impact the toxicity values or
characterization of potential for adverse human health effects and are revised as appropriate. It is
important to utilize the PPRTV database flittp://hhpprtv.ornl.gov) to obtain the current
information available. When a final Integrated Risk Information System (IRIS) assessment is
made publicly available on the Internet (www.epa.eov/iris). the respective PPRTVs are removed
from the database.
DISCLAIMERS
The PPRTV document provides toxicity values and information about the adverse effects
of the chemical and the evidence on which the value is based, including the strengths and
limitations of the data. All users are advised to review the information provided in this
document to ensure that the PPRTV used is appropriate for the types of exposures and
circumstances at the site in question and the risk management decision that would be supported
by the risk assessment.
Other U.S. Environmental Protection Agency (EPA) programs or external parties who
may choose to use PPRTVs are advised that Superfund resources will not generally be used to
respond to challenges, if any, of PPRTVs used in a context outside of the Superfund program.
QUESTIONS REGARDING PPRTVs
Questions regarding the contents and appropriate use of this PPRTV assessment should
be directed to the EPA Office of Research and Development's National Center for
Environmental Assessment, Superfund Health Risk Technical Support Center (513-569-7300).
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INTRODUCTION
Endosulfan sulfate, CASRN 1031-07-8, is a reaction product that forms due to oxidation,
biotransformation, or photolysis of endosulfan (ATSDR, 2000). Endosulfan is an organochlorine
insecticide that can be used on a wide variety of vegetables and fruits, cotton, and ornamental
plants. It has no residential uses. Table 1 provides physiochemical properties of endosulfan
sulfate and endosulfan. Chemical properties of endosulfan sulfate are similar to its parent
compound, endosulfan (ATSDR, 2000). The empirical formula for endosulfan sulfate is
C9H6CI6O4S (see Figure 1). The empirical formula for endosulfan is C9H6CI6O3S (see Figure 2).
Technical-grade endosulfan is a 7:3 mixture of conformational isomers a-endosulfan and
P-endosulfan arising from the pyramidal stereochemistry of sulfur.
Table 1. Physicochemical Properties of Endosulfan Sulfate (CASRN 1031-07-8)
and Endosulfan (CASRN 115-29-7)
Property (unit)
Value
Endosulfan Sulfate"
Endosulfanb
Boiling point (°C)
ND
ND
Melting point (°C)
181-182
106
Density (g/cm3)
ND (a solid)
1.745
Vapor pressure (Pa at 25°C)
1.0 x io~n
1.73 x 10 (approximate maximum of
0.2 ppm in air)
pH (unitless)
ND
7.2
Solubility in water (mg/L at 20°C)
0.48
0.32 (a-form); 0.33 ((3-form)
Relative vapor density (air =1)
ND
ND
Molecular weight (g/mol)
422.95
406.93
aHSDB (2009).
bHSDB (2010).
ND = no data.
Figure 1. Endosulfan Sulfate Structure
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Figure 2. Endosulfan Structure
No potentially relevant data investigating the effects of repeat-dose oral or inhalation
exposure in animals or humans have been identified for endosulfan sulfate. ATSDR (2000) has
noted that very little difference in toxicity exists between endosulfan and endosulfan sulfate as
indicated by acute and human case control studies. In addition, a structure similarity search
using the ChemlDplus database indicates that the two compounds are 93.07% similar (NLM)
with endosulfan being metabolized to endosulfan sulfate following absorption into the body
(ATSDR, 2000). Finally, both endosulfan and endosulfan sulfate appear to exert neurotoxicity
through a gar/wwa-aminobutyric acid (GABA)-antagonistic mode of action (ATSDR, 2000; Cole
and Casida, 1986). U.S. EPA (1994a) reports a reference dose (RfD) for chronic oral exposure
of 6 x 10 3 mg/kg-day for endosulfan. Because endosulfan and endosulfan sulfate appear to
share similar chemical, physical, and toxicological properties, endosulfan is considered a suitable
surrogate to develop toxicological values for endosulfan sulfate. A more detailed discussion is
provided in a following section.
A summary of available health-related values for endosulfan sulfate from U.S. EPA and
other agencies/organizations is provided in Table 2.
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Table 2. Summary of Available Toxicity Values for Endosulfan Sulfate (CASRN 1031-07-8)
and Endosulfan (CASRN 115-29-7)
Source/Parameter"
Value
(Applicability)
Notes
Reference
Date Accessed
Noncancer
ACGIH
8-hr TLV-TWA:
1 x 10 1 mg/m3 for
endosulfan0
TLV-TWA based on lower
respiratory tract irritation; liver and
kidney damage
ACGIH (2013)
NA
NV
NA
ACGIH (2013)
NA
ATSDRd
Oral Acute MRL:
7 x 10 3 mg/kg-d;
Oral Int. MRL:
5 x 10 3 mg/kg-d;
Oral Chr. MRL:
5 x 10 mg/kg-d
for endosulfan
NA
ATSDR (2013)
NA
NV for endosulfan
sulfate
NA
ATSDR (2013)
NA
Cal/EPA
NV
NA
Cal/EPA (2008,
2012)b
8-6-2013b
NIOSH
REL-TWA:
1 x 10 1 mg/m3 for
endosulfan
NA
NIOSH (2010)
NA
NV for endosulfan
sulfate
NA
NIOSH (2010)
NA
OSHA
NV
NA
OSHA (2006)
NA
8-hr PEL-TWA:
1 x 10 1 mg/m3 for
endosulfan
NA
OSHA (2011)
NA
NV for endosulfan
sulfate
NA
OSHA (2011)
NA
IRIS
RfD:
6 x 10 mg/kg-d
for endosulfan
NA
U.S. EPA
(1994a)
NA
NV for endosulfan
sulfate
NA
U.S. EPA
8-6-2013
Drinking water
NV
NA
U.S. EPA (2011)
NA
HEAST
Subchronic RfD:
6 x 10 mg/kg-d
for endosulfan
NA
U.S. EPA (2011)
NA
NV for endosulfan
sulfate
NA
U.S. EPA (2011)
NA
CARA HEEP
NV
NA
U.S. EPA
(1994b)
NA
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Table 2. Summary of Available Toxicity Values for Endosulfan Sulfate (CASRN 1031-07-8)
and Endosulfan (CASRN 115-29-7)
Source/Parameter"
Value
(Applicability)
Notes
Reference
Date Accessed
WHO
Temporary
acceptable daily
intake for man of
0-0.008
mg/kg-BW for
a-endosulfan,
(^-endosulfan. and
endosulfan sulfate
combined
NA
IPCS (1984)
NA
Cancer
IRIS
NV
NA
U.S. EPA
8-6-2013
HEAST
NV
NA
U.S. EPA (2011)
NA
IARC
NV
NA
IARC (2013)
NA
NTP
NV
NA
NTP (2010)
NA
Cal/EPA
NV
NA
Cal/EPA (2009)b
8-6-2013b
"Sources: Integrated Risk Information System (IRIS) database; Health Effects Assessment Summary Tables
(HEAST); International Agency for Research on Cancer (IARC); National Toxicology Program (NTP); California
Environmental Protection Agency (Cal/EPA); American Conference of Governmental Industrial Hygienists
(ACGIH); Agency for Toxic Substances and Disease Registry (ATSDR); National Institute for Occupational Safety
and Health (NIOSH); Occupational Safety and Health Administration (OSHA); Chemical Assessments and Related
Activities (CARA) list; Health and Environmental Effects Profile (HEEP); World Health Organization (WHO).
bThe Cal/EPA Office of Environmental Health Hazard Assessment (OEHHA) Toxicity Criteria Database
(http://www.oehha.ca.gov/risk/ChemicalDB/index.asp) was also reviewed and found to contain no information on
endosulfan sulfate and endosulfan.
°IFV = Inhalable Fraction and Vapor. ACGIH endnote used when material has sufficient vapor pressure to be
present in both particle and vapor phases, with each contributing a significant portion of the dose at the TLV-TWA
concentration. This endnote is typically used for substances with a Saturated Vapor concentration (SVC)/TLV ratio
between 0.1 and 10.
dFor duration, Acute = 1-14 d, Intermediate 15-364 d, and Chronic = >1 y.
BW = body weight; Chr. == chronic; IDLH = immediately dangerous to life or health; Int. Intermediate;
MRL = minimal risk level; NA = not applicable; NSRL = no significant risk level; NV = not available;
PEL = permissible exposure level; REL = recommended exposure level; TLV = threshold limit value; TWA = time
weighted average.
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Literature searches were conducted on sources published from 1900 through August 2013
for studies relevant to the derivation of provisional toxicity values for endosulfan sulfate
(CASRN 1031-07-8) and endosulfan (CASRN 115-29-7). The following databases were
searched by chemical name, synonyms, or CASRN: ACGIH, ANEUPL, ATSDR, BIOSIS, Cal
EPA, CCRIS, CD AT, ChemlDplus, CIS, CRISP, DART, EMIC, EPIDEM, ETICBACK,
FEDRIP, GENE-TOX, HAPAB, HERO, HMTC, HSDB, IARC, INCHEM IPCS, IP A, ITER,
IUCLID, LactMed, NIOSH, NTIS, NTP, OSHA, OPP/RED, PESTAB, PPBIB, PPRTV,
PubMed (toxicology subset), RISKLINE, RTECS, TOXLINE, TRI, U.S. EPA IRIS, U.S. EPA
HEAST, U.S. EPA HEEP, U.S. EPA OW, and U.S. EPA TSCATS/TSCATS2. The following
databases were searched for toxicity values or exposure limits: ACGIH, ATSDR, Cal EPA,
U.S. EPA IRIS, U.S. EPA HEAST, U.S. EPA HEEP, U.S. EPA OW, U.S. EPA
TSCATS/TSCATS2, NIOSH, NTP, OSHA, and RTECS.
BASIS FOR USE OF ENDOSULFAN TOXICITY DATA AS AN ESTIMATE OF
TOXICITY FOR ENDOSULFAN SULFATE
Two major public health assessments (ATSDR, 2000; IPCS, 1984) provide support that
the toxicity of endosulfan and endosulfan sulfate are similar. Metabolic data for humans cited in
these documents show detection of endosulfan sulfate as the primary metabolite in several
autopsy samples following acute ingestion of endosulfan, for example, in Boereboom et al.
(1998).
This is also demonstrated in a number of kinetic studies in animals cited in ATSDR
(2000) and IPCS (1984). Khanna et al. (1979) conducted an evaluation of the distribution of
endosulfan and endosulfan sulfate in the brains of cats given a single intravenous (i.v.) injection
of 3 mg/kg endosulfan. Peak concentrations of endosulfan in the brain were found at the earliest
time point examined (15 minutes after administration) and subsequently decreased whereas
endosulfan sulfate levels peaked in the brain at 1 hour postadministration and in the liver within
15 minutes postadministration. Based on the rapid appearance of endosulfan sulfate in the liver
following i.v. administration of endosulfan (Khanna et al., 1979), it is concluded that endosulfan
sulfate is a major metabolite of endosulfan and that the liver is a site of high metabolic activity.
Acute toxicity data in mice showed that the lethal dose for endosulfan sulfate was
comparable to that of the a-isomer of endosulfan at 8 mg/kg (Dorough et al., 1978). IPCS
(1984) described the study of NRCC (1975), in which endosulfan sulfate was noted as the only
compound detected in tissues of rats exposed in the diet to endosulfan sulfate for 3 months at
levels up to 500 mg/kg. No effects were detected other than increased liver or kidney weight.
IPCS (1984) stated that endosulfan sulfate was administered to dogs for 3 months at levels of
0.75-2.5 mg/kg-day. The lowest dose did not have any effect, but the highest dose was not
tolerated. The 1.5 mg/kg dose induced occasional signs of toxicity. Table 4 of this document
provides summaries of additional sub chronic-duration oral studies for endosulfan in rats and
mice with effects noted in this exposure range. A chronic-duration study of endosulfan in beagle
dogs showed neurological effects at the highest dose (approximately 2 mg/kg-day; Hoechst
Celanese Corporation, 1989b). According to NRCC (1975), endosulfan sulfate appeared to have
the same order of toxicity as endosulfan.
Endosulfan sulfate does not appear to be substantially more lipophilic than the parent
compound. Of all the metabolites of endosulfan, endosulfan sulfate accumulates predominantly
in the liver and kidneys (Hoechst Aktiengesellschaft, 1987).
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Therefore, endosulfan could be considered acceptable as a surrogate for developing
toxicity values for endosulfan sulfate.
BIODEGRADATION OF ENDOSULFAN
Appendix B provides information on the biodegradation of endosulfan.
REVIEW OF POTENTIALLY RELEVANT DATA
(CANCER AND NONCANCER)
Table 3 provides an overview of the relevant database for endosulfan and includes all
potentially relevant repeated short-term-, subchronic-, and chronic-duration studies. As
previously mentioned, no potentially relevant data investigating the effects of repeat-dose oral or
inhalation exposure were identified in animals or humans for endosulfan sulfate. The phrase
"statistical significance," used throughout the document, indicates ap-value of <0.05.
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NOAEL"
BMDL/
BMCLa
LOAEL"
Reference
(Comments)
Notesb
Human
1. Oral (mg/kg-d)a
Acute0
55-yr-old female, ingestion
case study
NR
Mortality
NDr
NA
NDr
Bernardelli and
Gennari (1987),
as reported in
ATSDR (2000)
PR

Female (age unknown),
accidental ingestion case
study
NR
Renal failure; disseminated
intravascular coagulation; thrombi in
pulmonary arteries and aorta;
cardiogenic shock; mortality 8 d after
exposure; postmortem examination
revealed bilateral pleural effusions,
congested and edematous lungs,
hyaline membranes, microatelectasia,
polymorphonuclear lymphocytes and
red cells in alveoli, and interstitial
fibrosis
NDr
NA
NDr
Blanco-
Coronado et al.
(1992), as
reported in
ATSDR (2000)
PR

Male (age unknown),
accidental ingestion case
study
NR
Muscle fasciculation; convulsions;
tubular necrosis of the kidney;
mortality 10 d following exposure
due to cardio-respiratory arrest/heart
failure and pulmonary edema
NDr
NA
NDr
Lo et al. (1995),
as reported in
ATSDR (2000)
PR

20-yr-old male, ingestion
case study
200 mL
Thionax, 30%
endosulfan
(-1,500 mg/kg)
Hypoxia; pulmonary edema;
tachycardia; hypertension;
cardiogenic shock; convulsions;
impaired psychomotor activity
NDr
NA
NDr
Shemesh et al.
(1988), as
reported in
ATSDR (2000)
PR
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NO A EL"
BMDL/
BMCLa
LOAEL"
Reference
(Comments)
Notesb
Acute0
Patient (sex and age
unknown), ingestion case
study
75 mL, 35% w/v
(-375 mg/kg)
Nausea; vomiting; diarrhea;
tonic-clonic seizures; myoclonic
jerks; psychosis; cortical blindness;
limb rigidity; reversible lesions of
basal ganglia and occipital cortex
NDr
NA
NDr
Pradhan et al.
(1997), as
reported in
ATSDR (2000)
PR
Short-termd
ND
Long-term6
ND
Chronic'
ND
2. Inhalation (mg/m3)a
Acute0
18 agricultural workers
(sex, age unknown),
application of endosulfan to
crops in absence of
protective equipment
NR
Nausea; vomiting; dizziness;
confusion; irritability; muscle
twitching; tonic/clonic convulsions;
conduction defects; increased
dyspnea and respiratory rate;
tachycardia; Bradycardia
NDr
NA
NDr
Chugh et al.
(1998), as
reported in
ATSDR (2000)
PR
22 agricultural workers
(sex, age unknown),
application of endosulfan to
crops
NR
Nausea; vomiting; abdominal pain;
diarrhea
NDr
NA
NDr
Singh et al.
(1992), as
reported in
ATSDR (2000)
(authors note
results possibly
due to dermal
exposure
because
workers who
suffered cuts on
legs had more
severe
symptoms)
PR
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NO A EL"
BMDL/
BMCLa
LOAEL"
Reference
(Comments)
Notesb
Acute0
Male (age unknown),
industrial worker, single
occupational exposure
NR
Repeated convulsions; impaired
consciousness; disorientation;
agitation; cognitive and emotional
deterioration; impaired memory;
impaired visual-motor coordination
NDr
NA
NDr
Aleksandrowicz
(1979), as
reported in
ATSDR (2000)
PR
Short-termd
ND
Long-term6
Children (number, sex, age
not reported), homes near
pesticide use
NR
No association with undescended
testes
NDr
NA
NDr
Garcia-
Rodriguez et al.
(1996), as
reported in
ATSDR (2000)
PR

269,746 children in the
Central Valley of California
potentially exposed to
endosulfan and other
pesticides during gestation
Wk 1-8, retrospective
case-control study
NR
Increased incidence of autism
spectrum disorder (ASD); exposure to
multiple pesticides occurred
simultaneously so was not possible to
determine the relationship between
ASD and endosulfan
NDr
NA
NDr
Roberts et al.
(2007)
PR
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NO A EL"
BMDL/
BMCLa
LOAEL"
Reference
(Comments)
Notesb
Long-term6
117/0(117 cases,
90 controls), schoolchildren
(10-19 yrs), retrospective
cohort study analyzing male
reproductive/developmental
effects in exposed children
NR
The R2 values corresponding to
sexual maturity rating (SMR) of
pubic hair, testes, and penis were
0.48, 0.43, and 0.43, respectively
(p < 0.001), indicating that significant
variance in SMR can be attributed to
age and exposure; R2 value of serum
testosterone was 0.61 (p < 0.001),
attributing 61% of variation to age,
exposure, and serum luteinizing
hormone (LH); increased (p < 0.001)
endosulfan residues detected in 78%
and 29% of the serum of exposed and
control groups, respectively
NDr
NA
NDr
Saiyed et al.
(2003);
critiqued by
Indulkar (2004)
PR
Chronic'
0/3 (3 cases, 7 controls),
population-based
occupational case-control
study, cases had "probable"
or "possible" exposure to
endosulfan plus other
xenoestrogens (no duration
reported)
NR
Adjusted (core confounders and
education) OR = 0.8 (CI, 0.2-3.2)
Given small sample size, no
significant conclusions drawn from
this study
NDr
NA
NDr
Aschengrau et
al. (1998)
PR
Animal
1. Oral (mg/kg-d)a
Subchronic8
10-12/0 per dose, Wistar
rat, diet, 8, 12, 18, or 22 wk
0,0.5,0.9, 1.8
(Adjusted)
Decreased serum antibody titer, IgG
concentrations, and LMI and MMI
factors
0.5
NDr
0.9
Baneijee and
Hussain (1986)
PR
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NO A EL"
BMDL/
BMCLa
LOAEL3
Reference
(Comments)
Notesb
Chronic
50/50 per dose, S-D rat,
diet, 2 yr
M: 0,0.1,0.3,
0.6, 2.9
F: 0,0.1,0.4,
0.7, 3.8
(Adjusted)
Decreased BW gain in males and
females; increased incidence of
marked progressive
glomerulonephrosis and blood vessel
aneurysms in males
0.6 (M)
0.7 (F)
NR
2.9 (M)
3.8 (F)
Hoechst
Celanese
Corporation
(1989a), as
summarized in
U.S. EPA
(1994a)
IRIS,
PR

6/6 per dose, beagle dog,
diet, 1 yr
M: 0, 0.2, 0.65,
2.1
F: 0,0.18,0.57,
1.9
(Adjusted)
Decreased weight gain in males;
neurological findings in males and
females
0.65 (M)
0.57 (F)
NR
2.1 (M)
1.9 (F)
Hoechst
Celanese
Corporation
(1989b), as
summarized in
U.S. EPA
(1994a)
IRIS,
PR
Developmental11
0/30, Wistar rat, diet,
GD 6-PND 21, pups
sacrificed on PND 21 or
PND 75
0, 3.74,10.8,
29.8
Developmental LOAEL: decreased
pup weight at PNDs 11 and 17
BMDL: decreased pup weight in
females at PND 11
NDr
0.29
3.74
Gilmore et al.
(2006)
PS

0/24, Wistar rat, gavage,
dams dosed on
GD 15-PND 21, offspring
sacrificed PND 65 or 140
0, 1.5, 3.0
Developmental LOAEL: increased
absolute and relative testis weight;
decreased sperm production and
percentage of seminiferous tubules
showing complete spermatogenesis at
puberty
BMDL: decreased daily sperm
production rate in male offspring
NDr
0.68
1.5
Dalsenter et al.
(1999)
PR
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry"
Critical Effects
NO A EL"
BMDL/
BMCLa
LOAEL"
Reference
(Comments)
Notesb
Reproductive
2-generation reproductive
study, 32/32 (F0), 28/28
(Fib), Crl:COBS
CD(SD)BR rat, diet
M: 0,0.2, 1.1,
5.4
F: 0, 0.25, 2.6,
6.6
Increased heart, liver, and kidney
weights in F0 males
1.1
NDr
5.4
Hoechst
Aktiengesell-
schaft (1984a),
as summarized
in U.S. EPA
(1994a)
PR
Carcinogenicity
Rat (number, strain, study
and duration not reported)
NR
No treatment-related increases in
tumors
NA
NA
NA
Hoechst
Celanese
Corporation
(1989a), as
summarized in
U.S. EPA
(2010)
PR
Mouse (number, strain,
study and duration not
reported)
NR
No treatment-related increases in
tumors
NA
NA
NA
Hoechst
Celanese
Corporation
(1988), as
summarized in
U.S. EPA
(2010)
PR
2. Inhalation (mg/m3)a
Short-term
Rat (number unknown),
SPF Wistar rats, nose-only
inhalation, 21 exposures
over 29 d
0,0.09, 0.18,
0.36
Decreased BW and leukocyte counts
in males; increased creatinine in
females
0.18
NDr
0.36
Hoechst
Aktiengesell-
schaft (1984b),
as summarized
in U.S. EPA
(2010)
PR
Subchronic
ND
Chronic
ND
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Table 3. Summary of Potentially Relevant Data for Endosulfan (CASRN 115-29-7)
Category
Number of Male/Female,
Strain, Species, Study
Type, Study Duration
Dosimetry3
Critical Effects
NOAEL3
BMDL/
BMCL3
LOAEL3
Reference
(Comments)
Notesb
Developmental
ND
Reproductive
ND
Carcinogenicity
ND
dosimetry: NOAEL, BMDL/BMCL, and LOAEL values are converted to an adjusted daily dose (ADD in mg/kg-d) for oral noncancer effects and a human equivalent
concentration (HEC in mg/m3) for inhalation noncancer effects. Values are converted to a human equivalent dose (HED in mg/kg-d) for oral carcinogenic effects. All
long-term exposure values (4 wk and longer) are converted from a discontinuous to a continuous (weekly) exposure. Values from animal developmental studies are not
adjusted to a continuous exposure.
HECer = (mg/m3) x (hours per day exposed ^ 24) x (days per week exposed ^ 7) x blood gas partition coefficient.
bNotes: IRIS = utilized by IRIS, date of last update; PS = principal study; PR = peer reviewed; NPR = not peer reviewed; NA = not applicable.
0 Acute = exposure for <24 hr (U.S. EPA, 2002).
dShort-term = repeated exposure for >24 hr <30 d (U.S. EPA, 2002).
"Long-term = repeated exposure for >30 d <10% lifespan (based on 70-yr typical lifespan) (U.S. EPA, 2002).
fChronic = repeated exposure for >10% lifespan (U.S. EPA, 2002).
8Table 4 summarizes additional subchronic-duration studies.
hTable 5 summarizes additional developmental studies.
BW = body weight; NA = not applicable; ND = no data; NDr = not determinable; NR = not reported; S-D = Sprague-Dawley.
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HUMAN STUDIES
Oral Exposures
The effects of oral exposure of humans to endosulfan have been evaluated in five
case-control studies, which are described in Appendix B (Bernardelli and Gennari, 1987, as
reported in ATSDR, 2000; Blanco-Coronado et al., 1992, as reported in ATSDR, 2000; Lo et al.,
1995, as reported in ATSDR, 2000; Shemesh et al., 1988, as reported in ATSDR, 2000; and
Pradhan et al., 1997, as reported in ATSDR, 2000).
Short-term Studies
No studies were identified.
Long-term Studies
No studies were identified.
Chronic-duration Studies
No studies were identified.
Inhalation Exposures
The effects of inhalation exposure of humans to endosulfan have been evaluated in three
case control studies, (Chugh et al., 1998, as reported in ATSDR, 2000; Singh et al., 1992, as
reported in ATSDR, 2000; and Aleksandrowicz, 1979, as reported in ATSDR, 2000), three
long-term studies (Garcia-Rodriguez et al., 1996, as reported in ATSDR, 2000; Roberts et al.,
2007; and Saiyed et al., 2003), and one chronic-duration study (Aschengrau et al., 1998).
Appendix B summarizes these studies.
ANIMAL STUDIES
Oral Exposures
The effects of oral exposure of animals to endosulfan have been evaluated in
16 sub chronic-duration studies (see Table 4), 2 chronic-duration studies (Hoechst Celanese
Corporation, 1989a,b), 13 developmental studies (see Table 5), and 1 reproductive study
(Hoechst Aktiengesellschaft, 1984a).
Subchronic-duration Studies
A total of 16 assays covering 2 species of animals (rats and mice) have been performed to
evaluate the subchronic effects of endosulfan. Subchronic administration of endosulfan to rats
and mice resulted in a number of effects with the most sensitive being immunological and
neurological. Table 4 provides a summary of the available literature concerning the subchronic
effects of endosulfan.
Banerjee and Hussain (1986)
Baneijee and Hussain (1986) conducted a peer-reviewed immunotoxicity study of
endosulfan using male Wistar albino rats. The authors did not report compliance with Good
Laboratory Practice (GLP) standards. Technical-grade endosulfan (70:30
a-endosulfan:P-endosulfan) was obtained from M/s Hindustan Insecticides Ltd. (India), and
purity was not reported. The rats weighed 85-90 g upon receipt and were fed a standard
laboratory diet containing 0, 5, 10, or 20 ppm endosulfan for 8-22 weeks. Interim sacrifices
were conducted on 20-24 rats/dose group at 8, 12, 18, and 22 weeks. Corresponding average
daily doses of 0, 0.5, 0.9, and 1.8 mg/kg-day are estimated based on standard food consumption
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and body-weight values (U.S. EPA, 1988). In order to prepare the treatment diet, known
quantities of endosulfan were first dissolved in groundnut oil, and then the mixture was manually
blended into the standard laboratory diet for at least 30 minutes. Control subjects received
standard laboratory diet mixed with the same quantity of groundnut oil. Water was provided ad
libitum. All rats were housed 4 per cage with 12 hours of light and 12 hours of darkness. The
temperature was maintained by air conditioning at 25°C. Body weights were recorded weekly.
Food consumption and clinical observations were recorded daily. A total of 10-12 of the
animals in each dose/control group from each exposure duration were immunized
subcutaneously with tetanus toxoid in Freund's complete adjuvant 20 days prior to termination
of treatment. An equal number from each group remained unimmunized. Blood samples were
taken from each animal after termination of treatment by cardiac puncture. Upon sacrifice,
peritoneal macrophages were collected from immunized rats only. The liver, spleen, and thymus
of immunized rats were removed and weighed. The serum protein content, albumin:globulin
ratio, and immunoglobulin (IgM and IgG) concentrations were determined for each rat. The
serum antibody titer to tetanus toxoid was estimated for immunized rats using an indirect
hemagglutination technique with microtiter plates. A solution of sheep red blood cells mixed
with tetanus vaccine was used as antigen-coated cells for antibody titration. Leukocyte-rich
plasma and peritoneal macrophages from immunized rats were used for the leukocyte migration
inhibition (LMI) and macrophage migration inhibition (MMI) tests. Statistical significance
between the treatment and control groups was determined using one-way analysis of variance
(ANOVA) (p < 0.05 or 0.01).
This study is summarized to evaluate the subchronic effects of endosulfan, and for
completeness, the study results that are considered of chronic duration (i.e., 18 and 22 weeks) are
included. Banerjee and Hussain (1986) noted that no treatment-related effects were observed for
clinical signs, mortality, growth rates, or food intake for any of the exposure durations (data not
reported). Relative spleen weight was significantly reduced by 13% relative to controls at
1.8 mg/kg-day in immunized rats following 22 weeks of treatment. No significant effects to
spleen weight were observed at any other dose level or exposure duration. Relative thymus
weight was unaltered by treatment, and the study authors did not report any findings for liver
weight. The serum globulin level was significantly decreased relative to controls (increased
albumin:globulin ratio) at 1.8 mg/kg-day in immunized rats following 12, 18, or 22 weeks of
treatment and at 0.9 mg/kg-day following 22 weeks of treatment (see Table C.4). No effects on
serum globulin, IgG, or IgM levels were observed in unimmunized rats. However, treated
immunized rats showed a significantly lower IgG level following immunization when compared
with control immunized rats. As shown in Table C.5, this effect was seen at concentrations
>0.9 mg/kg-day following 12, 18, or 22 weeks of treatment. The increase in IgM level following
immunization was unaffected by treatment. The serum antibody titer to tetanus toxoid was
significantly decreased in immunized rats compared with controls at concentrations
>0.9 mg/kg-day following 8, 12, 18, or 22 weeks of treatment. As shown in Table C.6, mean
values (expressed as ~log2 antibody titer) at these dose levels (>0.9 mg/kg-day) were affected in
a dose- and time-dependent manner by treatment. Treatment at these dose levels
(>0.9 mg/kg-day) also significantly decreased LMI/MMI responses in immunized rats following
8, 12, 18, or 22 weeks of treatment, indicating a possible effect on cell-mediated immunity (see
Tables C.7 and C.8). For the study considered subchronic in duration (8 and 12 weeks), a
NOAEL of 0.5 mg/kg-day and a LOAEL of 0.9 mg/kg-day are determined based on statistical
significance for decreased serum IgG concentration, decreased antibody titer to tetanus toxoid,
and decreased LMI/MMI response in immunized rats. However, the cutoff for consideration of
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toxicity for decreased serum IgG concentration is questionable and difficult to interpret.
U.S. EPA does not provide specific guidance on developing toxicity assessments for
immunologic endpoints, and there is no clear guidance on whether the statistically significant
change in the serum IgG concentration should be considered biologically significant
(IPCS/WHO, 1996, 2012), especially since other standard immunologic tests were not
performed. Consultation with U.S. EPA scientists that have expertise in this area suggests that a
30% change could be considered biologically significant (personal communication), which was
not attained in the Baneijee and Hussain (1986) study. Thus, decreased serum IgG concentration
was not considered as a plausible POD for deriving a toxicity value.
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
Rat
10-12/0, albino Wistar rat, diet, 8, 12,
18, or 22 wk; immunized with tetanus
toxoid 20 d prior to termination of
exposure or unimmunized; clinical signs
and food consumption monitored daily
(data not reported); BWs recorded
weekly (data not reported); liver (data
not reported), spleen, and thymus
weighed; serum globulin level, IgM and
IgG, and serum antibody titer against
tetanus toxoid measured; LMI and MMI
factors measured
0, 0.5, 0.9, or
1.8d
(0, 5, 10, or
20 ppm in diet)
(purity not
reported,
technical grade
used)
Decreased serum antibody titer to tetanus toxoid and depressed
LMI and MMI factors at >0.9 mg/kg-d after 8 or 12 wk;
decreased serum IgG concentration in immunized rats at
>0.9 mg/kg-d after 12 wk; decreased globulin level (increased
albumin: globulin ratio) in immunized rats at 1.8 mg/kg-d after
12 wk
(18- and 22-wk exposures considered chronic in duration
therefore results are not reported here)
LOAEL: decreased serum antibody titer and IgG concentration;
decreased LMI and MMI factors
0.5
0.9
Baneijee and
Hussain (1986)
25/25, CD S-D rat, diet, 13 wk; 5/5 kept
in a 4-wk recovery group after treatment
ended; ophthalmoscopic exams before
treatment and at Wk 13 in control and
high-dose rats; neurological
examinations (locomotor reflexes)
before treatment and at Wk 2, 6, and 13
in 10/10 from control and high-dose
rats; all animals examined for grip
reflex and ataxia at Wk 13; blood
sampled from 10/10 at each dose level
for hematological and clinical chemistry
examinations at Wk 0, 6, and 12/13
(standard battery of tests, not specified);
blood and plasma cholinesterase
estimations at Wk 5 and 12 in 10/10 at
each dose level; urine collected Wk 4
and 13 for standard urinalysis (specific
tests not reported); organ weights and
histopathological examination upon
sacrifice (specific organs not reported)
M: 0, 0.64, 1.92,
3.85, or 23.41s
F: 0, 0.75, 2.26,
4.59, or 27.17s
(purity 97.2%)
Increased hair loss in females at >4.59 mg/kg-d (reversed
during recovery); decreased water consumption (Wk 5) at
>1.92 mg/kg-d in males and 27.17 mg/kg-d in females;
decreased red blood cell count in males at >1.92 mg/kg-d after
6 wk and >3.85 mg/kg-d after 13 wk, and in females at
>4.59 mg/kg-d after 6 wk and at 27.17 mg/kg-d after 13 wk;
increased relative kidney weight in males at >3.85 mg/kg-d
(reversed after recovery at 3.85 mg/kg-d but not at
23.41 mg/kg-d) and in females at 27.17 mg/kg-d; increased
absolute liver weight (both sexes), decreased plasma and RBC
cholinesterase activities and dark urine with increased ketones
(females only), and increased epididymal weight at the
high-dose level; increased brain cholinesterase activity in
females at >4.59 mg/kg-d; granular/clumped pigmentation in
kidney cells in males at >3.85 mg/kg-d and in females at
27.1 mg/kg-d (no cell death associated with these findings,
decreases in pigmentation were seen during recovery); brown
pigment in scattered hepatocytes (males only) and enlargement
of hepatocytes (females only) at the high-dose level
LOAEL: hematological effects after 6 wk (males)
0.64
1.92
Hoechst
Aktiengesellschaft
(1985a), as
summarized in
U.S. EPA (1994a),
Cal/EPA (2008),
IPCS (1989), and
McGregor (1998)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
11-16/16-26, albino rat, gavage, 30 d;
terminal BWs and blood samples
(biochemical analysis: GOT, GPT,
alkaline phosphatase, protein, blood
sugar; haematological analysis: RBC,
WBC, Hb, DLC counts); weights and
microscopic exam of liver, kidney,
brain, spleen, testes, epididymis, ovary,
uterus, vagina, and cervix
M: 0, 0.75, 2.5,
or 5.0°
F: 0, 0.25, 0.75,
or 1.5°
(purity 98%)
Clinical signs at highest dose levels in males and females
included hyperexcitation, tremor, dyspnea, salivation during the
first 3-4 d of dosing; increased relative liver, kidney, and testes
weight in males at 5.0 mg/kg-d (data at lower doses not
reported); decreased relative kidney weight in females at
1.5 mg/kg-d (data at lower doses not reported); increased liver
and serum alkaline phosphatase, neutrophil, and RBC counts in
males at 5.0 mg/kg-d (data at lower doses not reported);
increased liver alkaline phosphatase and decreased serum
alkaline phosphatase in females at 1.5 mg/kg-d; increased liver
and serum protein in females at 1.5 mg/kg-d (data at lower
doses not reported)
LOAEL: biochemical changes and decreased relative kidney
weight in females
0.75
1.5
Dikshith et al.
(1984)
0/8, Wistar rat, gavage, 30 d; all animals
were ovariectomized before treatment
began; positive control group received
1 |ig estradiol diproprionate
intraperitoneally; treatment groups
received either endosulfan alone or
endosulfan plus 1 |ig estradiol
diproprionate daily; negative control
group received vehicle alone; terminal
BWs recorded; uterus, cervix, vagina,
and pituitary weighed; microscopic
examination of pieces of uterus and
vagina and whole cervix; glycogen
content of uterus, cervix, and vagina
measured
0, 1.5 with or
without 1 |ig
estradiol
diproprionate0
(purity not
reported)
No effects following treatment with endosulfan alone; treatment
with endosulfan and estradiol dipropionate caused increased
relative uterus, cervix, vagina, and pituitary weights, and
increased glycogen levels in the uterus, cervix, and vagina
1.5
NDr
Raizada et al.
(1991)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
16/0, albino Wistar rat, diet, 6 wk; each
rat immunized with tetanus toxoid 25 d
after beginning exposure; clinical signs
and food consumption monitored daily;
BWs recorded weekly (only final BWs
reported); liver, spleen, and thymus
weighed; serum globulin fractions, IgM
and IgG concentrations, and serum
antibody titer against tetanus toxoid
measured; leukocyte and macrophage
migration inhibition (LMI and MMI)
factors measured
0, 1.8, 5.5, 9.3d
(0, 10, 30, or
50 ppm in diet)
(purity 98%)
Decreased serum antibody titer to tetanus toxoid and decreased
LMI and MMI factors at >5.5 mg/kg-d; increased relative liver
weight at 9.3 mg/kg-d; decreased serum IgM, IgG and
y-globulin levels at 9.3 mg/kg-d
LOAEL: decreased serum antibody titer to tetanus toxoid and
decreased LMI and MMI factors
1.8
5.5
Baneijee and
Hussain (1987)
12/12, Wistar rat, diet, 13 wk; clinical
observations and BWs recorded
(interval not reported); neurotoxicity
examined with functional observational
battery (FOB), motor activity,
locomotor activity, measured grip
strength, foot splay, and neuropathology
examination; plasma cholinesterase
activity measured; histopathological
examination of 6 animals/dose group
(specific organs not reported)
M: 0,2.11, 13.7,
or 37.2f
F: 0, 2.88, 16.6,
or45.5f
(purity 98.1%
and 96.5%)
Convulsions/death observed in one female and red nasal stain
observed in 3 females at 45.5 mg/kg-d; decreased BWs on
Day 7 in females at >16.6 mg/kg-d possibly due to palpability;
decreased food consumption at Wk 1 in females at
>16.6 mg/kg-d and males at 37.2 mg/kg-d; decreased plasma
cholinesterase activity in females at >16.6 mg/kg-d; increased
absolute and relative kidney and liver weights in females at
>16.6 mg/kg-d and in males at >13.7 mg/kg-d
LOAEL: increased absolute and relative kidney and liver
weights in males
2.11
13.7
Sheets et al.
(2004), as
summarized in
Cal/EPA (2008)
and U.S. EPA
(2010)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
10-12/0, Wistarrat, gavage, 7 d/wk,
90 d; food consumption and BWs
recorded every 15 d (n = 12);
spontaneous motor activity and muscle
coordination measured every 15 d
(n = 10 for each); learning and memory
tested using pole-climbing test 24 hr
after last treatment (n = 24); 5-HT
(5-hydroxytryptamine) concentration in
the cerebrum and midbrain, brain
protein concentration, and
acetylcholinesterase activity measured
(n = 10 each)
0 or 2°
(purity 95%)
Increased spontaneous motor activity on Days 75 and 90;
learning and memory deficits (manifested as decreased number
responding and increased response time); increased 5-HT
concentration in the cerebrum and midbrain
LOAEL: increased motor activity; memory and learning
deficits; and increased 5-HT levels in the cerebrum and
midbrain
NDr
2
Paul et al. (1994)
10/10, Wistar rat, gavage, 90 d; BWs
and behavior recorded (interval not
reported); motor coordination measured
every 15 d using rota-rod apparatus;
unconditioned and conditioned
avoidance test (pole-climbing)
performed at the end of treatment
0 or 2°
(purity 95%)
Decreased number of animals responding to simultaneous
unconditioned and conditioned stimuli (impaired avoidance
response to shock) in both sexes at 2 mg/kg-d
LOAEL: impaired avoidance response to shock
NDr
2
Paul et al. (1992)
15-16/0, Long-Evans hooded rat,
gavage, 3 d/wk for 7 wk, 5 mg/kg-d for
20 d or 10 mg/kg-d 3 d/wk to total
10 dosing days then challenged 14-16 d
later with matching dose; detailed
behavioral observations 30 min and 1 hr
following the 1st, 10th, 21st, and
challenge doses; electrical kindling
performed 1-2 wk after challenge dose
to measure threshold for inducing an
after-discharge (AD), duration of
development of an AD, and rate of
kindling
0,2.1, or 4.3e
(purity not
reported)
Enhanced seizure score (increased number of animals
expressing myoclonic jerks) after challenge dose as compared
with after 1st dose at both dose levels; decreased kindling rate
(number of stimulation sessions required to produce the first
stage 5 seizure) as compared with control at both dose levels
LOAEL: increased incidence of myoclonic jerks observed
following repeated doses; decreased kindling rate
NDr
2.1
Gilbert (1992)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,"
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
10/10, Wistar rat, diet, 30 d; mortality
and initial and final BWs recorded; liver
weighed; liver and serum concentrations
of glutamic oxaloacetic transaminase,
glutamic pyruvic transaminase,
acetylcholinesterase, and alkaline
phosphatase determined; spontaneous
motor activity and motor coordination
tested; additional group of 10/10 at low
dose tested for learning and memory
processes
0, 3, or 6°
(purity 95%)
Increased relative liver weight in both sexes at >3 mg/kg-d
(more markedly in females); increased motor activity in both
sexes at >3 mg/kg-d (more markedly in males); increased liver
and serum glutamic oxaloacetic transaminase and glutamic
pyruvic transaminase in males at >3 mg/kg-d (these enzymes
increased in females in liver samples at 6 mg/kg-d); increased
liver and serum alkaline phosphatase in both sexes at
>3 mg/kg-d; learning and memory deficits at >3 mg/kg-d in
both sexes
LOAEL: increased relative liver weight in both sexes; increased
activities of liver enzymes indicative of liver injury; increased
motor activity; memory and learning deficits
NDr
3
Paul et al. (1995)
6/0, Wistar rat, gavage, 7 d/wk, 30 d;
additional 6/0 treated for 30 d followed
by 7-d recovery period; BWs recorded
(interval not reported); testes removed,
weighed, and homogenized for analysis;
measured testicular and plasma
testosterone levels, plasma
gonadotrophins (FSH and LH) levels,
and activities of testicular enzymes:
microsomal mixed function oxidases
(MFO), steroidogenic enzymes, and
glutathione -S -transferase
0, 7.5, 10, or
10 (30 d plus 7 d
recovery)0
(purity not
reported,
technical grade
used)
Decreased plasma testosterone, FSH, LH, and testicular
testosterone at >7.5 mg/kg-d; decreased activities of testicular
steroidogenic enzymes, MFO system, and glutathione-
S-transferase at >7.5 mg/kg-d; decreased testicular testosterone
at 10 mg/kg following 7-d recovery period
LOAEL: decreased plasma and testicular testosterone levels;
decreased plasma FSH and LH levels; decreased activities of
testicular enzymes
NDr
7.5
Singh and Pandey
(1990)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
20/0, SPF Wistar rat, diet, 30 d, with
4-wk recovery period in half of animals;
rats monitored daily for behavior,
appearance, and general health
condition (including neurological signs,
ophthalmologic changes, dental health);
BWs and food consumption recorded
weekly; gross examination at necropsy;
liver, kidney, and brain weighed;
histological examination of left kidney,
liver, and brain using light or electron
microscopy; residues of endosulfan
measured in liver, kidney, and brain
tissue
0, 34, or 67.8f
(purity 97.9%)
Increased absolute and relative liver weight at >34 mg/kg-d
(data not reported; effect not observed after recovery);
increased absolute and relative kidney and brain weights at
67.8 mg/kg-d (data not reported; effect not observed after
recovery); darkened kidneys, granular pigmentation and
proliferation, and enlargement of lysosomes in renal proximal
tubule cells at >34 mg/kg-d (data not reported; effect decreased
after recovery)
LOAEL: increased relative liver weight; histological changes in
renal proximal tubule cells
NDr
34
Leist and Mayer
(1987), as
summarized in
U.S. EPA (1994a),
IPCS (1989) and
McGregor (1998)
Unreported number (19 in high-dose
group)/0, albino rat, gavage, 60 d; BWs
recorded daily; liver, brain, spleen,
kidney, lung, heart, testes, epididymis,
ventral prostate, and seminal vesicles
collected and weighed
0,2.5, or 7.5C
(purity not
reported)
Mortality observed in all groups (2/unknown number in control
group, 2/unknown number in mid-dose group, 8/19 in high-dose
group); hyperactivity observed at 7.5 mg/kg-d; clonic
tremors/convulsions observed in animals that died at
7.5 mg/kg-d; increased liver and lung weights were reported by
the study authors; however, data not reported and it is unclear at
what dose level the increased organ weights were seen
LOAEL (frank-effect level [FEL]): mortality
NDr
2.5 (FEL)
Ansari et al.
(1984)
Unreported number/0, albino rat,
gavage, 30 d; BWs recorded (interval
not reported); organs weighed (specific
organs not reported); blood chemistry
examined (specific tests not reported);
histopathological examination (specific
organs/tissues not reported)
0 or llf
(purity not
reported)
Mortality observed at 11 mg/kg-d (3 animals, cause of death not
reported)
LOAEL (FEL): mortality
NDr
11 (FEL)
Nath et al. (1978),
as summarized in
McGregor (1998)
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Table 4. Summary of Oral Subchronic-Duration Studies for Endosulfan (CASRN 115-29-7)
Number of Male/Female, Strain,
Species, Route of Administration,
Study Duration, Methods
Dosimetry,3
Purity of Test
Compound
Critical Effects
NOAELa'b
LOAELab
Reference
Mouse
20/20, CD-I mouse, diet, 13 wk; clinical
signs, food consumption, and BWs
recorded (interval not reported);
hematology and clinical chemistry
parameters examined (specific tests not
reported); microscopic examination
upon sacrifice (specific organs/tissues
not reported)
M: 0, 0.24, 0.74,
2.13, or7.3f
F: 0, 0.27, 0.8,
2.39, or7.52f
(purity 97.2%)
Increased mortality in both sexes at the high dose (data not
reported); decreased glucose levels in females at >0.8 mg/kg-d
(data not reported); increased hemoglobin levels and decreased
mean corpuscular hemoglobin concentration in females at all
dose levels (data not reported); reduced neutrophil count and
spleen weight in males at 7.3 mg/kg-d (data not reported);
increased serum lipid concentration in females at 7.52 mg/kg-d
(data not reported)
LOAEL (FEL): mortality
2.13
7.3 (FEL)
Hoechst
Aktiengesellschaft
(1985b), as
summarized in
U.S. EPA (1994a),
ATSDR (2000)
and McGregor
(1998)
10/10, Hoe:NMRKf (SPF 71) mouse,
diet, 6 wk; clinical signs, food
consumption, and BWs recorded
(interval not reported); organs weighed
and macroscopically examined (specific
organs not reported); eyes
microscopically examined upon
sacrifice
M: 0 or 3.7f
F: 0 or 4.6f
(purity not
reported)
Mortality observed in females at 4.6 mg/kg-d (2/10; cause of
death unable to be determined); increased absolute and relative
liver weights in females (data not reported)
LOAEL (FEL): mortality in females
NDr
4.6 (FEL)
Donaubauer et al.
(1985) Hoechst
Aktiengesellschaft
(1985b), as
summarized in
McGregor (1998)
and ATSDR
(2000)
""Dosimetry: NOAEL and LOAEL values are adjusted daily doses in mg/kg-d. No useful data were available to perform BMD modeling. Values are based on a 7:3 mixture
of a-endosulfan and (3-endosulfan unless otherwise noted.
bDU = data unsuitable; NA = not applicable; NV = not available; ND = no data; NDr = not determinable; NI = not identified; NP = not provided; NR = not reported; NR/Dr
= not reported but determined from data; NS = not selected.
Daily doses provided by the study author(s).
dDaily doses were calculated using the following equation: Doseadj = concentration in food (ppm or mg/kg) x Food Consumption per Day (kg/d) x (1 -f- BW [kg]) x (Days
Dosed Total Days).
eDoses provided by the study author(s) were adjusted for continuous exposure using the following equation: Doseadj = dose (mg/kg) x (Days Dosed Total Days)
fDaily doses as reported in the secondary source(s).
8Achieved daily doses as reported in Cal/EPA (2008).
BW = body weight; S-D = Sprague-Dawley.
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Chronic-duration Studies
U.S. EPA (1994a) reviewed and provided study summaries for chronic-duration studies
in rats and beagle dogs (Hoechst Celanese Corporation, 1989a,b). Table 3 provides the
information for these studies. In addition, U.S. EPA (2010) evaluated the carcinogenic potential
of endosulfan. No evidence of carcinogenicity was found in rats or mice exposed for 2 years to
endosulfan via the oral route (Hoechst Celanese Corporation, 1988; 1989a, as reported by
U.S. EPA, 2010). Details of the studies were not provided. U.S. EPA (2010) concluded that the
doses were adequate in both studies and that endosulfan is classified as "Not Likely to be
Carcinogenic to Humans." Endosulfan sulfate is also classified as 'Not Likely to be
Carcinogenic to Humans.
Developmental and Reproductive Studies
A total of 14 studies have been performed to evaluate the developmental and
reproductive effects of endosulfan in rats and rabbits. A number of effects on the male
developmental system were reported including decreases in sperm production, spermatogenesis,
and testis weight, and increases in morphological abnormalities in sperm. Table 5 provides a
summary of the available literature concerning the developmental effects of endosulfan. In
addition, the selected principal study for the screening subchronic p-RfD is summarized below.
U.S. EPA (1994a) also reviewed and provided a summary for a two-generation reproductive
study in rats (Hoechst Aktiengesellschaft, 1984a; see Table 3).
Gilmore et al. (2006) is selected as the principal study for deriving the subchronic
p-RfD. In a developmental neurotoxicity study, Gilmore et al. (2006) administered doses of 0,
50, 150, or 400 ppm of endosulfan (purity of 99.1%; dissolved in acetone) via diet to groups of
30 female Wistar Cr:WI (Han) rats (Charles River Laboratories, Raleigh, NC) on Gestation Day
(GD) 6 though Postnatal Day (PND) 21. Because this study contained confidential business
information (CBI), the original report was not available for review. However, a U.S. EPA Office
of Pesticide Program (OPP) Data Evaluation Record (DER) that provided the detailed results
data was available. In addition, the study was evaluated by U.S. EPA. U.S. EPA (2010).
However, there is no evidence of a formal external review which is a requirement for
development of a provisional toxicity value. Therefore, as explained later in this document, a
screening level toxicity value is developed.
The DER provided average daily doses of 0, 3.74, 10.8, or 29.8 mg/kg-day for the 0-, 50-,
150-, and 400-ppm groups, respectively; it is unclear if the study authors or DER reviewer
converted the doses. Males were at least 15 weeks old at study initiation; it is unclear what the
average male rat weighed. Females were at least 12 weeks old and weighed 159.2-218.9 g at
study initiation. All animals were given 7 days to acclimate to test room conditions before
treatment initiation. Dams were housed individually in plastic cages with bedding during
gestation and lactation. The room was maintained at a temperature of 18-26°C, with
30-70% humidity, and a 12-hour light/dark cycle. Animals were allowed food (Purina Mills
Rodent Diet 5002) and water (Kansas City municipal water) ad libitum. Gilmore et al. (2006) is
an acceptable reproductive/developmental study for development of toxicity values.
Parental animals were observed once daily in their cages for clinical signs of toxicity,
mortality, morbidity, and behavioral changes. Dams were examined in more detail once daily
from GD 6 through Lactational Day (LD) 21. Body weight and food consumption were recorded
weekly during gestation and lactation. A functional observational battery (FOB) was completed
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on GDs 13 and 20. Another set of 10 dams/dietary level was also examined using a FOB on
LDs 11 and 21. Each dam was examined for delivery beginning on GD 20; the day of delivery
was designated LD 0 for each dam and PND 0 for pups. Litters that contained fewer than three
pups at delivery or less than seven pups by PND 4 were sacrificed and not necropsied. Litters
were culled on PND 4 to yield four males and four females per litter (when possible).
After parturition, authors measured anogenital distance (AGD) and weight of individual
pups. On Days 0, 4, 11, 17, and 21, authors recorded the number of live pups, sex, and
individual weights. Pups were examined daily during lactation for signs of mortality and
morbidity. Detailed observations for clinical signs of toxicity and body weights were recorded
daily before weaning and once a week after weaning. On PND 21, authors examined all pups for
pupil constriction. Beginning on PND 38 for males and PND 29 for females, authors examined
animals daily and recorded the first observation of vaginal patency or balanopreputial separation.
After PND 21, authors examined all animals twice daily for mortality and once daily for clinical
signs. Weights were recorded once weekly. The authors did not record food consumption after
weaning. The authors calculated the mating, live birth, and lactation indices.
When animals were approximately 50-60 days old, authors performed ophthalmic
examinations (minimum of 10/sex/dose representing at least 20 litters/dose) of animals selected
for perfusion at study termination. The pupillary reflex was tested, and the conjunctiva, cornea,
lens, vitreous humor, retina, choroid, and optic disc were examined.
Males were sacrificed immediately after mating, and dams were sacrificed on LD 21
(after weaning). Routine necropsies were not performed on F0 generation males or females.
Animals in the F1 generation were sacrificed on either PND 21 or 75 ± 5 days and given a gross
necropsy that involved examination of all organs, body cavities, cut surfaces, external orifices
and surfaces, and gross abnormalities. Lesions of the neural tissues or skeletal muscle were
examined microscopically. All animals found dead also underwent necropsy. Animals selected
for perfusion on PND 21 were anesthetized and then perfused via the left ventricle with a sodium
nitrite flush followed by in situ fixation. The authors collected the brain with olfactory bulbs on
PND 21 and collected the brain, spinal cord, both eyes (with optic nerves), selected peripheral
nerves (sciatic, tibial, and sural), the gasserian ganglion, gastrocnemius muscle, and both
forelimbs at study termination. Brain tissues from perfused animals and gross lesions from all
animals were examined microscopically.
The authors evaluated continuous data for equality of variance using Bartlett's test. An
ANOVA was completed for group means with equal variances. If the ANOVA was significant,
the data were evaluated using a Dunnett's test. Nonparametric data were analyzed using the
Kruskal-Wallis ANOVA followed by the Mann-Whitney U test. FOB tests were analyzed using
ANOVA and Dunnett's test (continuous data) or General Linear Modeling and Categorical
Modeling (CATMOD) procedures followed by Dunnett's test and an Analysis of Contrasts
(categorical data). Pathology data were analyzed using a number of statistical tests, including
Bartlett's test for homogeneity with ANOVA, the Kruskal-Wallis test (organ weight data and
gross brain measurements), ANOVA, and/or Mests (microscopic brain measurements).
The authors reported that statistically significant maternal clinical observations (hair loss,
rearing) could not be definitively attributed to treatment with the test substance due to a lack of a
dose-response relationship. Furthermore, the observations did not always occur in the same
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dams. There were no treatment-related effects in FOB results. Table C.9 provides data on
maternal body weight and food consumption. Dams experienced statistically significant,
dose-dependent decreases in body weight throughout gestation. Gestational food consumption
was also decreased in a statistically significant, dose-dependent manner. Food efficiency was
nominally decreased in the mid- and high-dose groups on Days 6-13, but efficiency in these
groups was comparable to or increased when compared with control values on GDs 13-20.
Maternal body weight was significantly decreased on LDs 0, 4, and 7 in the mid- and high-dose
groups. However, maternal food consumption was not significantly altered at any point during
lactation.
Table C.10 provides pup body weights. Female pup weight was significantly decreased
at the highest dose on PND 4 (before culling but not after). On PNDs 11, 17, and 21, statistically
significant (p < 0.01), dose-dependent decreases occurred in both male and female pup weights
in all litters from dams treated with endosulfan. Postweaning pup weight was significantly
reduced in mid- and high-dose males from PNDs 28-70 and in high-dose females from
PNDs 28-49. The authors also noted a significant decrease in days to sexual maturation
(preputial separation) in male pups of the mid- and high-dose groups (see Table C. 11). It is
unclear whether the pup weight decrements at the low dose on PND 11 (9% relative to controls)
and PND 17 (7% relative to controls) were due to unpalatable endosulfan in the dam's milk, a
reduced milk supply, or a toxic effect of endosulfan in the milk. Either approach would yield an
appropriate point of departure (POD) for derivation of a toxicity value (i.e., unpalatability of the
dam's milk would be a biologically relevant response to dosing the dam, as would reduced milk
supply or some toxicity to the pups associated with the milk itself). A significant decrease in
vaginal opening in females was observed at 3.74 and 10.8 mg/kg-day but not at 29.8 mg/kg-day
(see Table C.l 1). Rearing in high-dose males at PND 45 was increased in a dose-dependent
manner (significant at the high dose only), but the authors did not consider this effect to be
treatment related. Based on the dam and pup weight decreases, a developmental and maternal
LOAEL of 3.74 mg/kg-day is identified. A NOAEL cannot be identified because the lowest
dose was a LOAEL.
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d) ,a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Rat
Developmental, 0/30, Wistar Crl:WI
(Han) rat, diet, dams and offspring
provided treatment diet ad libitum on
GD 6-PND 21 (weaning); dams and
pups sacrificed onPND 21 or
PND 75 ± 5; daily clinical
observations and weekly recordings of
maternal BW and food consumption
during dosing; FOB of dams on GDs
13, 20 and LDs 11, 21; selected
offspring from each group evaluated
for B W, food consumption, onset of
sexual maturation (balanopreputial
separation/vaginal patency), FOB,
motor activity, auditory startle
habituation, learning and memory,
ophthalmic examination, brain weight
and neuropathology, and sperm
analysis (testes and epididymal sperm)
0, 3.74, 10.8,
or 29.8°
(purity 99.1%)
Decreased maternal BW from GD 13-LD
7 at 10.8 mg/kg-d and decreased food
consumption for GDs 6-13 at
>3.74 mg/kg-d and for GDs 13-20 at
>10.8 mg/kg-d
Litter-based decreased pup weight at
>3.74 mg/kg-d on PND 11 (both sexes)
and PND 17 (males only); decreased pup
weight on PNDs 35-70 in males at
>10.8 mg/kg-d and in females on
PNDs 28-49 at 29.8 mg/kg-d; delayed
sexual development (day of preputial
separation) in males at >10.8 mg/kg-d;
increased rearing in females at
10.8 mg/kg-d on PND 21 and in males at
29.8 mg/kg-d onPND 45; decreased
perfused fixed brain weight in PND-21
males at 29.8 mg/kg-d (relative fixed
brain weight unaffected); decreased
hippocampal gyrus (10% smaller than
control) in females at 29.8 mg/kg-d
Developmental LOAEL: decreased pup
weight at PNDs 11 and 17
BMDL: Decreased pup BW in females at
PND 11
Maternal: NDr
Developmental: NDr
0.29
Maternal: 3.74
Developmental: 3.74
Gilmore et
al. (2006)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/24, Wistar rat,
gavage, dams dosed GD 15-PND 21;
maternal BWs during dosing and pup
BWs during lactation recorded daily;
litter size and number of viable
offspring assessed; male offspring
(1—2/litter; 15/group): investigated for
age of testes descent and preputial
separation, sacrificed on PND 65
(puberty) or PND 140 (adulthood) and
investigated for changes in absolute
and relative testes, epididymis,
seminal vesicle, and ventral prostate
weights; sperm and spermatid counts;
daily sperm production rate; serum
testosterone level; and histology of
testes; different male offspring
(15/group) mated with control virgin
females on PND 120: maternal and
fetal BW and pregnancy outcomes
analyzed (mating/pregnancy/fertility
rates)
0, 1.5, or3.0c
(purity 97%)
Reduced maternal BW on GDs 16, 17,
and 18 at 3.0 mg/kg-d; increased absolute
and relative testicular weights and
decreased daily sperm production rate in
male offspring at >1.5 mg/kg-d at puberty
and 3.0 mg/kg-d at adulthood; decreased
percentage of seminiferous tubules
showing complete spermatogenesis at
>1.5 mg/kg-d at puberty
Maternal LOAEL: reduced maternal BW
during gestation
Developmental LOAEL: increased
relative testis weight; decreased sperm
production and percentage of seminiferous
tubules showing complete
spermatogenesis at puberty
BMDL: Decreased daily sperm production
rate in male offspring
Maternal: 1.5
Developmental: NDr
0.68
Maternal: 3.0
Developmental: 1.5
Dalsenter et
al. (1999)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/20-24, Wistar rat,
gavage, dams dosed on GDs 7-16;
clinical observations and BWs
recorded (frequency not reported); sex
ratio and reproductive parameters
examined (specific parameters not
reported); fetuses examined for
skeletal and other abnormalities
(further details not reported)
0,0.66, 2, or6e
(purity 97.3%)
Maternal: mortality (4/20-24) and clinical
signs of toxicity (convulsions,
hypersalivation) observed at 6 mg/kg-d;
Decreased maternal BW (data not
reported)
Developmental: increased incidence of
fragmented thoracic vertebral centra at
6 mg/kg-d (data not reported)
Maternal LOAEL (FEL): mortality
Developmental LOAEL: not determinable
due to effects seen only in the presence of
mortality
Maternal: 2
Developmental: 2
NDr
Maternal: 6 (FEL)
Developmental: NDr
Albrecht and
Baeder
(1993), as
summarized
in McGregor
(1998)
Developmental, 0/10, Wistar rat,
gavage, dams dosed throughout entire
gestation period and through PND 28;
maternal BWs recorded (frequency
not reported); offspring examined for
litter size, sex ratio, birth weight, and
crown-to-rump length; offspring
weights recorded during postnatal
period (frequency not reported); male
offspring examined for anogenital
distance (PNDs 1, 28 and 90),
cryptorchidism, hypospadia, incidence
of apoptosis of testis germ cells, testis
histology, daily sperm production,
epididymal sperm count and
morphology, and fertility
0, 0.5, 1.0, or
2.5°
(purity not
reported)
Maternal: mortality observed at
2.5 mg/kg-d (4/10)
Developmental: no significant effects
observed with any of the parameters
examined
Maternal LOAEL (FEL): mortality
Developmental LOAEL: NDr
Maternal: 1.0
Developmental: 2.5
NDr
Maternal: 2.5 (FEL)
Developmental: NDr
Zhu et al.
(2000)
(abstract
only) and as
summarized
in Cal/EPA
(2008)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/number of females
not reported, Druckrey rat, gavage,
dams dosed GD 12 through
parturition; offspring sexed and size
and weight of litters were recorded;
male offspring were fostered to
untreated females that had given birth
the prior day; male offspring
monitored for dietary intake, B W,
clinical signs and behavior (intervals
not reported), and sacrificed at 100 d
of age; epididymis, testes, seminal
vesicles, prostate glands removed and
weighed; sperm and spermatid counts
and testicular marker enzyme levels
(LDH, SDH, GGT, G6PDH) analyzed
0, 1.0, or 2.0°
(purity
95.32%)
Decreased absolute and relative weights
of testes, epididymis, and seminal vesicle
at >1.0 mg/kg-d; decreased sperm count
(epididymis) and spermatid count (testis)
at >1.0 mg/kg-d; increased LDH activity
and decreased SDH activity at
>1.0 mg/kg-d
Developmental LOAEL: decreased
relative testes, epididymis, and seminal
vesicle weights; decreased sperm and
spermatid counts; increased testicular
LDH activity; decreased testicular SDH
activity
Developmental: NDr
NDr
Developmental: 1.0
Sinha et al.
(2001)
Male developmental, 15/0, Druckrey
rat, gavage, 5 d/wk, 70 d; BW
recorded twice weekly; testes and
epididymis weighed and analyzed;
activities of testicular enzymes
(marker enzymes of spermatogenesis:
LDH, SDH, GGT, G6PDH) measured;
cauda epididymis sperm count and
morphology analyzed; intratesticular
spermatid count analyzed
0, 1.8, 3.6, or
7.1d
(purity
95.32%)
Increased testicular LDH, SDH, GGT, and
G6PDH activities at >1.8 mg/kg-d;
decreased cauda epididymis sperm counts
at >1.8 mg/kg-d; decreased testis
spermatid counts and sperm production
rate at >3.6 mg/kg-d; increased altered
sperm morphology (% sperm abnormality)
at >3.6 mg/kg-d
LOAEL: increased activities of testicular
LDH, SDH, GGT, and G6PDH; decreased
sperm counts in cauda epididymis
NDr
NDr
1.8
Sinha et al.
(1995)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 5/0, weaned (3-wk
old) Druckrey rat, gavage, 5 d/wk,
69 d; BWs recorded twice weekly;
upon sacrifice at 90 d of age, testes
and epididymis removed and weighed;
sperm and spermatid counts, sperm
morphology, and testicular marker
enzyme levels (LDH, SDH, GGT,
G6PDH) analyzed
0, 1.8,3.6, or
7.1d
(purity
95.32%)
Decreased sperm count (epididymis),
decreased spermatid count (testis), and
increased percentage of sperm
morphological abnormalities at
>1.8 mg/kg-d; increased LDH, GGT, and
G6PDH activity and decreased SDH
activity at >1.8 mg/kg-d
Developmental LOAEL: decreased sperm
and spermatid counts; increased
morphological abnormalities in sperm;
increased LDH, GGT, and G6PDH
activity; decreased SDH activity
Developmental: NDr
NDr
Developmental: 1.8
Sinha et al.
(1997)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/25, CD S-D rat,
gavage, dams dosed GDs 6-19; 10
additional animals were added to the
high-dose group due to mortality, 5
additional animals were added to the
control group; maternal BWs recorded
on GDs 0 and 20 (additional intervals
not reported); gravid uterine weight,
corrected BW, corrected BW gain,
percentage live fetuses, number of
resorptions per litter, percentage
resorbed fetuses, and mean fetal
weight and length assessed; fetuses
examined for developmental
abnormalities (full range of
abnormalities not reported)
0, 0.66, 2.0, or
6.0e
(purity 97.3%)
Maternal: mortality (7/25) and clinical
signs of toxicity (face rubbing [20/35],
brown exudates [4/35], rough coat [5/35],
flaccidity [8/35], hyperactivity [11/35])
observed at 6.0 mg/kg-d; face rubbing
(6/25) observed at 2.0 mg/kg-d; reduced
maternal BW (GD 20) at 6.0 mg/kg-d
Developmental: decreased mean fetal BW
and crown-rump length at 6.0 mg/kg-d;
reduction in percentage of live fetuses and
an increase in the number of resorbed
fetuses at 2.0 mg/kg-d only; increase in
misaligned sternebrae at >0.66 mg/kg-d;
increased incidence of litters with extra
ribs and poorly ossified and unossified
sternebrae at 6.0 mg/kg-d
Maternal and offspring LOAEL:
precluded due to replacement of animals
during or after the study, which made
interpretation difficult
Maternal: NDr
Developmental: NDr
NDr
Maternal: NDr
Developmental: NDr
FMC (1980),
as
summarized
in Cal/EPA
(2008),
U.S. EPA
(1994a),
ATSDR
(2000) and
McGregor
(1998)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/6, CD S-D rat,
gavage, dams dosed GDs 6-19;
maternal BWs and clinical signs of
toxicity recorded (intervals not
reported); details of offspring
parameters not reported
0, 1.25,2.5,
5.0, 10, 20, or
40e
(purity not
reported)
Maternal: mortality observed at
>10 mg/kg-d; clinical signs (salivation,
piloerection, hyperactivity, head-rubbing,
hostility, spasticity, tremors, and
convulsions) observed at >2.5 mg/kg-d;
decreased BW gain at >1.25 mg/kg-d
(data not reported)
Developmental: results not reported
Maternal LOAEL: decreased BW gain
Maternal: NDr
Developmental: NDr
NDr
Maternal: 1.25
Developmental: NDr
Fung (1980),
as
summarized
in Cal/EPA
(2008)
Developmental, 0/18-21, albino rat,
gavage, dams dosed GDs 6-14;
maternal BWs recorded GD 0, daily
during dosing, and before and after
c-section; c-section and sacrifice on
GD 21; maternal viscera and uteri
examined for gross pathology and
resorptions; fetuses weighed and
examined for external abnormalities;
half of fetuses examined for skeletal
abnormalities/variations, other half
examined for soft-tissue abnormalities
0, 5.0, or 10.0C
(purity not
reported)
Mortality observed at >5.0 mg/kg-d (1/20,
5/21); increased percentage of litters with
resorptions, percentage of fetuses with
skeletal abnormalities, and incidence of 5th
absent sternebrae at >5.0 mg/kg-d;
increased incidence of fetuses with
incomplete calcification and percentage of
litters with skeletal or soft-tissue
abnormalities at 5.0 mg/kg-d only
Maternal LOAEL: mortality and increased
resorptions sites
Developmental LOAEL: not determinable
due to effects seen only in the presence of
mortality
Maternal: NDr
Developmental: NDr
NDr
Maternal: 5.0 (FEL)
Developmental: NDr
Gupta (1978)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Rabbit
Developmental, 0/20-26, New
Zealand white rabbit, gavage, dams
dosed on GDs 6-28; maternal BWs
and clinical observations recorded
during gestation (frequency not
reported); number of implantations,
litter size, sex ratio, mean fetal weight
and length, and numbers of live and
resorbed fetuses were analyzed;
offspring examined for external,
soft-tissue, and skeletal
abnormalities/variations
0, 0.3, 0.7, or
1.8e
(purity 97.3%)
Mortality observed at 1.8 mg/kg-d (4/26);
increased incidence of clinical signs of
toxicity (convulsions/thrashing,
noisy/rapid breathing, hyperactivity,
salivation, and nasal discharge) at
1.8 mg/kg-d; decreased maternal BW gain
during GDs 19-29, and BW gain
corrected for gravid uterine weight at
sacrifice at 1.8 mg/kg-d (data not
reported)
Maternal LOAEL: increased mortality and
clinical signs of toxicity; decreased BW
gain
Developmental: no developmental effects
observed
Maternal: 0.7
Offspring: 1.8
NDr
Maternal: 1.8 (FEL)
Offspring: NDr
Nye (1981),
as
summarized
in Cal/EPA
(2008),
U.S. EPA
(1994a) and
McGregor
(1998)
Developmental, 0/unreported number
of females, New Zealand White
rabbit, gavage, dams dosed on
GDs 6-18; range-finding study; dams
observed for clinical signs and
mortality
0, 0.5, 0.625,
1.0, 1.25,2.0,
2.5, 5.0, 10, 20,
40, or 80e
(purity not
reported)
Mortality observed in dams at
2.0 mg/kg-d (2/6) and at >5.0 mg/kg-d (all
animals died); clinical signs of
neurotoxicity in dams (hyperactivity,
opisthotonos, convulsions, and paralysis)
observed at >1.25 mg/kg-d (data not
reported)
Maternal LOAEL: mortality and clinical
signs of neurotoxicity
Developmental LOAEL: no results
reported
Maternal: 1.0
Developmental: NDr
NDr
Maternal: 1.25
Developmental: NDr
Fung
(1981a), as
summarized
in Cal/EPA
(2008)
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Table 5. Summary of Developmental Studies for Endosulfan (CASRN 115-29-7)
Study Type, Number of
Male/Female, Strain, Species, Route
of Administration, Study Duration,
Methods
Dosimetry
(mg/kg-d),a
Purity of Test
Compound
Critical Effects
NOAELa'b
BMDL
LOAELab
Reference
Developmental, 0/3-10, New Zealand
White rabbit, gavage, dams dosed on
GDs 6-28; range-finding study; dams
observed for clinical signs and
mortality
0, 1, 2, 4, 8, or
12e
(purity not
reported)
Mortality observed in dams at >4 mg/kg-d
(4/8 at 4 mg/kg-d, all animals died at
higher doses); clinical signs of
neurotoxicity in dams (hyperactivity,
opisthotonos, convulsions, and paralysis)
observed at >2 mg/kg-d (data not
reported)
Maternal LOAEL: mortality and clinical
signs of neurotoxicity
Developmental LOAEL: no results
reported
Maternal: 1
Developmental: NDr
NDr
Maternal: 2 (FEL)
Developmental: NDr
Fung
(1981b), as
summarized
in Cal/EPA
(2008)
Reproductive studies are presented as duration-adjusted doses (from 5-6 d/wk to continuous 7 d/wk). Doses for oral developmental studies are not adjusted beyond
continuous daily dose as dosing is typically every day throughout the developmental period.
bDU = data unsuitable; NA = not applicable; NV = not available; ND = no data; NDr = not determinable; NI = not identified; NP = not provided; NR = not reported;
NR/Dr = not reported but determined from data; NS = not selected.
"Daily doses provided by the study author(s).
dDoses provided by the study author(s) were adjusted for continuous exposure using the following equation: Doseadj = dose (mg/kg) x (Days Dosed Total Days).
"Daily doses as reported in the secondary source(s).
BW = body weight; S-D = Sprague-Dawley.
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Inhalation Exposures
The effects of inhalation exposure of animals to endosulfan have been evaluated in one
short-term study (Hoechst Aktiengesellschaft, 1984b, as summarized in U.S. EPA, 2010).
Short-term Studies
Hoechst Aktiengesellschaft (1984b) evaluated the effects of endosulfan in a 21-day
inhalation study in rats. The original study contained CBI and was not available for review.
However, the study was summarized by U.S. EPA (2010) and is, therefore, considered peer
reviewed. According to U.S. EPA (2010), male and female rats were exposed to 0, 0.0005,
-3
0.0010, or 0.0020 mg/L (calculated to be equivalent to 0, 0.5, 1, or 2 mg/m ) endosulfan
(97.2% purity) by nose-only inhalation for 6 hours/day for 21 exposures over 29 days. Estimated
human equivalent concentrations (HECs) based on default body-weight data (U.S. EPA, 1988)
are 0, 0.09, 0.18, and 0.36 mg/m3. However, because the vapor pressure of endosulfan is very
low (2.7 x 10~7 Pa at 25°C; HSDB, 2009, 2010), the exposure system could be generating
particles and not vapor. According to U.S. EPA protocol (U.S. EPA, 1994c), the dosage must be
calculated based on particulate characteristics, which are not provided, thus precluding an
accurate interpretation of the study's exposure atmosphere and accurate dosimetry (i.e.,
calculation of HECs).
(Hoechst Aktiengesellschaft, 1984b, as reported in U.S. EPA, 2010) The
concentration and endpoint for risk assessment are based on a systemic NOAEL
of 0.0010 mg/L [NOAELhec of 0.18 mg/m3], and a LOAEL of 0.0020 mg/L
[LOAELhec of 0.36 mg/m ], based on decreased body weight in males, decreased
leukocyte counts in males, and increased creatinine values in females. It should
be noted that decreased body weight in adult males was more sensitive than body
weight loss in adult females in the inhalation study. The protection of adult male
body weight loss via the inhalation route therefore protects adult females and the
young.
Subchronic-duration Studies
No studies were identified.
Chronic-duration Studies
No studies were identified.
Developmental Studies
No studies were identified.
Reproductive Studies
No studies were identified.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
Appendix B provides other data for endosulfan and endosulfan sulfate.
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DERIVATION OF PROVISIONAL VALUES
Tables 6 and 7 present summaries of noncancer and cancer reference values, respectively.
IRIS data are indicated in the tables, if available. As explained above, these values are based on
effects from exposure to the parent compound, endosulfan, and are considered to be appropriate
for endosulfan sulfate, its principal metabolite in mammalian systems.
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Table 6. Summary of Reference Values for Endosulfan Sulfate (CASRN 1031-07-8)
Toxicity Type (units)
Species/Sex
Critical Effect
p-Reference
Value
POD
Method
POD
UFC
Principal Study
Screening subchronic p-RfD
(mg/kg-d)
Rat/F pups
Endosulfan exposure resulted in a litter-based
decrease in pup B W
3 x 1(T3
BMDL05
0.29a
100
Gilmore et al. (2006)
Chronic RfD for endosulfan
(mg/kg-d)
IRIS (U.S. EPA, 1994a)
Rat/M
Dog/F
Endosulfan exposure resulted in decreased
BW gain, increased incidence of marked
progressive glomerulonephrosis, and blood
vessel aneurysms
Neurological findings
6 x 10 3 (IRIS)b
NOAF.I,/
LOAEL
0.6
100
Hoechst Celanese
Corporation (1989a,b)
Subchronic p-RfC
(mg/m3)
NDr
Hoechst
Aktiengesellschaft
(1984b) was
determined to be
inadequate for
development of a
p-RfC
Chronic p-RfC
(mg/m3)
NDr
^Because the POD is based on a study evaluating the effects of endosulfan, a molecular weight conversion for endosulfan to endosulfan sulfate was applied to the POD
during the determination of the screening subchronic p-RfD.
bA molecular weight conversion for endosulfan to endosulfan sulfate was applied to the IRIS RfD. However, after rounding, the value remained unchanged.
BW = body weight; NDr = not determined.
Table 7. Summary of Cancer Values for Endosulfan Sulfate (CASRN 1031-07-8)
Toxicity Type
Species/Sex
Tumor Type
Cancer Value
Principal Study
p-OSF
NDr
p-IUR
NDr
NDr = not determined.
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DERIVATION OF ORAL REFERENCE DOSES
Derivation of Subchronic Provisional RfD (Subchronic p-RfD)
Gilmore et al. (2006) is selected as the principal study for derivation of the
subchronic p-RfD. Although this study was accepted by an agency of the United States
government, it was not subjected to external review by independent scientists, which is a
requirement for utilization of a study for development of provisional values. However, the study
provides information that appears to be reasonably complete and reputable. Therefore, according
to the PPRTV protocol, a screening level provisional RfD is presented in Appendix A. Please
refer to this appendix for the screening p-RfD.
Derivation of Chronic RfD (Chronic RfD)
A chronic RfD of 0.006 mg/kg-day is available in IRIS (U.S. EPA, 1994a) for endosulfan
based on a 2-year study in rats (Hoechst Celanese Corporation, 1989a) and a 1-year feeding
study in dogs (Hoechst Celanese Corporation, 1989b). A UF of 100 was applied by IRIS (10 for
intraspecies variability and 10 for interspecies extrapolation).
Because the principal study focused on the exposure of the parent compound, endosulfan,
an MW conversion for consideration of endosulfan sulfate is applied to the IRIS chronic RfD
(note the value does not change for endosulfan sulfate after rounding to one significant figure).
Chronic RfDen(j0Sl]ifan sulfate — MWmetabolite ~ MW parent x RfDparent
= 422.95 -h 406.93 x 0.006 mg/kg-day
= 6 x 10 3 mg/kg-day
DERIVATION OF INHALATION REFERENCE CONCENTRATIONS
Derivation of Subchronic Provisional RfC (Subchronic p-RfC)
No repeat-dose studies investigating toxicological effects following inhalation exposure
of endosulfan sulfate are available. Additionally, no studies investigating the effects of
inhalation exposure to endosulfan in humans are considered appropriate for derivation of a
subchronic p-RfC. Available endosulfan human inhalation studies are limited by poor exposure
characterization and coexposures with other chemicals. The database of inhalation studies on
endosulfan in animals is limited to a single, unpublished 21-day study in rats (Hoechst
Aktiengesellschaft, 1984b). This study contains CBI and was unobtainable at the time of this
assessment. The study is summarized in an assessment conducted by U.S. EPA (2010).
However, no detailed information regarding the exposure delivery system is given. Because the
vapor pressure of this compound is very low, the delivery system could provide particles and not
vapor. Particle size information is not provided, which is necessary to calculate HECs. Without
this dosimetry information, a reliable p-RfC cannot be developed.
Derivation of Chronic Provisional RfC (Chronic p-RfC)
As indicated above, no value can be derived.
CANCER WOE DESCRIPTOR
Table 8 identifies the cancer WOE descriptor for endosulfan sulfate.
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Table 8. Cancer WOE Descriptor for Endosulfan Sulfate
Possible WOE Descriptor
Designation
Route of Entry
(Oral, Inhalation, or Both)
Comments
"Carcinogenic to
Humans "
NS
NA
NA
"Likely to Be
Carcinogenic to Humans"
NS
NA
NA
"Suggestive Evidence of
Carcinogenic Potential"
NS
NA
NA
"Inadequate Information
to Assess Carcinogenic
Potential"
Selected
Inhalation and Oral
No data were available concerning the
carcinogenic potential of endosulfan or
endosulfan sulfate via the inhalation
route. U.S. EPA (2010) evaluated data
on the oral carcinogenicity of
endosulfan and concluded that it is
"Not Likely to be Carcinogenic to
Humans". Because endosulfan is
metabolized to endosulfan sulfate
following absorption, the same
conclusion is drawn for the metabolite.
However, on further review, based on
the paucity of data, "inadequate" is a
more appropriate descriptor.
"Not Likely to Be
Carcinogenic to Humans "
NS
NA
NA
NA = not applicable; NS = not selected.
DERIVATION OF PROVISIONAL CANCER POTENCY VALUES
Derivation of Provisional Oral Slope Factor (p-OSF)
U.S. EPA (2010) evaluated data on the oral carcinogenicity of endosulfan and concluded
that it is "Not Likely to be Carcinogenic to Humans." No treatment-related neoplasms were
observed in any of the combined chronic/carcinogenicity feeding studies on rats and mice. No
further information was found indicating carcinogenic effects following oral exposure to
endosulfan. Therefore, derivation of the p-OSF for endosulfan sulfate is precluded.
Derivation of Provisional Inhalation Unit Risk (p-IUR)
The lack of data on the carcinogenicity of endosulfan precludes the derivation of
quantitative estimates for inhalation (p-IUR) exposure.
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APPENDIX A. PROVISIONAL SCREENING VALUES
For reasons noted in the main PPRTV document, it is inappropriate to derive provisional
toxicity values for endosulfan sulfate. However, information is available for this chemical
which, although insufficient to support derivation of a provisional toxicity value, under current
guidelines, may be of limited use to risk assessors. In such cases, the Superfund Health Risk
Technical Support Center summarizes available information in an appendix and develops a
"screening value." Appendices receive the same level of internal and external scientific peer
review as the PPRTV documents to ensure their appropriateness within the limitations detailed in
the document. Users of screening toxicity values in an appendix to a PPRTV assessment should
understand that there is considerably more uncertainty associated with the derivation of an
appendix screening toxicity value than for a value presented in the body of the assessment.
Questions or concerns about the appropriate use of screening values should be directed to the
Superfund Health Risk Technical Support Center.
Gilmore et al. (2006) is selected as the principal study for derivation of the screening
subchronic p-RfD. The critical effect is decreased body weight in female pups. Details are
provided in the "Review of Potentially Relevant Data" section. Based on the information
provided in the Introduction section of this report, it is considered to be a reasonable assumption
that studies performed using endosulfan are predictive of the toxicological effects that would
occur following exposure to endosulfan sulfate. Tables 4 and 5 summarize the available
databases of subchronic and developmental studies, respectively. The effects in fetal animals are
seen at relatively lower doses when compared with adult animals. Table D. 1 lists the BMD
output models for all endpoints considered for derivation of the screening subchronic p-RfD with
curve and BMD output text provided for the selected model in Appendix D (see Figure D.l and
the text output that follows the figure). Among the studies considered for derivation of the
screening subchronic p-RfD, the Gilmore et al. (2006) data for decreased body weight in female
pups on PND 11 provide the most sensitive POD (BMDL05 = 0.29 mg/kg-day). Other reported
developmental effects provide supporting evidence for the POD include delayed sexual
development, decreased sperm production and spermatogenesis, and changes in reproductive
organ weights (see Table 5). In addition, both neurological and immunological effects were
reported at similar doses (0.9-2 mg/kg-day). In this regard, selecting the BMDL05 of
0.29 mg/kg-day for decreased body weight in female pups as the POD will protect against these
other identified effects. The Gilmore et al. (2006) rat data for decreased body weight on PND 11
gives a BMDL05 of 0.29 mg/kg-day and provides evidence of the most sensitive indicator of
toxicity among the available studies. Other potential endpoints from this study occurred at
higher doses. Possible endpoints from other studies were considered, including the NOAEL of
0.5 mg/kg-day from Baneijee and Hussain (1986), but not selected because of lack of a clear
toxicity threshold for these effects and the fact that the BMDL05 of 0.29 for Gilmore et al. (2006)
would be protective for the potential immunotoxicity endpoint. The use of the benchmark
response (BMR) of 5% is appropriate for effects on early life periods. Data on a per-litter basis
for this study are not available. Instead, the results were reported as litter-based means. Thus,
the use of nested models provided by BMD software (BMDS) is not possible (U.S. EPA, 2012).
Instead, continuous BMD models are used to determine the POD. The continuous data models
in the U.S. EPA BMDS (version 2.1.2) were fit to litter-based means for pup body weights on
PND 11 following exposure of maternal rats to endosulfan by diet from GD 6-PND 21 (see
Table A. 1). The Hill constant variance model provides the best model fit (see Table A.2).
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Table A.l. Litter-based Body Weights (PND 11) of Female Pups from Female Wistar
Rats Exposed to Endosulfan from GD 6-PND 21—Used for BMD Analysis"
Dose (mg/kg-d)
Number of Litters
Mean
Standard Deviation
0
23
23.6
1.726
3.74
23
21.7*
2.206
10.8
23
20.9*
2.590
29.8
21
20.4*
2.200
aGilmore et al. (2006).
* Significantly different from control at p < 0.01: statistical test run was not reported.
Table A.2. Model Predictions for Female Pup Bodt Weight (PND ll)a
Model
Homogeneity
Variance />-valuc
Goodness-of-Fit
/>-valucb
AIC for Fitted
Model
BMD0S
(mg/kg-d)
BMDL0S
(mg/kg-d)
Hill
(constant variance)
0.298
0.960
235.99
1.63
0.29
Exponential (M4)
(constant variance)
0.298
0.653
236.19
2.07
0.77
Exponential (M5)
(constant variance)
0.298
0.653
236.19
2.07
0.77
Exponential (M2)
(constant variance)
0.298
0.010
243.28
12.28
8.68
Exponential (M3)
(constant variance)
0.298
0.010
243.28
12.28
8.68
Linear
(constant variance)
0.298
0.008
243.62
13.01
9.41
Polynomial
(constant variance)
0.298
0.008
243.62
13.01
9.41
Power
(constant variance)
0.298
0.008
243.62
13.01
9.41
'Gilmorc et al. (2006).
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
AIC = Akaike's Information Criteria; BMD = benchmark dose; BMDL = lower confidence limit (95%) on the
benchmark dose.
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The screening subchronic p-RfD for endosulfan sulfate is based on the BMDL05 of
0.29 mg/kg-day based on decreased female pup body weight in rats (Gilmore et al., 2006).
Dosimetry
Molecular Weight (MW) Correction:
Because the principal study was performed with endosulfan, an MW conversion is
necessary to convert the BMDL05 for endosulfan sulfate as follows.
BMDLo5(MW adj)	— MWmetabolite ~ MWparent x BMDL05 parent
= 422.95 -h 406.93 x 0.29 mg/kg-day
= 0.30 mg/kg-day
HED Conversion is not appropriate for a developmental endpoint:
EPA endorses body-weight scaling to the % power to extrapolate toxicologically
equivalent doses of orally administered agents from all laboratory animals to humans for the
purpose of deriving an RfD under certain exposure conditions. The use of BW3 4 scaling for
deriving an RfD is recommended when the observed effects are associated with the parent
compound or a stable metabolite but not for portal-of-entry effects or developmental endpoints.
In this case (a developmental endpoint: pup weights), the adjustment is not recommended or
applied since exposure to the chemical occurred through a sequential combination of in utero,
lactational, and direct exposure to neonatal and juvenile animals post-weaning.
Screening Subchronic p-RfD = POD ^ UFc
= 0.30 mg/kg-day -M00
= 3 x 10~3 mg/kg-day
Table A.3 summarizes the uncertainty factors (UFs) for the screening subchronic p-RfD
for endosulfan sulfate.
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Table A.3. UFs for Screening Subchronic p-RfD for Endosulfan Sulfate
UF
Value
Justification
Notes
UFa
10
A UFa of 10 is applied for interspecies extrapolation to account
for potential toxicokinetic and toxicodynamic differences
between rats and humans. No dosimetric adjustment factor
was utilized because a developmental endpoint was used for
derivation.
No information is available
regarding extrapolation from
extrapolation from animals to
humans.
ufd
1
A UFd of 1 is selected because there is an acceptable
two-generation reproduction study in rats (Hoechst
Aktiengesellschaft, 1984a) and multiple acceptable
developmental studies via the oral route (see Table 5) for the
endosulfan surrogate used in this assessment.
Developmental/Reproductive
studies are available to evaluate
these endpoints.
UFh
10
A UFh of 10 is applied for intraspecies differences to account
for potentially-susceptible individuals in the absence of
information on the variability of response to humans.
No information is for human
variability for exposure to this
compound.
ufl
1
A UFl of 1 is applied because the POD is developed using a
BMDL.
The use of benchmark dose analysis
at a justifiable response level of a
5% B W decrement supports the
value of 1 for the UFL.
UFS
1
A UFS of 1 is applied because further adjustment for exposure
duration is not warranted when developmental toxicity data are
used to develop a POD.
None.
UFC
100


BW = body weight.
The confidence descriptors for the screening subchronic p-RfD for endosulfan sulfate are
explained in Table A.4 below.
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Table A.4. Confidence Descriptors for Screening Subchronic p-RfD for Endosulfan
Sulfate
Confidence Categories
Designation"
Discussion
Confidence in study
H
Confidence in the principal study is high. The study of
Gilmore et al. (2006), although on endosulfan rather than
endosulfan sulfate, was adequate in design for a developmental
neurotoxicity study in rats. While the original study is not
available due to the inclusion of CBI, both a DER and a review of
the data by U.S. EPA (2010) provide sufficient review of the data.
The study is GLP compliant. Several studies report effects at
doses similar to those seen in the principal study providing
supporting evidence.
Confidence in database
H
The database (based on endosulfan) includes multiple subchronic -
duration studies in rats and mice (see Table 4), multiple
developmental studies in rats and rabbits (see Table 5), and a
two-generation reproductive study in the rat (Hoechst
Aktiengesellschaft, 1984a).
Confidence in screening
subchronic p-RfDb
H
The overall confidence in the screening subchronic p-RfD is high.
aL = low; M = medium; H = high.
bThe overall confidence cannot be greater than the lowest entry in the table.
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APPENDIX B. ADDITIONAL INFORMATION
ATSDR (2000) reported the following information regarding the biodegradation of
endosulfan in soil:
Endosulfan released to soil is most likely subjected to photolysis (on soil
surfaces), hydrolysis (under alkaline conditions), or biodegradation. Endosulfan
has been shown to be biodegraded by a wide variety of soil microorganisms in
numerous studies. Sixteen of 28 species offungi, 15 of 49 species of soil bacteria,
and 3 of 10 species of actinomycetes metabolized radiolabeled endosulfan in a
laboratory study under aerobic conditions (Martens 1976). Endosulfan sulfate
was the major product of the fungal metabolism, whereas the bacterial
transformation produced endosulfan diol. Degradation of endosulfan by soil fungi
and bacteria has also been reported (El Beit et al. 1981b). Biotransformation
occurs under both aerobic and anaerobic conditions. Aerobic incubation of soil
with endosulfan yielded mainly endosulfan sulfate (30-60%), some endosulfan
diol (2.6%), and endosulfan lactone (1.2%) (Martens 1977). Flooded (anaerobic)
incubation produced mainly endosulfan diol (2-18%), endosulfan sulfate
(3-8%), and endosulfan hydroxyether (2-4%). In aqueous nutrient media (20EC)
containing a mixed culture of microorganisms isolatedfrom a sandy loam soil,
endosulfan was reported to be transformed to endosulfan diol with half-lives of
about 1.1 and 2.2 weeks for the a- and ft-isomers, respectively (Miles andMoy
1979).
A two-membered bacterial coculture was found to aerobically degrade a- and
fi-endosulfan efficiently without accumulating any of its metabolites. However, the
degradation of soil-bound endosulfan was slower by 4-fold than in culture media;
only 50% of the material (initially at 50 ppm) was degraded in 4 weeks
(Awasthi et al. 1997). A field study report stated that endosulfan was transformed
to endosulfan sulfate following incorporation of 6.7 kg/hectare of the pesticide
into sandy loam soil (Stewart and Cairns 1974). The half-lives for the a- and
^-isomers were reported to be 60 and 800 days, respectively. Pseudomonad
microbes have been reported to isomerize ft-endosulfan to a-endosulfan and
biodegrade both isomers to endosulfan alcohol and endosulfan ether (U.S.
Department of Interior 1978). In a field study conductedfrom 1989 1990 in
northern India, dissipation of endosulfan in sandy loam soil was examined
(Kathpal et al. 1997). It was found that a-endosulfan could be detected up to 14
and 28 days in two different soil plots, while ft-endosulfan could be detected up to
70 and 238 days. An overall half-life for endosulfan degradation ranged from
39.5 to 42.1 days. Endosulfan residues dissipated to an extent of92-97% in the
first 4-week period of application and by about 99% in 238 days. A residue
half-life of 15 days for endosulfan (unspecified isomer) has been reported in
Australian black soil when incubated at 30 EC at field capacity moisture level
(Kathpal et al. 1997). Fate and movement of endosulfan isomers and endosulfan
sulfate under field application conditions have been studied (Antonious and Byers
1997). New modes of cultivation showed reduced runoff water and sediment loss
and reduced endosulfan movement from the site of application to the surface
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water runoff. Results indicated vertical movement of the pesticide through the
vadose zone at a concentration of 0.63 /ug/L. Soil core data shows endosulfan
leaches from 23 to 46 cm into the soil (Antonious and Byers 1997).
On plant surfaces, as in soils, numerous studies have demonstrated that
endosulfan is oxidized to endosulfan sulfate. Initial residues of endosulfan on
treated vegetables generally range from 1 to 100 mg/kg. However, residue levels
typically decrease to less than 20% of initial levels within 1 week after treatment
(NRCC 1975). Residues of endosulfan isomers are generally negligible after
2-3 weeks; the a-isomer is much less persistent than the ft-isomer. In most plant
residue studies, endosulfan sulfate residue levels tend to increase relative to the
parent isomers and other metabolites and appear to be very persistent (Coleman
and Dolinger 1982).
HUMAN STUDIES
Oral Exposures
Acute
Bernardelli and Gennari (1987)
Bernardelli and Gennari (1987, as summarized in ATSDR, 2000) described the case of a
55-year-old woman who died after taking endosulfan orally (amount unspecified) in a colorless
liquid containing 55% xylene. No gross anatomical or histological abnormalities were found.
The authors indicated that a malignant melanoma and the coexposure of xylene may have
contributed to her death.
Blanco-Coronado et al. (1992)
Blanco-Coronado et al. (1992, as summarized in ATSDR, 2000) reported a case of
poisoning in a woman who ingested an unknown amount of endosulfan in food. The woman
experienced tonic-clonic convulsions, nausea, vomiting, headache, and dizziness 1-4 hours after
eating the endosulfan-contaminated food. When admitted to the hospital, the endosulfan
concentrations (both isomers) in the stomach, blood, and urine were 55.4, 2.4, and 3 mg/L,
respectively. The patient suffered from renal failure, disseminated intravascular coagulation,
thrombi in the pulmonary arteries and aorta, and cardiogenic shock; she died 8 days later from
these complications. Postmortem examination revealed bilateral pleural effusions, congested and
edematous lungs, hyaline membranes, microatelectasia, polymorphonuclear lymphocytes, red
cells in the alveoli, and interstitial fibrosis.
Lo et al. (1995)
Lo et al. (1995, as summarized in ATSDR, 2000) presented the case of a man who
ingested an unknown amount of endosulfan and died 10 days later. The man suffered from
muscle fasciculations and episodes of convulsions. The authors indicated that the cause of death
was cardio-respiratory arrest/heart failure and pulmonary edema. The patient had an elevated
white blood cell count. Mucosal inflammation of the stomach and proximal small intestine,
centrilobular congestion of the liver, slight prominence of the bile canaliculi, and extensive
tubular necrosis of the kidney were noted in postmortem examinations.
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Shemesh et al. (1988)
Shemesh et al. (1988, summarized in ATSDR, 2000) described a case in which a
20-year-old man attempted suicide through the ingestion of 200 mL of Thionax
(30% endosulfan). He presented with respiratory effects including hypoxia due to alveolar
hypoventilation and pulmonary edema. Within the first 16 hours, he also experienced episodes
of tachycardia and hypertension followed by cardiogenic shock. These respiratory and
cardiovascular symptoms occurred even though his stomach was pumped and he was given
activated charcoal during the first 16 hours after exposure. During the 2 subsequent weeks, the
man experienced recurrent aspiration pneumonia and consistently required mechanical
ventilation. The patient also experienced recurrent convulsions during this period. A year after
the exposure, his mental activity (presumably his psychomotor activity) was still impaired, and
he took medicine to control his seizures. The authors stated that the respiratory effects were
likely secondary to the direct effects of endosulfan on the central nervous system (CNS) rather
than a direct action of the substance on the lungs. The authors were unsure whether the
endosulfan was directly responsible for the cardiovascular effects. It was unclear if other
ingredients in Thionax may have contributed to the man's symptoms.
Pradhan et al. (1997)
Pradhan et al. (1997, summarized in ATSDR, 2000) reported on a patient who ingested
around 75 mL of liquid endosulfan (35% w/v). The patient suffered from nausea, gagging,
vomiting, and diarrhea. The patient also had tonic-clonic seizures and myoclonic jerks,
psychosis, cortical blindness, and limb rigidity. Reversible lesions of the basal ganglia and
occipital cortex were apparent on magnetic resonance images.
Inhalation Exposures
Acute
Chugh et al. (1998)
In an occupational study, Chugh et al. (1998, summarized in ATSDR, 2000) reported on
18 cases of endosulfan poisoning between October 1995 and September 1997 in agricultural
workers in India who applied endosulfan to crops but did not use protective equipment to limit
dermal or inhalation exposure to the chemical. Exposed workers experienced gastrointestinal
symptoms including discomfort after meals, nausea, and vomiting. Neurological symptoms
included dizziness, confusion, irritability, muscle twitching, tonic-clonic convulsions, and
conduction defects. Respiratory effects included an increase in dyspnea and respiratory rate.
The authors also reported cardiovascular effects including tachycardia and bradycardia.
Singh et al. (1992)
Singh et al. (1992, summarized in ATSDR, 2000) reported on 22 workers who applied
endosulfan to cotton and rice fields and experienced gastrointestinal effects. The workers
suffered from nausea, vomiting, abdominal pain, and diarrhea. The authors reported that the
effects were most likely the result of dermal exposure to endosulfan because workers who
suffered cuts on the legs from the plants had more severe symptoms. Three of the 22 workers
exhibited tremors, and 11/22 experienced convulsions although all patients recovered from these
conditions.
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Aleksandrowicz (1979)
Aleksandrowicz (1979, as summarized in ATSDR, 2000) described a case of long-term
(possibly permanent) brain damage in an industrial worker occupationally exposed to endosulfan
while cleaning vats containing residues. Acute effects included repeated convulsions and
impaired consciousness; afterward, he was disoriented and agitated. The man showed cognitive
and emotional deterioration, impaired memory, and impaired visual-motor coordination 2 years
after the exposure. The authors noted that the man consumed 1 L of wine per week, which may
have contributed to the impairment and decreased endosulfan metabolism in the liver.
Short-term Studies
No studies were identified.
Long-term Studies
dare la-Rodr Iguez et al. (1996)
In an epidemiologic study, Garcia-Rodriguez et al. (1996, summarized in ATSDR, 2000)
examined the association between the geographic use of pesticides in relation to the homes of
children and incidence of cryptorchidism (undescended testes) in Granada, Spain.
Cryptorchidism incidence was ascertained from records of surgical correction for the disorder.
Exposure levels were not available in this study although ATSDR (2000) reported that other
studies indicated there was significant endosulfan exposure in the region. However, the authors
did not find any clear association between local pesticide use and incidence of cryptorchidism.
Roberts et al. (2007)
Using a retrospective case-control study design, Roberts et al. (2007) investigated the
association between maternal ambient pesticide exposure near agricultural fields and autism
spectrum disorders (ASDs) among children in the Central Valley of California. The study
population included 269,746 singleton births between January, 1, 1996, and December 31, 1998,
to mothers that lived in one of the 19 counties of the Sacramento River Valley and San Joaquin
River Valley (also known as the Central Valley).
Authors specifically investigated the risk of ASDs among children that were not born
prematurely (i.e., not born <37 weeks gestation or weighing <2,500 g). ASD cases were
ascertained using the California Department of Developmental Services (DDS); cases included
all children reported by DDS as receiving services for autism or who have an ASD diagnostic
code from the Diagnostic and Statistical Manual of Mental Disorders, 4th edition. DDS ran
regional centers (RCs) that provided voluntary services for people with autism, mental
retardation, and other developmental disabilities. DDS services were used by people of many
racial/ethnic groups and socioeconomic levels; however, there was some possibility of disparity
and lack of case identification. For example, children with milder forms of developmental
disabilities, such as Asperger's Syndrome, may not be eligible for services at the centers.
Staff of the California Center for Autism and Developmental Disabilities Research and
Epidemiology collected demographic information for the cases identified through DDS live birth
vital records (e.g., first name, last name, date of birth, sex). The control:case ratio was 15:1, with
control births randomly selected from the study population of full-term singleton births described
above. The DDS RC was used as a covariate; 6 centers served the 19 counties identified in the
study population. For controls, RCs were simulated based on the assumption that migration
during the first years of life would be the same for cases and controls.
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Pesticide data were obtained from the California Department of Pesticide Regulation
(DPR), which included agricultural pesticide applications from January 1995 to January 1999
(total number of applications: 6,710,727). Data are reported to DPR by county agriculture
commissioners and referenced to public land survey sections. The authors refined these data
through land-use survey field polygons data provided by the California Department of Water
Resources. Exposure determination was based on both spatial and temporal data. Residence
address at time of birth and last maternal menstrual cycle (to estimate gestation time) were
compared with temporal and spatial proximity to the pesticide applications reported to DPR.
The authors estimated the pounds of pesticides applied during temporal windows (Days 7-49
[CNS development], Days 4-24 [neural tube development], and Day 14-date of birth
[gestation]) within a specified radius of the mother's residence. The pesticide use was divided
into quartiles based on the estimated pounds of exposure. The authors selected pesticides for
inclusion based on plausibility of biological connection to autism, physical characteristics (e.g., a
widely used pesticide), and community concerns expressed through a series of meetings with
local governmental and nongovernmental organizations. A total of 249 combinations of
compounds, buffer radii, and temporal periods met the requirement of five exposed cases and
controls that were initially identified.
Authors used a conditional logistic regression model for analysis, controlling for maternal
race/ethnicity, education, and RC of diagnosis. Final analysis indicated that 465 ASD cases and
6,975 matched controls were retained in the final study population; 85.2% of cases were male
versus 51.4% of cases in controls. Results indicated that the fourth (highest) quartile of pesticide
exposure was statistically significantly associated with applications occurring during the CNS
development period (odds ratio [OR] = 4.2; 95% confidence interval [CI]: 1.7-10.9;p < 0.05).
All other neural tube, CNS, and gestation periods/exposure levels did not yield significant ORs.
The authors reported that organochlorine pesticides were associated with ASD regardless of the
size of the buffer radius between application site and residence; however, the effects were
smaller as the buffer radius increased (with the OR finally becoming nonsignificant with a buffer
around 1,750 m [data not reported]). The authors also reported that there was a significant OR of
7.6 (95% C.I.: 3.1-18.6;p < 0.05) for 26-81 days postfertilization in the fourth quartile of
pesticide application. The authors concluded that these 8 weeks represent the maximum
embryonic susceptibility to organochlorine pesticides.
In the study area, dicofol and endosulfan accounted for more than 98% of the
organochlorine pesticides applied. During the a posteriori time period (26-81 days
postfertilization), 88 subjects (cases and controls) lived within 500 m of a dicofol application,
and 27 lived within 500 m of an endosulfan application. Due to a small sample size, however,
authors could not calculate ORs specific to endosulfan. The authors indicated that the
magnitudes of association were slightly higher for endosulfan compared with dicofol (data not
reported).
An important strength of this study was its ability to estimate both space and timing of
pesticide application with relatively high confidence. The authors were also able to assess the
biological plausibility of certain compounds' ability to interfere with neurological development.
The DDS diagnosis system has also been used in prior studies and has proven to be a good
measure of autism. In some exposure categories, however, the number of cases was small (e.g.,
the fourth quartile of exposure had only 29 subjects, 8 of which had ASD). Misclassification of
exposure may have occurred because an estimated 1 in 3 mothers changed addresses during
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pregnancy. It was also not possible to separate the effects of individual pesticides, so no
definitive conclusions can be reached for the relationship between endosulfan exposure and
ASDs. Furthermore, authors were not able to address all confounders such as use of prenatal
vitamins, alcohol consumption, smoking, and familial history of cognitive disorders.
Saiyedetal. (2003)
Saiyed et al. (2003) conducted a study to analyze the relationship between endosulfan
exposure and reproductive development in male children and adolescents (10-19 years old).
Subjects included 117 male schoolchildren in a village near cashew plantations where endosulfan
had been aerially sprayed for more than 20 years and 90 comparable controls from another
village 20 km away without a history of endosulfan exposure.
The study collected clinical history and included a physical examination, assessment of
sexual maturity rating (SMR) according to Tanner's classification, serum levels of testosterone,
luteinizing hormone (LH), follicle-stimulating hormone (FSH), and endosulfan residues. Serum
samples were drawn at approximately the same time on examination day, centrifuged, separated,
stored, and analyzed with a gas chromatography-electron capture detector (GC-ECD) to quantify
endosulfan residues. Hormones were estimated by radioimmunoassay.
Descriptive statistics were measured for subjects and controls. Multiple regression
analysis was performed using age and aerial endosulfan exposure (AEE; AEE subjects = 1;
controls = 0) to endosulfan as independent variables while SMR and serum hormone levels
served as dependent variables. The multiple regression equation below was used to analyze
variance in dependent variables attributable to independent variables. SPSS (version 6.1.4) was
used for statistical analysis.
SMR score = bo + ^i(age) + b2 (AEE)
Where:
bo = regression constant
b\ = regression coefficient of age
62 = regression coefficient of exposure
The regression coefficients of age and exposure were fitted for SMR scores for pubic
hair, testes, and penis. Other multiple regression equations were fitted to testosterone, LH, and
FSH serum levels. Another multiple regression analysis was conducted to analyze the
relationship of serum testosterone versus age, AEE, and serum LH levels.
There were no significant differences in the descriptive statistics (age, height, weight,
Basal Metabolic Index [BMI], skin fold thickness) of participating subjects and controls and
nonparticipating subjects and controls (Saiyed et al., 2003). Six cases (5.1%; not statistically
significant) of congenital malformations were observed in the study group, including
undescended testis (2), congenital hydrocele (3), and congenital inguinal hernia (1). Table C.l
summarizes results of multiple regression analysis. The R (coefficient of determination) values
corresponding to SMR of pubic hair, testes, and penis were 0.48, 0.43, and 0.43, respectively
(p < 0.01). These values indicated that a significant proportion of variance in SMR scores could
be attributed to age and positive endosulfan exposure. The exposure (AEE) coefficient (b2) was
negative in all equations, indicating delayed sexual maturity associated with positive exposure to
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endosulfan. Positive age coefficients (b\) were also observed as age was expected to increase
with sexual maturity. Multiple regression analysis of serum testosterone levels resulted in an R2
value of 0.61, indicating 61% of the variation of testosterone levels was expected to be attributed
to age, AEE, and serum LH (p < 0.001). Regression of serum testosterone against age and serum
LH produced positive coefficients for age and serum LH levels. This confirmed that age and
serum LH increased, as expected, with serum testosterone levels. The negative AEE coefficient
of-0.62 indicated that testosterone levels in exposed individuals were statistically lower than
expected by age and LH levels. Endosulfan was detected in serum of 78% of the
endosulfan-exposed study group and 29% of the control group participants. Table C.2
summarizes the mean serum endosulfan levels for exposed and control groups. A significant
(p < 0.001) increase in serum endosulfan residues in endosulfan-exposed study males was
observed when compared with the control group. The study authors concluded that endosulfan
exposure may delay sexual maturity and affect hormone synthesis and that a larger sample study
and a long-term follow up should be conducted to validate these findings.
While SMR study nonparticipation rates were high (57% for exposed, 33% for control),
growth-related descriptive statistics for participants and nonparticipants were comparable (see
Table C.3). The study authors stated that random variability in hormone levels would have
increased exposure misclassification (testosterone levels) and decreased the power of the study
by biasing the results towards the null. To minimize the effect of diurnal variation in hormone
secretion and, thus, hormone serum levels, all blood samples were collected within the same
2-hour window, 1,000-1,200 hours.
A Critique noting deficiencies in the study design have been reported (Indulkar, 2004).
According to the critiques, Saiyed et al. (2003) incorrectly stated that endosulfan was the only
pesticide sprayed for decades when in fact other pesticides were also used in exposed and control
study areas. In addition, it was noted that the SMR and hormone level data displayed a poor
correlation with age, the sample size was too small, and normal biological SMR and hormone
ranges were not reported in the study for reference.
Chronic-duration Studies
Aschengrau et al. (1998)
In a population-based case-control study, Aschengrau et al. (1998) studied the association
between breast cancer incidence in females and exposure to suspected estrogenic chemicals,
including endosulfan. Incident cases were Cape Cod, Massachusetts permanent residents from
five towns who were diagnosed with breast cancer between 1983 and 1986 and registered in the
Massachusetts Cancer Registry. Controls were also permanent residents of similar age and race
from the same population. Random digit dialing, Medicare beneficiary lists, and death
certificates were used to identify controls. Controls were gathered for a larger study of nine
cancers. The controls for the breast cancer study were selected from this pool by stratification of
breast cancer cases by age, gender, vital status, and, if applicable, year of death. Controls were
then selected if they fell into a stratum with one or more cases.
Exposure was assessed for each case or control in a stepwise process (Aschengrau et al.,
1998). Exposure for each job held by a study subject was determined using the National Institute
for Occupational Safety and Health National Occupational Exposure Survey (NIOSH/NOES)
database, chemical production and usage information, and the expert judgment of a certified
industrial hygienist. Exposure was assessed for 18 substances showing estrogenic activity,
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including a- and P- endosulfan, by cross-referencing job types held by women in the study with
information from the NIOSH/NOES database. An extensive database and literature search on
the production and usage of the 18 suspected xenoestrogens were used to identify jobs in which
women were not likely to have been exposed. Occupational exposure estimates were assigned a
level of confidence (probable or possible) by an industrial hygienist based on interview
information on specific job duties.
Subjects were categorized as having one or more jobs with probable or possible exposure
to suspected xenoestrogens overall as well as to specific chemicals such as endosulfan
(Aschengrau et al., 1998). ORs for probable exposure were calculated to assess the relative risk
of breast cancer by exposure categories. Crude OR 95% confidence intervals (CIs) were
calculated (Miettinen's test, >5 exposed cases; Fisher's exact method, <5 exposed cases).
Multivariate logistic regression models were used to calculate ORs adjusted for confounders, and
95% CIs were calculated using the maximum likelihood estimate of standard errors. The authors
noted that the number of subjects evaluated was insufficient to examine by exposure duration.
Only a few subjects were occupationally exposed to endosulfan plus other xenoestrogens
(3 cases, 7 controls), and no cases or controls were exposed to only endosulfan
(Aschengrau et al., 1998). The crude OR for endosulfan plus other suspected xenoestrogens was
1.3 (95% CI: 0.2-1.2). The adjusted OR was 0.8 (95% CI: 0.2-3.2) after adjusting for core
confounders (age at diagnosis or index year, vital status at interview, family history of breast
cancer, age at first birth, personal history of breast cancer, benign breast disease) and education
level. Given the small study size for endosulfan (3 exposed) and exposure to multiple
compounds, it is difficult to draw any conclusions about endosulfan from the findings of this
study.
OTHER DATA (SHORT-TERM TESTS, OTHER EXAMINATIONS)
The genotoxicity of endosulfan has been studied in bacteria, yeast, mammalian cells in
culture, and in laboratory animals (see Table B. 1). Selected data on the acute toxicity,
toxicokinetics, and mode of action of endosulfan are present. As shown in Table B. 1, numerous
genotoxicity studies have been conducted in bacteria, yeast, mammalian cells, and in laboratory
animals. These data are inconclusive as both positive and negative results are seen. In addition,
it is possible that some of these studies used formulations of endosulfan that may have contained
epichlorohydrin, a known genotoxic compound, as a stabilizer (ATSDR, 2000). U.S. EPA
(2010) noted that chronic animal bioassays in rats and mice provided no evidence that
endosulfan is carcinogenic and concluded that endosulfan is neither mutagenic nor carcinogenic.
The limited information available on endosulfan sulfate (Bajpayee et al., 2006) also indicates
that this metabolite is neither mutagenic nor carcinogenic. Based on the WOE, it is concluded
that endosulfan is not a genotoxic compound. Because of the similarities in structure and
chemical characteristics between endosulfan and endosulfan sulfate, it is also concluded that
endosulfan sulfate is not a genotoxic compound.
Short-term Studies
Dorough et al. (1978) conducted an acute toxicity test on female albino mice; however,
the discussion of this experiment, including a description of the mice (such as weight gain), was
limited, making the study methodology difficult to ascertain (see Table B.2). For endosulfan
isomers and metabolites, authors reported dosing female mice with initial concentrations of
120 mg/kg, which were then adjusted throughout the study. Results indicated that the acute
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toxicity of endosulfan sulfate (LD50 = 8 mg/kg) was similar to the toxicity of a-endosulfan
(LD50 = 11 mg/kg).
Metabolism/Toxicokinetic Studies
Table B.2 reports data concerning the kinetics of endosulfan. Endosulfan was absorbed
following exposure, with the highest levels of accumulation taking place in the liver and kidneys
(Dorough et al., 1978). Endosulfan and endosulfan sulfate have also been detected in human
placenta, umbilical cord serum, and breast milk, indicating the likelihood of the compounds to be
passed from mother to fetus and/or child (Campoy et al., 2001; Cerillo et al., 2005). Figure B. 1
shows the metabolic pathway for endosulfan. Endosulfan is readily metabolized to endosulfan
sulfate and endosulfan diol and then further metabolized to endosulfan lactone either directly
from the sulfate or indirectly via the corresponding ether and hydroxyether from endosulfan diol
(ATSDR, 2000). Studies have demonstrated that elimination of endosulfan and its metabolites
occurs via renal and biliary excretion (Dorough et al., 1978; Wilson and Leblanc, 1998; ATSDR,
2000). In addition, endosulfan is eliminated via breast milk in lactating women (Campoy et al.,
2001; Cerillo et al., 2005).
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S=0
CI
Endosulfan
CI
Ck
CI-C-CI
CI
:so„
ci
Endosulfan sulfate
C
CI H2
Endosulfan lactone
CI
CI
CI'
,ch2oh
ch2oh
CI
Endosulfan diol
CI " 2
Endosulfan hydroxyether
C
CI H*
Endosulfan ether
Figure B.l. Proposed Metabolic Pathway for Endosulfan
Source: ATSDR (2000)
Mode of Action/Mechanistic Studies
ATSDR (2000) summarizes the available evidence on the mode of action for endosulfan.
Table B.2 also summarizes these studies, which indicate that the neurotoxicity induced by
endosulfan involves a GABA-antagonism mechanism of toxicity via binding of endosulfan at
multiple receptors in neurons and inhibiting the GABAergic function. While no data on the
immunotoxic mode of action for endosulfan were identified, a close dynamic relationship exists
between the neurological and immunological systems (Banerjee and Hussain, 1987). Therefore,
it is possible that different modes of action exist for the neurotoxicity and immunotoxicity of
endosulfan and that they are linked.
Limited data exist concerning the mode of action for the developmental effects caused by
endosulfan. Wilson and LeBlanc (1998) reported an increased testosterone biotransformation in
male and female mice fed endosulfan for 7 days. An in vitro study with human sperm indicated
that at 1 nM, endosulfan inhibited the acrosome reaction (AR) initiated by progesterone
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(ATSDR, 2000). Because chloride channels activated by GABA are involved in the AR
(ATSDR, 2000), it is possible that the modes of action for the neurological, immunological, and
reproductive effects of endosulfan are linked.
A full evaluation of these data is provided in ATSDR (2000).
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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
Genotoxicity studies in prokaryotic organisms
Reverse
mutation
Salmonella typhimurium strains
TA97a, TA98, TA100, TA102,
TA104, TA1535, TA1537,
TA1538, and TA1978 with and
without S9 activation
3,256 mg/L
(Pednekar et al.,
1987)
5,000 (ig/plate
(Shirasu et al.,
1978; Moriya et
al., 1983)


Endosulfan was not found to be
mutagenic with or without metabolic
activation; cell growth was inhibited
by 90% at 1,650-3,256 mg/L among
the different strains (Pednekar et al.,
1987)
Pednekar et al. (1987),
Moriya et al. (1983),
Doroughetal. (1978),
Shirasu et al. (1978),
Quinto et al. (1981),
Adams (1978), and
Shirasu et al. (1982), as
reported in Cal/EPA
(2008)

S. typhimurium strain TA98 with
and without S9 activation;
commercial endosulfan (7:3 o:\\
isomers), a-endosulfan,
(^-endosulfan. endosulfan diol,
endosulfan ether, endosulfan
hydroxyether, endosulfan lactone,
and endosulfan sulfate were tested;
S. typhimurium strains TA97a,
TA100, TA102, and TA104 also
were tested
1 (ig/plate
+ (all
compounds
were tested
excluding
commercial
endosulfan)
+ (all
compounds
were tested
excluding
commercial
endosulfan)
Reverse mutations were increased in
TA98 for all compounds except for the
commercial endosulfan formulation
(7:3 a:(3 isomers); more revertants
were observed in TA98 than in any
other strain tested; Bajpayee et al.
(2006) suggested an interaction
between the isomers could have
inhibited the induction of a frame-shift
mutation in TA98
Bajpayee et al. (2006)

S. typhimurium strains TA97a,
TA100, and TA102 with and
without S9 activation; commercial
endosulfan (7:3 a:(3 isomers),
a-endosulfan, (3-endosulfan,
endosulfan diol, endosulfan ether,
endosulfan hydroxyether,
endosulfan lactone, and endosulfan
sulfate were tested; S. typhimurium
strains TA98 and TA104 also were
tested
DU
±
±
Reverse mutations were increased in
TA97a, TA100, and TA102 with and
without S9 activation; the increases
were not concentration-dependent and
are, thus, equivocal
Bajpayee et al. (2006)
58
Endosulfan sulfate

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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
Reverse
mutation
S. typhimurium strain TA104 with
and without S9 activation;
commercial endosulfan (7:3 a:(3
isomers), a-endosulfan,
fi-c ndosul fan. endosulfan diol,
endosulfan either, endosulfan
hydroxyether, endosulfan lactone,
and endosulfan sulfate were tested;
S. typhimurium strains TA97a,
TA98, TA100, and TA102 also
were tested
20 (ig/plate


No increase in reverse mutations
relative to controls in strain TA104
caused by treatment with any of the
compounds tested
Bajpayee et al. (2006)

Escherichia coli K12 strainAB1157
(repair proficient) was treated with
various concentrations of
endosulfan with and without
ampicillin
10 iig/niL
+
NR
Mutation index increased with dose to
a maximum of 14.4
Chaudhuri et al. (1999)

E. coli (strain not specified)
NR

NR
Results reported by Cal/EPA (2008) in
summary table only
Fahrig (1974), as
reported in Cal/EPA
(2008)

E. coli WP2 her
NR

NR
Concentration-specific data have not
been provided for endosulfan although
Moriya et al. (1983) reported testing
up to a maximum concentration of
500 (ig/plate for all pesticides
examined
Moriya et al. (1983)
SOS repair
induction
E. coli WP2 prophage 1 induction
assay
150 (ig/mL
+
NR
Endosulfan induced prophage 1 with
maximum induction of 3.5-fold higher
than spontaneous induction
Chaudhuri et al. (1999)
59
Endosulfan sulfate

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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
SOS repair
induction
S. typhimurium TA1535/pSK 1002
induction of iiiriii
150 (ig/mL
+
NR
Endosulfan induced umu gene
expression with maximum induction
of 4.2-fold higher than spontaneous
induction
Chaudhuri et al. (1999)
Genotoxicity studies in nonmammalian eukaryotic organisms
Mutation
Saccharomyces cerevisiae strain D7
without metabolic activation; cells
treated with 1% endosulfan
dissolved in acetone at exposure
times of 10, 20, and 30 min; treated
colonies compared with 10% (v/v)
acetone controls
1% (w/v)
+
NR
Endosulfan induced reverse mutations;
these effects increased with exposure
time
Yadav et al. (1982)

S. cerevisiae strain D4; gene
conversion assay at the Ade 2 and
Trp 5 loci, treated for 4 hr
5,000 ng/mL


Cal/EPA (2008) reported that there
was no treatment-related increase in
gene conversion when compared with
controls; Cal/EPA (2008) stated the
study was acceptable under Federal
Insecticide, Fungicide, and
Rodenticide Act (FIFRA) Guidelines
Milone and Hirsch
(1984), as summarized in
Cal/EPA (2008)

S. cerevisiae Tl/PG-154,
T2/PG-155
10 (ig/mL
+
NR
Results were reported by Cal/EPA
(2008) in summary table only;
therefore, it is unknown at which
doses a significant effect occurred;
Cal/EPA (2008) stated the study was
not acceptable under FIFRA
Guidelines
L'vova (1984), as
reported in Cal/EPA
(2008)

Schizosaccharomyces pombe
haploid 4-hr exposure
500 ng/L


Results reported by Cal/EPA (2008) in
summary table only; Cal/EPA (2008)
stated the study was not acceptable
under FIFRA Guidelines
Mellano (1984), as
reported in Cal/EPA
(2008)
60
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Table B.l. Summary of Endosulfan Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Mutation
Drosophila melanogaster fed
endosulfan dissolved in dimethyl
sulfoxide (DMSO) and diluted with
5% sucrose solution; induction of
sex-linked recessive lethals
(SLRLs) measured in males
exposed as larvae (at 50 or
100 ppm) and in adult male germ
cells (3 broods) exposed for 48 hr
(at 150 or 200 ppm)
100 ppm (larvae)
200 ppm (adult)
+
NR
Significant increases in SLRLs in
larvae at 100 ppm and in the combined
data for the 3 adult male broods at
200 ppm; data suggested a
dose-response induction of SLRLs
Velazquez et al. (1984)
Recombination
induction
S. cerevisiae strain D7 without
metabolic activation; cells treated
with 1% endosulfan dissolved in
acetone at exposure times of 10, 20,
and 30 min; treated colonies
compared with 10% (v/v) acetone
controls
1% (w/v)

NR
Endosulfan did not induce mitotic
cross-over
Yadav et al. (1982)
S. cerevisiae strain D7 without
metabolic activation; cells treated
with 1% endosulfan dissolved in
acetone at exposure times of 10, 20,
and 30 min; treated colonies
compared with 10% (v/v) acetone
controls
1% (w/v)
+
NR
Endosulfan induced mitotic gene
conversion; effects increased with
exposure time
Yadav et al. (1982)
Chromosomal
aberration
S. cerevisiae strain D7 without
metabolic activation; cells treated
with 1% endosulfan dissolved in
acetone at exposure times of 10, 20,
and 30 min; treated colonies
compared with 10% (v/v) acetone
controls
1% (w/v)
+
NR
Endosulfan increased the percentage
of aberrant colonies that formed at the
ade 2 locus; effects increased with
exposure time
Yadav et al. (1982)
61
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Table B.l. Summary of Endosulfan Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Chromosomal
malsegregation
ND
ND
ND
ND
NA
NA
Mitotic arrest
ND
ND
ND
ND
NA
NA
Sex
chromosome
loss
Induction of sex chromosome loss
in germ cells (3 broods) of adult
males exposed for 24 hr
50 ppm
+
NDr
Significant increases in frequency of
sex chromosome loss resulted in
exceptional offspring from the germ
cells of the adult male broods when
data from all three broods combined
Velazquez et al. (1984)
Genotoxicity studies in mammalian cells—in vitro
Mutation
L5178Y Ik Ik mouse lymphoma
cell forward mutation assay; treated
for 4 hr without rat S9 metabolic
activation
25 (ig/mL
+
NR
In the first test, the lowest observed
effective dose was 18.6 |ig/mL without
metabolic activation; 25 |ig/mL
reduced the relative total growth to
5%, and there was a 21-fold increase
in mutant fraction relative to controls;
in the second test, there was moderate
toxicity, and mutagenic responses
were 2- and 4-fold above controls
McGregor (1988)
Chromosomal
aberrations
ND
ND
ND
ND
NA
NA
Sister chromatid
exchange (SCE)
Human lymphoid cells, LAZ-007
cell line, incubated with
10 6—10 4 M endosulfan for 48 hr
without rat S9 metabolic activation,
and for 1 hr with or without
metabolic activation
10~6 M (48 hr)
+
NR
Significant increase in SCE frequency
in cells exposed to 10 6-10 4M
endosulfan without metabolic
activation for 48 hr; no significant
difference was observed for cells
exposed with or without metabolic
activation for 1 hr
Sobtietal. (1983)
10~4M(1 hr)


62
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Table B.l. Summary of Endosulfan Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
Sister chromatid
exchange (SCE)
Human HepG2 cells were treated
with
1 x 10 12—1 x 10 5IVT a-cndosulfan
or
l x ict5m
(a-endosulfan)

NR
Significant increase in SCE observed
for (^-endosulfan at 1 x 10 -1 x 10 5
M; a nonsignificant increase in SCE
was observed for a-endosulfan
Lu et al. (2000)
(^-Endosulfan for 48 hr and
examined for SCE using single-cell
gel electrophoresis (SCG) assays
1 x l(T7M
((^-endosulfan)
+


DNA damage
Human HepG2 cells treated with
2 x 10 5—1 x 10 3 M a-endosulfan
or (^-endosulfan for 1 hr and
examined for DNA strand breaks
using SCG assays
2 x 1(T4M
(a-endosulfan)
+
NR
Significant increase in DNA strand
breaks observed for a-endosulfan at
2 x 10 4-l x 10~3M andfor
(^-endosulfan at 1 x 10 3 M
Lu et al. (2000)
1 x l(T3M
((^-endosulfan)
+
Chinese Hamster ovary (CHO) cells
using the alkaline Comet assay with
endosulfan, a-or (^-endosulfan.
endosulfan diol, endosulfan ether,
endosulfan hydroxyether,
endosulfan lactone, or endosulfan
sulfate, as well as positive and
negative controls
0.25 nM
+
NR
Significant increase in olive tail
movement (OTM; the product of the
distance of DNA migration from the
body of nuclear core and the total
fraction of DNA in the tail) produced
by all compounds tested at
0.25-10.0 ^M, except for
(^-endosulfan and endosulfan parent
compound, which were significant at
>1.0 |iIVI; all compounds tested had
significant concentration-dependent
increase in % tail DNA at
0.25-10 |iIVI. except for endosulfan,
which was significantly increased
>1.0 |iM; a-endosulfan and endosulfan
lactone produced the greatest amount
of damage, and the isomeric mixture
(parent compound) produced the least
Bajpayee et al. (2006)
63
Endosulfan sulfate

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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
DNA damage
Human lymphocyte cells (from
single male donor) using the Comet
assay
0.25 nM
+
NR
All test compounds produced
significant concentration-dependent
increase in OTM and % tail DNA at
>0.25 |iIVI. except for a-endosulfan,
which was significantly increased at
>1.0 |iM
Bajpayee et al. (2006)

Human lymphocytes
100 iig/mL

NR
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred; Cal/EPA (2008) stated
the study was not acceptable under
FIFRA Guidelines
Shirasu et al. (1978), as
reported in Cal/EPA
(2008)

NMRI mice (5/sex/dose); after 6 hr,
bone marrow removed and assessed
for induction of micronuclei
5.0 mg/kg

NR
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred; Cal/EPA (2008) stated
the study was not acceptable under
FIFRA Guidelines
Cifone (1983), as
reported in Cal/EPA
(2008)

Rat bone marrow and
spermatogonia; rats administered
treatment by gavage for 5 d
11 mg/kg

NR
Results reported by Cal/EPA (2008) in
summary table only; only one dose
tested; Cal/EPA (2008) stated the
study was not acceptable under FIFRA
Guidelines
Dikshith and Datta
(1978), as reported in
Cal/EPA (2008)

F344 male rat primary hepatocytes,
autoradiographic unscheduled DNA
synthesis (UDS) assay; 3
cultures/dose and 50 cells/culture
were analyzed
51.0 ng/mL

NR
Cal/EPA (2008) reported that there
was no UDS observed at any
concentration tested, but there was
toxicity observed at 51.0 (ig/mL;
Cal/EPA (2008) stated the study was
acceptable under FIFRA Guidelines
Hoechst
Aktiengesellschaft
(1984b), as reported in
Cal/EPA (2008) and
ATSDR (2000)
64
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Table B.l. Summary of Endosulfan Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation
DNA damage
Human lung carcinoma (A 549
cells) UDS assayed using liquid
scintillation counting
NR
±
±
ATSDR (2000) concluded the study
was inconclusive because the author
did not present any evidence that DNA
synthesis was inhibited, and there were
high background levels of DNA
synthesis
Hoechst Celanese
Corporation (1988), as
reported in ATSDR
(2000)
DNA adducts
Cultured fetal rat liver, fetal quail
liver, and human liver
hepatoblastoma (Hep G2) cells
incubated for 72 hr with 50 |iIVI
endosulfan prepared in DMSO
(<0.1% v/v medium) and 10 6 M
dexamethasone (to maintain
cytochrome P450 expression and
promote cell survival); DNA-adduct
formation measured using the
32P-postlabeling method; mRNA
extracted and hybridized (Northern
blot) to cDNA probes coding for
human CYP1 Al, CYP2E, and
CYP3A4 and rat CYP1A1,
CYP2B1, and CYP3A1 as well as
human glyceraldehydes
3-phosphate dehydrogenase
(GADPH); real-time PCR of Hep
G2 cell mRNA for expression of
human CYP3A7
50 nM
+
NR
DNA adducts formed in rat and human
hepatic cells, likely by selectively
inducing expression of CYP3 A family
enzymes (CYP3 Al mRNA in rat liver
cells and CYP3 A7 mRNA in Hep G2
human cells); no DNA adducts were
observed in quail hepatocytes
Dubois et al. (1996)
Frequency of
micronuclei
Human HepG2 cells treated with
1 x 10 —1 x 10 3IVT a-cndosulfan
or (^-endosulfan for 48 hr and
examined for increased frequency
of micronuclei using SCG assays
1 x 10~3M
(a-endosulfan)
—
NR
Significant increase in frequency of
micronuclei observed for (S-endosulfan
at 5 x 10 5—1 x 10 3 M; nonsignificant
increase in frequency of micronuclei
observed for a-endosulfan
Lu et al. (2000)
5 x 10~5M
((^-endosulfan)
+
65
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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
Genotoxicity studies in mammals—in vivo
Chromosomal
aberrations and
DNA damage
0/6, Syrian hamsters, single
intraperitoneal injection of a
commercial insecticide containing
35% endosulfan at 8, 16, 40, or
80 mg/kg-BW; recorded number of
chromosomal aberrations induced
in bone marrow cells and compared
with negative controls (no
treatment) and positive controls
(treated with 40 mg/kg-BW
cyclophosphamide)
8 mg/kg-BW
+
NA
Significant increase in the number of
aberrations observed at all doses tested
Dzwonkowska and
Hiibner (1986)

Mouse bone marrow
0.2, 1.0,
5.0 mg/kg
+
NA
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred
Kurinnyi et al. (1982), as
reported in Cal/EPA
(2008)

Mouse bone marrow
1.75, 3.5,
5.25 mg/kg
+
NA
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred
Sharma and Guatam
(1991), as reported in
Cal/EPA (2008)

Mouse bone marrow
1.0, 10 mg/kg
+
NA
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred
L'vova (1984), as
reported in Cal/EPA
(2008)
Chromosomal
aberrations and
DNA damage
Alkaline Comet assay of DNA
damage following occupational
application of pesticide mixture
(including endosulfan) compared
with DNA levels prior to
application
NR
+
NA
DNA damage in mononuclear
leukocytes increased in 2 of 4 workers
(pesticide mixtures)
Lebailly et al. (1998), as
reported in ATSDR
(2000)
66
Endosulfan sulfate

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Table B.l. Summary of Endosulfan Genotoxicity



Resultsb


Endpoint
Test System
Dose
Concentration"
Without
Activation
With
Activation
Comments
References
Chromosomal
aberrations and
DNA damage
Cytochalasin-B method of arresting
cytokinesis assessment of the
frequency of micronuclei in
peripheral blood lymphocytes of
Chilean pesticide sprayers
NR

NA
Endosulfan reportedly applied by
workers only 3.7% of the time
(pesticide mixtures)
Venegas et al. (1998), as
reported in ATSDR
(2000)

5 -bromodeoxyuridine
DNA-labeling technique assessment
of frequency of micronuclei in
Italian greenhouse workers
NR
+
NA
Exposed to mixtures
Falck et al. (1999), as
reported in ATSDR
(2000)

Cytochalasin-B method of arresting
cytokinesis assessment of the
frequency of micronuclei in
peripheral blood lymphocytes
assessment of occupational
exposure
NR

NA
Exposed to mixtures
Scarpato et al. (1996a,b)
and Scarpato et al.
(1997), as reported in
ATSDR (2000)
Sister chromatid
exchange (SCE)
ND
DNA adducts
ND
Mouse
biochemical or
visible specific
locus test
ND
Dominant lethal
Swiss albino mice
9.8, 12.7, 16.6,
21.6 mg/kg
+
NA
Results reported by Cal/EPA (2008) in
summary table only; therefore, it is
unknown at which doses a significant
effect occurred; Cal/EPA (2008) stated
the study was acceptable under FIFRA
Guidelines
Milone and Hirsch,
(1986), as reported in
Cal/EPA (2008)
67
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Table B.l. Summary of Endosulfan Genotoxicity
Endpoint
Test System
Dose
Concentration"
Resultsb
Comments
References
Without
Activation
With
Activation

Albino mice, intraperitoneal
injection
5, 10 mg/kg

NA
Results reported by Cal/EPA (2008) in
summary table only; Cal/EPA (2008)
stated the study was not acceptable
under FIFRA Guidelines
Arnold (1972), as
reported in Cal/EPA
(2008)
Genotoxicity studies in subcellular systems
DNA binding
ND
aLowest effective dose for positive results; highest dose tested for negative results.
b+ = positive; ± = equivocal or weakly positive; - = negative; T = cytotoxicity; DU = data unsuitable; NA = not applicable; NV = not available; ND = no data; ND = not
determinable; NI = not identified; NP = not provided; NR = not reported; NR/Dr = not reported but determined from data; NS = not selected.
68
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Acute
Female albino mice were dosed orally with a-
or (3- endosulfan isomers or endosulfan
metabolites in a 1:1 mixture of Tween80 and
water. An initial dose of 120 mg/kg was
administered and then increased or decreased
according to the up-and-down method.
LD50 (mg/kg) values were as follows:
a-endosulfan =11
(^-endosulfan = 36
endosulfan sulfate = 8
endosulfan hydroxyl ether = 120
endosulfan lactone = 120
endosulfan ether = 270
endosulfan diol = >2,000
a-Endosulfan and endosulfan
sulfate were the most acutely
toxic.
Dorough et al.
(1978)
Metabolism/
toxicokinetic
In an occupational exposure to endosulfan, a
worker applied 300 L of 0.7 g/L endosulfan in
a greenhouse. 10 urine samples were taken for
3 d following exposure, and urine extracts were
analyzed using gas chromatography tandem
mass spectrometry (GC-MS/MS) to identify a-
and (^-endosulfan and endosulfan metabolites.
The peak endosulfan concentration in the urine of
5,368 pg/mL was reached 0.2 d after exposure and
concentration decreased to near-control levels
after 1.5 d (2,239-2,535 pg/mL). The half lives of
a-endosulfan and (3-endosulfan were 1.35 and 1.67
d, respectively, by first-order kinetics.
a-Endosulfan was excreted
more quickly than (^-endosulfan.
Both were excreted via first
order kinetics.
Arrebola et al.
(1999), as reported
in Cal/EPA (2008)

In an occupational exposure to endosulfan,
workers applied endosulfan for 2-5 hr/d
without protective equipment or clothing,
during either the day or week prior to providing
urine samples. Urine samples were analyzed
for endosulfan and metabolites using
GC-MS/MS (Vidal et al., 1997). The amounts
of endosulfan applied were not reported.
In workers applying endosulfan during the week
prior to providing urine samples, 4/5 of the
workers' urine contained a-endosulfan
(84-123 pg/mL), (S-endosulfan (<18-169 pg/mL),
endosulfan sulfate (amount not reported), and
endosulfan lactone (amount not reported). In
workers applying endosulfan during the day prior
to providing urine samples, 4/4 of the workers'
urine contained a-endosulfan (787-894 pg/mL),
(^-endosulfan (801-896 pg/mL), endosulfan
sulfate (amount not reported), and endosulfan
lactone (amount not reported).
Although the amounts applied
were not reported, workers
exposed to endosulfan in the
previous day had greater
amounts of endosulfan and
endosulfan metabolites in their
urine than those who were
exposed 1 wk earlier.
Vidal etal. (1998),
Vidal (1997), as
reported in Cal/EPA
(2008) and ATSDR
(2000)

3 fatal human poisoning cases; blood and
tissues were analyzed for combined a- and
(^-endosulfan concentration using
gas-chromatography-electron capture detection
(GC-ECD). The amounts ingested were not
reported.
Blood endosulfan concentrations ranged from
0.4-0.8 mg/100 mL blood. Liver, kidney, and
brain concentrations ranged from 0.08-0.14,
0.24-0.32, and 0.025-0.03 mg/100 g tissue,
respectively.
Highest levels of endosulfan
were found in the kidney and
blood. The amount ingested
was not reported.
Coutselinis et al.
(1978); Coutselinis
et al. (1976), as
reviewed in ATSDR
(2000)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
Women environmentally exposed to
contaminants in rural Kazakhstan; the levels of
endosulfan in breast milk were measured.
Two out of 19 breast milk samples had detectable
endosulfan, but the specific concentrations were
not reported.
Endosulfan is detectable in
human breast milk, indicating
that infants of exposed mothers
may be exposed through breast
milk.
Lutter et al. (1998),
as reported in
ATSDR (2000)

Male albino rats were administered 2.5 or
7.5 mg/kg/d of a 2:1 a-:(3-endosulfan mixture
orally for 60 d. a- and (^-endosulfan
concentrations were measured in the testis,
epididymis, seminal vesicles, ventral prostate,
liver, brain, kidney, spleen, lung, and heart
using gas-liquid chromatography coupled with
an electron capture detector.
a-Endosulfan was measured at the highest
concentration in the kidneys (574 and 1,655 ng/g,
respectively, in the 2.5- and 7.5-mg/kg-d groups).
(^-Endosulfan was measured at the highest
concentration in the seminal vesicle (960 and
1,344 ng/g, respectively, in the 2.5- and
7.5-mg/kg-d groups). Combined a- and
(^-endosulfan was greatest in the seminal vesicle
and kidney at 1,008 and 587 ng/g, respectively, in
the 2.5 mg/kg-d group. The kidney had the
greatest concentration of combined endosulfan
isomers in the 7.5 mg/kg-d group with 1,676 ng/g,
followed by the seminal vesicle at 1,434 ng/g.
The concentration of (^-endosulfan was higher
than a-endosulfan in the seminal vesicle,
epididymis, heart, and liver in both dose groups.
a- and (^-endosulfan distributed
to the greatest extent into the
kidneys and seminal vesicle.
There were some differences in
the relative distribution of a-
and (3-endosulfan between the
two dose levels, indicating a
different pattern of distribution
of the two isomers.
Ansari et al. (1984)
70
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
Female albino rats (number not specified)
administered a single gavage dose of
approximately 2 mg/kg [14C]-labeled
endosulfan in corn oil, and then feces and urine
were collected.
5 d after dosing, 88% and 87% of the
[14C]-labeled a- and (S-endosulfan administered
via gavage was eliminated in the urine (13-19%)
and feces (68-75%).
5 d after dosing via gavage, kidney and liver
tissues showed 0.35 and 1.66 ppm of
[14C] -endosulfan residue, respectively, after
treatment with a-endosulfan, and 0.22 and
1.13 ppm, respectively, after treatment with
(^-endosulfan (both were equal to a combined
1.5% of the gavage dose). Animals receiving
endosulfan in the diet had the greatest distribution
of [14C]-endosulfan residues in the kidney and
liver, where endosulfan accumulated but had a
half-life of about 7 d.
a- and (^-endosulfan
administered via gavage were
mainly eliminated through the
feces and urine.
Dorough et al.
(1978)

Male rats (number not specified) had a cannula
surgically implanted in their bile ducts and then
received a single oral dose of 1.2 mg/kg of
[14C]-labeled a- or (3-endosulfan.
47% and 29% of [14C]-labeled a- and
(^-endosulfan administered via gavage,
respectively, was collected in the bile via the
implanted cannula after 48 hr.
Collecting the bile decreased
elimination in the feces but did
not alter urinary excretion of a-
and (3- endosulfan, suggesting
that metabolites were excreted
from the liver into the intestine
without allowing for resorption
and elimination by the kidney.
Dorough et al.
(1978)

Female albino rats (number not specified) were
fed 5 ppm of either a- or (}-|MC| endosulfan for
up to 14 d, 25 ppm of a-[14C]endosulfan for
14 d, or 25 ppm of 7:3 mixture of
a-:P-[14C]endosulfanfor 14 d. Urine and feces
were collected daily. The kidney, liver,
visceral fat, subcutaneous fat, muscle, and brain
were removed and analyzed for [14C] residue
content.
Animals receiving endosulfan in the diet
eliminated 61-65% of the dose after 14 d of
feeding (56-57% in the feces and 7-9% in the
urine). Afterwards, the animals eliminated an
additional 8% during the 14 d after stopping
treatment. Kidney tissues had greatest [14C]
residues followed by the liver. [14C] residues
were detected in the kidney, liver, visceral fat,
subcutaneous fat, muscle, and brain.
Animals receiving a- and
P-[14C]endosulfan in the diet
over 14 d eliminated the
majority of endosulfan in the
feces. The kidneys and liver
contained the greatest amount of
noneliminated endosulfan
residues.
Dorough et al.
(1978)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
Female mice (number not specified) were fed
endosulfan at a dose of 7.5 mg/kg-d for 7 d,
and then for an additional 36 h after a Day-7
intraperitoneal injection with 125,000 dpm
[14C]testosterone in 100 |iL corn oil. Feces and
urine were collected 8, 24, 32, and 48 h after
injection and assessed for elimination of
[14C]testosterone. Urine samples were brought
up to equal volume using distilled water, and
[14C]testosterone was measured using
scintillation counting in a 100 |iL aliquot.
Ground up dried feces were oxidized to release
[14C] into scintillation cocktail that was then
quantified via liquid scintillation spectroscopy.
94% and 97% of [14C] was eliminated after 48 hr
in controls and treated animals, respectively. 70%
and 30% of the total recovered radioactivity were
eliminated in the feces and urine, respectively.
The [14C] clearance rate in the feces was not
affected by endosulfan treatment, but the
clearance rate in the urine was increased
~3.6-fold, and the total rate of elimination was
increased 2.3-fold. There was no significant
effect on serum testosterone or 17(3-estradiol
levels caused by endosulfan treatment.
While feces is the primary route
of elimination of endosulfan in
mice, endosulfan treatment
increased the rate of clearance
of [14C] administered as
[14C]testosterone. However,
these effects were not sufficient
to alter hormone homeostasis in
treated mice.
Wilson and Leblanc
(1998)

Male and female cats (n = 28) were
administered a single i.v. injection of 3 mg/kg
endosulfan dissolved in propylene glycol and
sacrificed at 15 min, 30 min, or at 1, 2, 4, or
6 hr by air administered directly to the heart.
Blood was drawn, and plasma was separated.
Tissue samples were taken from the liver,
spinal cord, cerebral cortex, cerebellum, and
brain stem. Tissues and plasma were evaluated
for identification of endosulfan and endosulfan
sulfate.
Peak concentrations of endosulfan in the brain
were found at the earliest time point examined
(15 min after administration) and then decreased.
Endosulfan sulfate levels peaked in the brain at
1 hr postadministration and in the liver within
15 min postadministration.
Endosulfan sulfate is a major
metabolite of endosulfan, and
the liver is a site of high
metabolic activity for this
conversion.
Khanna et al. (1979)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Metabolism/
toxicokinetic
Healthy, breast-feeding volunteers (17-35 yr)
were randomly selected from two hospitals in
Southern Spain. Milk samples were drawn
1-7 d postdelivery (colostrum), 6-12 d
(transition), and 13-35 d postdelivery transition
(mature) between 11 and 12 am. For each
sample, 5-10 mL was collected from the first
breast, the baby was fed for 5-10 min, and
more milk was drawn, and this was repeated
with the other breast and combined for
analysis. A liquid-liquid extraction was
conducted, purified, and analyzed via gas
chromatography.
Results indicated that endosulfan and endosulfan
sulfate were present in human breast milk in both
agricultural and urban settings and in each type of
milk, allowing it to be passed from mother to
child during breastfeeding.
Results indicated that
endosulfan and endosulfan
sulfate can be passed from
mother to child during
breastfeeding.
Campoy et al. (2001)

Women of reproductive age in Southern Spain:
Adipose tissue analysis: 149 women
undergoing various surgeries, samples
(subcutaneous abdominal fat or mammary
tissue) collected during surgery.
Placenta and umbilical cord blood analysis:
200 women, sampled at term deliveries.
Breast milk analysis: 23 breast feeding women
volunteers selected randomly from placenta
volunteers; mature milk drawn Days 13-35
postdelivery.
Samples were extracted and eluted using
HPLC, fractions containing pesticides were
analyzed by gas chromatography and
electron-capture detection and then by gas
chromatography and mass spectrometry.
Adipose tissue: endosulfan ether most frequently
detected (49.6%); endosulfan sulfate highest mean
concentration (16.16 ± 92.56 ng/g fat; 12.8% of
samples).
Placenta: endosulfan sulfate most frequently
detected (67.5%); endosulfan diol highest mean
concentration (15.62 ± 19.23 ng/g placenta).
Umbilical cord serum: endosulfan diol most
frequently detected (81%), a-endosulfan (76.5%);
endosulfan diol highest mean concentration
(13.23 ± 11.34 ng/mL serum).
Human milk: endosulfan ether (100%) and
endosulfan lactone (91.3%) most frequently
detected; (3-endosulfan highest mean
concentration (10.70 ± 8.71 ng/mL human milk).
Highest combined endosulfan (a- and (3-) was in
adipose tissue and then human milk samples.
Endosulfan sulfate was found to be the main
metabolite, present in all analyzed tissues.
Endosulfan and its metabolites
are present in adipose tissue,
placenta, umbilical cord serum,
and human milk in women of
reproductive age in southern
Spain.
Cerillo et al. (2005)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode of
action/
mechanistic
Male S-D rats were partially hepatectomized
and treated for 70 d according to the following
protocol: Group 1 was administered a vehicle
control; Group 2 was administered technical
grade endosulfan (ENDOtech) at 1 mg/kg-d;
Group 3 was administered ENDOtech at 5
mg/kg-d; Group 4 (control) was not partially
hepatectomized but was administered with
ENDOtech at 5 mg/kg/d; Groups 5-8 were
partially hepactectomized and injected with 30
mg nitrosodiethylamine (NDEA) before being
administered (2 mL/kg corn oil, 1 mg/kg, and
5 mg/kg, or 500 ppm of Phenobarbital,
respectively). Treatment was carried out for
10 d, and discontinued for two prior to
sacrifice.
No clinical signs of toxicity observed; no
significant difference was observed between test
Groups 2-4 and controls in terms of body-weight
gain, relative liver weights, and plasma
transaminase activities; treatment Groups 6 and 7
had significantly increased relative liver weights;
all rats treated with endosulfan showed congestion
of the peritoneum and inner organs. No
significant differences were observed between
Groups 6, 7 (treatments), and 5 (control) in terms
of the number of y-glutamyltranspeptidase
(GGT)-positive enzyme altered foci per cm3, and
percentage liver tissue occupied by foci. Groups 6
and 7 showed significantly decreased mean focal
volume compared with Group 5. Treatment
Groups 1-4 (not treated with NDEA) showed low
incidence of GGT-positive hepatocyte foci.
No dose-related increase in
enzyme-altered foci incidence
after induction with NDEA.
Flodstrom et al.
(1988)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode of
action/
mechanistic
Male S-D rats were partially hepatectomized
and treated according to the following protocol:
Group 1 served as a control and was fed a
normal diet; Group 2 was the positive control
and was fed a diet of 750 ppm DDT; Groups
3-5 were initiated by injection of 30 mg/kg
NDEA and were fed diets of 30, 100, or 300
ppm a-endosulfan (Groups 3a-3c),
(3-endosulfan (Groups 4a-4c), or 73:27
(a:(3)-endosulfan as an isomeric mixture
(Groups 5a-5c) for 20 wk. Groups Id, 3d, 4d,
and 5d were not initiated and fed standard diet
or 300 ppm a-endosulfan, (3-endosulfan, or
73:27 (a:(3)-endosulfan.
No clinical signs of toxicity were observed. A
statistically significant decrease in body-weight
gain was observed in all initiated groups exposed
to (3-endosulfan and 73:27 (a:(3)-endosulfan
(Groups 4a-4c and 5a-5c, respectively) during
the promotion period and in the uninitiated
(3-endosulfan group (4d). Absolute and relative
liver and kidney weights were increased in a
dose-related manner by a-endosulfan,
(S-cndosulfan. and 73:27 (a:(^-endosulfan.
Relative liver weight was significantly increased
in all high-dose groups (3c~d, 4c~d, 5c~d)
regardless of initiation. Relative kidney weight
was significantly increased in the mid-dose groups
(3b-d, 4b-d, 5b-d). Blood plasma alanine
aminotransferase (ALT) activity was significantly
increased in the initiated, high-dose a-endosulfan
group (3c), and aspartate aminotransferase (AST)
activity was significantly decreased in the
initiated, mid-dose a-endosulfan group (3b) and
the initiated, low-dose 73:27 (a:(3)-endosulfan
group (5a). A significant increase in GGT activity
was observed in blood plasma in the initiated,
high-dose a-endosulfan group and in the initiated,
mid-dose 73:27 (a:(3)-endosulfan group. All test
substances caused a marginal (2-3 x control)
induction of both forms of hepatic cytochrome
P450-dependent mono-oxygenases, induced a
dose-dependent, nonfocal diffuse expression of
GGT in hepatocytes, and enhanced development
of altered hepatic foci (AHF) (initiated, high-dose
a-endosulfan group only).
Endosulfan enhances clonal
expansion of
carcinogen-induced,
phenotypically altered
hepatocytes, indicating that
endosulfan has
tumor-promoting ability, but it
requires initiation by other
compounds.
Fransson-Steen
(1992a)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode of
action/
mechanistic
WBF344 rat liver epithelial cells and male S-D
primary rat hepatocytes tested endosulfan
isomers for the following:
(1)	the effect of a- and (^-endosulfan on gap
junction intercellular communication
(GJIC);
(2)	the influence of dibuturyl cyclic AMP
(dB-cAMP) on GJIC inhibition induced by
a- and (^-endosulfan;
(3)	the effect of 7:3 (a:(S)-cndosulfaa a-, or
(^-endosulfan on intracellular concentration
of cyclic AMP ((cAMP]i);
(4)	concentration-response and kinetics
(recovery time) of 7:3 (a:(S)-cndosulfan. a-,
or (3-endosulfan, and endosulfan metabolites
on GJIC in WBF344 rat liver cells (30 min
treatment).
(1)	In WBF344 liver cells, a- and (^-endosulfan
treatment resulted in a concentration-dependent
decrease in GJIC, but there was no difference
in inhibition between the two isomers. In
primary hepatocytes, (S-c ndosulfan was a more
potent inhibitor of GJIC compared with
a-endosulfan (40% inhibition with 10 |iIVI (3
compared with 40% inhibition with 50 |iIVI a).
(2)	In WBF344 liver cells, pretreatment with
0.1-0.5 mM dB-cAMP significantly enhanced
GJIC by approximately 25%. dB-cAMP was
unable to counteract the GJIC-inhibitory effect
of a- or (^-endosulfan at 5 |iM In primary rat
hepatocytes, no increase in GJIC was observed
with 0.1-0.5 mM dB-cAMP pretreatment.
However, pretreatment with 0.25 and 0.5
mM dB-cAMP significantly prevented the
inhibitory effect of GJIC by 75|iIVI
(^-endosulfan but not 75 a-endosulfan.
(3)	In WBF344 liver cells, significant increase of
(cAMPJj was observed after exposure to 5 |iIVI
7:3 (a:(3)-endosulfanfor 10 min; however, after
30 min of exposure, (cAMPji returned to
normal.
(4)	Endosulfan sulfate and 7:3 (a:(3)-endosulfan
strongly inhibited GJIC at >5-10 |iM
(complete inhibition at 25 |iM); however, the
effects were reversible and returned to control
levels after 30 min. Endosulfan ether inhibited
GJIC at >10 (iM (complete inhibition at
100(iM). Endosulfan lactone inhibited GJIC at
>100 pM.
Differences in GJIC induced by
a- and (^-endosulfan in primary
rat hepatocytes and rat liver
epithelial cells suggest different
mechanisms of inhibition in the
two cell types.
Endosulfan, its isomers, and
metabolites are unlikely to
inhibit GJIC by decreasing
intracellular cAMP.
Fransson-Steen
(1992b)
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Table B.2. Other Studies
Test
Materials and Methods
Results
Conclusions
References
Mode of
action/
mechanistic
5-10 male or female mice were fed endosulfan
at doses of 0, 3.8, 7.5, or 15 mg/kg-d for
approximately 7 d. Livers were removed, and
microsomal and cytosolic cell fractions were
isolated. Testosterone hydroxylase,
17 p-hydroxysteroid dehydrogenase,
UDP-glucuronosyltransferase activities were
assayed using microsomal protein and
[14C]testosterone. Sulfotransferase activity was
assayed using cytosolic protein
[14C]testosterone. Thin-layer chromatography
was used to isolate metabolites, and liquid
scintillation spectroscopy and liquid
scintillation counting were used to quantify the
products of the various enzymatic reactions
assayed.
BW was decreased in males at 15 mg/kg-d.
Endosulfan treatment resulted in a significant
dose-dependent increase in testosterone hydroxyl
metabolite formation in female mice, with the
most frequent hydroxylation occurring at the op-
position. In males, treatment significantly
reduced the rate of conversion of testosterone to
androstenedione and increased hydroxylation at
the 16p- position (significantly at only
15 mg/kg-d). Glucuronic acid and sulfate
conjugation rates were unaffected. Serum
testosterone and 17p-estradiol levels were slightly
decreased although the difference was not
statistically significant.
Endosulfan treatment resulted in
increased testosterone
biotransformation in male and
female mice although the
increased rate of elimination of
testosterone compensated for
these effects. There was no
effect on serum testosterone
levels.
Wilson and Leblanc
(1998)

3H-dihydropicrotoxinin and GABA receptors
were used. No other methodological details
were provided.
The ability of endosulfan to induce convulsions
was correlated with its potency as a
noncompetitive GABA antagonist acting at the
chloride channel within the GABA receptor. By
inhibiting GABA-induced chloride flux into the
neurons, the membranes become hyperpolarized,
and cell firing is inhibited.
Endosulfan acts as a
noncompetitive GABA
antagonist at the chloride
channel within the GABA
receptor in brain synaptosomes.
Abalis et al. (1986);
Cole and Casida
(1986);	Gant et al.,
(1987);	and Ozoe
and Matsumura
(1986), as reported
in ATSDR (2000)

Primary cultures of cortical neurons from
15-d-old mice fetuses were used. No other
methodological details were provided.
a-Endosulfan blocked the chloride uptake induced
by GABA by interacting with the
t-butylbicyclophosphorothionate binding site.
GABA-antagonism mechanism
of toxicity via binding of
endosulfan at multiple receptors
in neurons and inhibiting
GABAergic function.
Pomes et al. (1994),
as reported in
ATSDR (2000)
BW = body weight; DU = data unsuitable; NA = not applicable; NV = not available; ND = no data; NDr = not determinable; NR = not reported; NR/Dr = not reported by
the study author but determined from data; NS = not selected.
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APPENDIX C. DATA TABLES
Table C.l. Summary of Multiple Regression Analysis of SMR (Sexual Maturity Rating)
Parameters Against Age and Endosulfan Aerial Spray Exposure"
SMR Parameter
R2
Intercept (b0)
Age
AEE
(Aerial Endosulfan Exposure)
bi
SE

SE
Pubic hair
0.48**
-2.54**
0.36**
0.03
0.43**
0.11
Testes
0.43**
-2.07**
0.32**
0.03
0.32*
0.11
Penis
0.43**
-2.00**
0.32**
0.03
-0.37*
0.12
"Sayied et al. (2003).
* Significantly different from controls at p< 0.05; multiple regression analyses performed by the study authors.
**Significantly different from controls at p < 0.01; multiple regression analyses performed by the study authors.
Table C.2. Serum Endosulfan Levels in Study and Control Subjects"
Parameterb
Controls (n = 45)
Study (n = 70)
a-Endosulfan (ppb)
0.87 ±0.23
4.24 ± 0.74** (487)
(^-Endosulfan (ppb)
0.40 ±0.17
1.77 ±0.36** (443)
Endosulfan sulfate (ppb)
0.10 ±0.08
1.47 ±0.33** (1,470)
Total endosulfan (ppb)
1.37 ±0.40
7.47 ± 1.19** (545)
aSayied et al. (2003).
bMean ± SE (% of controls).
**Significantly different from controls at /? < 0.01: multiple regression analyses performed by the study authors.
Table C.3. Growth-Related Parameters in Study and Control Subjects"
Parameterb
Controls (n = 90)
Study (n = 117)
Age (years)
13.10 ± 2.12
12.80 ± 2.07 (98)
Height (cm)
141 ± 10.60
139 ± 13.30 (99)
Weight (kg)
30.70 ±7.44
29.50 ± 8.93 (96)
Body mass index
15.30 ± 1.98
15.00 ±2.11 (98)
Skin-fold thickness (mm)
7.31 ± 2.15
7.40 ±2.28 (101)
aSayied et al. (2003).
bMean ± SD (% of controls).
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Table C.4. Albumin Versus Globulin Ratio of Serum in Tetanus Toxoid-Stimulated and
Unstimulated Rats Exposed to Various Levels of Endosulfan for 8-22 Weeksa'b
Parameter
Exposure Group (mg/kg-d)
0
0.5
0.9
1.8
Stimulated
Albumin:
globulin
ratio0
8 wk
1.00 ±0.14
1.00 ±0.20 (100)
1.10 ±0.21 (110)
1.08 ±0.14 (108)
12 wk
0.94 ±0.17
1.05 ±0.10 (112)
1.05 ±0.05 (112)
1.11 ±0.12* (118)
18 wk
0.96 ±0.11
1.02 ±0.10 (106)
1.00 ±0.25 (104)
1.10 ± 0.15* (115)
22 wk
0.85 ± 0.11
1.00 ±0.14 (118)
1.14 ±0.20* (134)
1.15 ±0.10* (135)
Unstimulated
Albumin:
globulin
ratio0
8 wk
1.26 ±0.26
1.23 ±0.11 (98)
1.17 ±0.15 (93)
1.20 ±0.12 (95)
12 wk
1.25 ±0.10
1.36 ±0.21 (109)
1.21 ±0.04 (97)
1.18 ±0.14 (94)
18 wk
1.15 ±0.14
1.14 ±0.17 (99)
1.22 ±0.07 (106)
1.30 ±0.05 (113)
22 wk
1.25 ±0.14
1.16 ±0.21 (93)
1.14 ±0.16 (91)
1.20 ±0.15 (96)
aBaneijee and Hussain (1986).
Stimulated rats were immunized with tetanus toxoid in Freund's complete adjuvant 20 d prior to termination of
treatment. Unstimulated rats were treated in a manner similar to stimulated rats except for immunization.
°Albumin:globulin ratios were calculated from percentage of total protein content. Values are expressed as mean ±
standard deviation (% relative to controls) of 10-12 rats per group.
* Significantly different from controls at p< 0.05; ANOVA performed by the study authors.
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Table C.5. Serum Immunoglobulin Concentrations in Tetanus Toxoid-Stimulated and
Unstimulated Rats Exposed to Various Levels of Endosulfan for 8-22 Weeksa'b
Parameter
Exposure Group (mg/kg-d)
0
0.5
0.9
1.8
Stimulated0
Serum IgM
(mg/mL)
8 wk
0.70 ±0.12
0.66 ±0.12 (94)
0.64 ±0.15 (91)
0.63 ±0.10 (90)
12 wk
0.72 ±0.11
0.64 ±0.12 (89)
0.64 ±0.10 (89)
0.60 ±0.15 (83)
18 wk
0.68 ±0. 13
0.64 ±0.10 (94)
0.60 ±0.10 (88)
0.57 ±0.14 (84)
22 wk
0.70 ±0.12
0.64 ±0.15 (91)
0.58 ±0.18 (83)
0.55 ±0.10 (79)
Serum IgG
(mg/mL)
8 wk
15.56 ± 1.50
14.88 ±2.30 (96)
14.61 ±2.22 (94)
14.05 ± 2.07 (90)
12 wk
15.11 ± 1.20
14.00 ±2.80 (93)
13.00 ±2.08* (86)
12.75 ± 1.15* (84)
18 wk
15.01 ±2.62
14.08 ± 2.20 (94)
12.70 ± 1.60* (85)
12.15 ± 1.30* (81)
22 wk
15.20 ± 1.20
14.00 ±2.12 (92)
12.10 ±2.10** (80)
12.56 ± 1.45** (83)
Unstimulated0
Serum IgM
(mg/mL)
8 wk
0.52 ±0.10
0.49 ±0.16 (94)
0.44 ±0.12 (85)
0.45 ±0.15 (87)
12 wk
0.51 ± 0.13
0.50 ±0.15 (98)
0.45 ± 0.06 (88)
0.44 ±0.15 (86)
18 wk
0.50 ±0.10
0.49 ±0.13 (98)
0.44 ± 0.20 (88)
0.45 ±0.10 (90)
22 wk
0.52 ±0.15
0.48 ±0.12 (92)
0.42 ±0.16 (81)
0.42 ±0.13 (81)
Serum IgG
(mg/mL)
8 wk
12.50 ±2.53
11.15 ± 1.03 (89)
12.19 ± 1.50 (98)
11.60 ± 1.30 (93)
12 wk
12.40 ±2.15
12.00 ± 2.00 (97)
12.17 ± 1.60 (98)
10.11 ±2.50 (82)
18 wk
11.24 ± 1.88
10.18 ± 1.81 (91)
10.18 ± 1.50 (91)
10.35 ± 1.66 (92)
22 wk
11.50 ±2.20
10.57 ±2.50 (92)
10.43 ± 1.70 (91)
10.00 ± 1.30 (87)
aBaneijee and Hussain (1986).
Stimulated rats were immunized with tetanus toxoid in Freund's complete adjuvant 20 d prior to termination of
treatment. Unstimulated rats were treated in a manner similar to stimulated rats except for immunization.
°Values are expressed as mean ± standard deviation (% relative to controls) of 10-12 rats per group.
* Significantly different from controls at p< 0.05; ANOVA performed by the study authors.
**Significantly different from controls at p < 0.01: ANOVA performed by the study authors.
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Table C.6. Antibody Response of Male Wistar Albino Rats to Tetanus Toxoid After
8-22 Weeks of Treatment to Endosulfana'b
Parameter
Exposure Group (mg/kg-d)
0
0.5
0.9
1.8
No. of animals
10-12
10-12
10-12
10-12
-Log2
antibody
titer0
8 wk
14.53 ± 1.05
13.72 ±2.17 (94)
12.72 ± 1.30* (88)
10.30 ± 1.73** (71)
12 wk
14.80 ±0.99
13.81 ± 1.55 (93)
12.32 ± 1.61* (83)
12.13 ± 1.67** (82)
18 wk
14.90 ±0.93
13.96 ± 1.92 (94)
10.99 ± 1.49** (74)
8.57 ± 1.36** (58)
22 wk
14.80 ± 1.18
15.17 ±0.74 (103)
9.66 ± 1.67** (65)
8.79 ± 1.11** (59)
aBaneijee and Hussain (1986).
bData digitized for this review.
°Values are expressed as mean ± S.D (% relative to controls).
* Significantly different from controls at p< 0.05; ANOVA performed by the study authors.
**Significantly different from controls at p < 0.01; ANOVA performed by the study authors.
Table C.7. Leukocyte Migration Inhibition (LMI) Response of Male Wistar Albino Rats to
Tetanus Toxoid After 8-22 Weeks of Treatment to Endosulfana'b
Parameter
Exposure Group (mg/kg-d)
0
0.5
0.9
1.8
No. of animals
10-12
10-12
10-12
10-12
Leukocyte
migration
inhibition (%)°
8 wk
45.37 ±3.08
40.23 ± 9.74 (89)
38.42 ±7.44* (89)
34.31 ±7.69** (76)
12 wk
50.18 ±6.15
49.91 ±6.92 (99)
40.16 ±8.21* (80)
32.97 ±5.64** (66)
18 wk
48.07 ±5.38
43.70 ± 11.28 (91)
30.35 ±6.92** (63)
24.70 ± 13.85** (51)
22 wk
46.21 ±4.87
40.30 ±6.15 (87)
30.29 ±5.13** (66)
30.54 ±4.62** (66)
aBaneijee and Hussain (1986).
bData digitized for this review.
°Values are expressed as mean ± S.D (% relative to controls).
* Significantly different from controls at p < 0.05; ANOVA performed by the study authors.
**Significantly different from controls at p < 0.01: ANOVA performed by the study authors.
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Table C.8. Macrophage Migration Inhibition (MMI) Response of Male Wistar Albino Rats
to Tetanus Toxoid After 8-22 Weeks of Treatment to Endosulfana'b
Parameter
Exposure Group (ppm)
0
0.5
0.9
1.8
No. of animals
10-12
10-12
10-12
10-12
Macrophage
migration
inhibition (%)°
8 wk
35.36 ±6.11
32.52 ± 5.86 (92)
27.91 ± 5.35* (79)
25.58 ±5.09** (72)
12 wk
31.87 ±7.64
30.82 ±5.61 (97)
23.40 ±4.84** (73)
21.33 ±4.84** (67)
18 wk
31.18 ± 5.60
26.31 ± 5.09 (84)
16.60 ± 5.09** (53)
18.35 ±4.84** (59)
22 wk
35.84 ±5.09
30.21 ± 8.92 (84)
14.89 ± 12.73** (42)
10.27 ±9.68** (29)
aBaneijee and Hussain (1986).
bData digitized for this review.
°Values are expressed as mean ± S.D (% relative to controls).
* Significantly different from controls at p< 0.05; ANOVA performed by the study authors.
**Significantly different from controls at p < 0.01: ANOVA performed by the study authors.
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Table C.9. Maternal Body Weight and Food Consumption for Female F344 Rats After Oral
Exposure to Endosulfan from GD 6-PND 21a
Observation/
Study Week0
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
50 (3.74)
150 (10.8)
400 (29.8)
Mean body weight (g)
GD 0
202.5 ± 2.44
196.5 ±2.71 (97)
198.7 ±2.91 (98)
198.5 ±2.16 (98)
GD 6
221.8 ±3.99
213.9 ±3.76 (96)
220.1 ±3.16 (99)
220.0 ±2.12 (99)
GD 13
250.7 ±3.16
238.3 ±3.11* (95)
226.6 ± 3.00* (90)
209.7 ± 2.60** (84)
GD 20
311.6 ±4.25
293.6 ±4.24* (94)
282.8 ±4.11** (91)
268.2 ± 3.36** (86)
LD0
241.1 ±3.74
231.2 ± 3.55 (96)
219.1 ±3.27** (91)
210.7 ±3.64** (87)
LD 4
253.0 ±3.61
241.4 ± 3.25 (95)
234.0 ±4.00** (92)
226.0 ±2.51** (89)
LD 7
262.0 ±3.62
255.7 ±2.79 (98)
245.3 ±4.04* (94)
241.6 ± 3.53** (92)
Mean food consumption (g/animal/d)
GDs 6—13d
19.8 ±0.39
17.5 ±0.54** (88)
12.8 ±0.31** (65)
9.5 ±0.32** (48)
GDs 13-20
21.2 ±0.43
19.7 ± 0.55 (93)
18.1 ±0.53** (85)
17.5 ±0.53** (83)
LDs 0-7
34.2
32.1 (94)
31.4(92)
32.2 (94)
LDs7-14
50.6
49.1 (97)
48.3 (95)
48.8 (96)
LDs14-21
61.7
58.5 (95)
60.7 (98)
60.5 (98)
LDs0-21
146.5
139.7 (95)
140.4 (96)
141.5 (97)
aGilmore et al. (2006).
bDoses reported in data evaluation record; unclear if converted by authors or reviewers.
°Values expressed as mean ± SD (% of control); % was calculated.
dNo standard deviations provided in data evaluation record.
* Significantly different from control at p< 0.05; test was not reported.
**Significantly different from control at p < 0.01: test was not reported.
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Table C.10. Litter-based Body Weights of Pups from Female F344 Rats Exposed to
Endosulfan from GD 6-PND 21a
Observation/
Study Dayc
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
50 (3.74)
150 (10.8)
400 (29.8)
No. of litters
23
23
23
21
Preweaning mean body weight (g)—male
PNDO
5.8 ±0.09
5.8 ±0.11 (100)
5.9 ±0.09 (102)
5.9 ±0.12 (102)
PND 4d
9.2 ±0.18
9.1 ±0.21 (99)
8.7 ±0.19 (95)
8.5 ± 0.25 (92)
PND 4e
9.3 ±0.18
9.1 ±0.21 (98)
8.8 ±0.18 (95)
8.5 ±0.26 (91)
PND 11
24.3 ±0.42
22.3 ± 0.49** (92)
21.5 ±0.50** (88)
21.1 ±0.52** (87)
PND 17
37.6 ±0.67
35.0 ±0.82* (93)
34.3 ±0.68** (91)
33.3 ±0.61** (89)
PND 21
47.5 ±0.78
44.5 ± 1.10(94)
43.9 ±0.81** (92)
42.5 ±0.86** (89)
Preweaning mean body weight (g)—female
PNDO
5.5 ±0.08
5.5 ±0.10 (100)
5.6 ±0.08 (102)
5.6 ±0.10 (102)
PND 4d
8.9 ±0.17
8.8 ±0.17 (99)
8.4 ±0.17 (94)
8.2 ± 0.24* (92)
PND 4e
8.9 ±0.17
8.7 ±0.17 (98)
8.5 ±0.18 (96)
8.2 ± 0.23 (92)
PND 11
23.6 ±0.36
21.7 ±0.46** (92)
20.9 ±0.54** (89)
20.4 ±0.48** (86)
PND 17
36.5 ±0.63
34.1 ±0.78 (93)
33.5 ±0.70** (92)
32.5 ±0.59** (89)
PND 21
45.9 ±0.62
43.0 ±0.97* (94)
42.7 ±0.90 (93)
41.3 ±0.83** (90)
Postweaning mean body weight (g)—male
PND 28
77.0 ± 10.4
75.0 ± 7.6 (97)
71.5 ±6.9 (93)
69.1 ±7.8* (90)
PND 35
125.4 ± 13
117.7 ± 113.2 (94)
111.3 ±10.5* (89)
110.1 ±11.7* (88)
PND 42
171.6 ± 14.4
162.2 ± 15.7(95)
154.7 ± 12.8* (90)
154.0 ± 14.6* (90)
PND 49
214 ± 15.6
203 ± 17.6 (95)
194.5 ± 14.2* (91)
193.2 ± 18.1* (90)
PND 56
257 ± 17.9
245.7 ± 20 (96)
236.9 ± 16.6* (92)
234.9 ±21* (91)
PND 63
289.3 ± 19.3
277.8 ± 24 (96)
269.5 ± 17* (93)
267.2 ±23.2* (92)
PND 70
317.6 ±22.7
304.8 ± 26.7 (96)
297.0 ± 19.1* (94)
294.0 ±25.1* (93)
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Table C.10. Litter-based Body Weights of Pups from Female F344 Rats Exposed to
Endosulfan from GD 6-PND 21a
Observation/
Study Dayc
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
50 (3.74)
150 (10.8)
400 (29.8)
Postweaning mean body weight (g)—female
PND28
75.5 ± 10.3
73.3 ±6.7 (97)
70.5 ±6.6 (93)
67.5 ±7.6* (89)
PND 35
111.7 ± 9.8
108.5 ± 8.3 (97)
105.7 ±7.7 (95)
102.2 ±9.0* (91)
PND42
136.8 ±9.4
134.6 ±8.7 (98)
130.8 ±7.2 (96)
126.6 ±9.8* (93)
PND 49
152.1 ±9.9
149.1 ±9.7 (98)
146.0 ± 8.4 (96)
142.6 ± 11.2* (94)
PND 56
171.3 ± 11.6
167.2 ± 11.5 (98)
166.4 ± 8.9 (97)
161.9 ± 12.5 (95)
PND 63
181.8 ± 11.5
178.2 ±11.5 (98)
178.0 ±9.4 (98)
172.9 ± 12.9(95)
PND 70
191.0 ± 11.4
187.6 ± 11.4(98)
188.2 ± 10.2 (99)
182.9 ± 13.7 (96)
aGilmore et al. (2006).
bDoses reported in data evaluation record; unclear if converted by authors or reviewers.
°Values expressed as mean ± SE (% of control); % was calculated.
dBefore standardization (culling).
eAfter standardization (culling).
* Significantly different from control at p< 0.05; test was not reported.
**Significantly different from control at /? < 0.01: test was not reported.
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Table C.ll. Mean Age of Sexual Maturation in Offspring Female Wistar Rats After Oral
Exposure to Endosulfan from GD 6-PND 21a
Observation/
Study Week0
Exposure Group, ppm (Adjusted Daily Dose, mg/kg-d)b
0
50 (3.74)
150 (10.8)
400 (29.8)
Number of animals (M/F)
66/77
67/69
69/69
63/63
Day of preputial separation
(males)
44.9 ±0.40
44.8 ±0.29 (100)
47.1 ±0.49* (105)
46.8 ±0.43* (104)
Day of vaginal opening
(females)
33.0 ±0.27
34.0 ±0.30* (103)
34.2 ±0.40* (104)
34.0 ±0.40 (103)
'Gilmorc et al. (2006).
bDoses reported in data evaluation record; unclear if converted by authors or reviewers.
°Values expressed as mean ± SD (% of control); % was calculated.
* Significantly different from control at p< 0.05; test was not reported.
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APPENDIX D. BMD OUTPUTS
Table D.l. Summary of the Viable BMD Models for the Screening Subchronic p-RfD for
Endosulfan Sulfate
Study and
Year
Endpoint
Gender/
Species
Model Name
BMD
BMDL
Goodness
of Fit
/7-Value
AIC
Scaled
Residual of
Interest
Gilmore
et al.
(2006)
PupBWPND 11
F
Hill (constant
variance)
1.6
0.29
0.9598
235.99
0.003
Gilmore et
al. (2006)
PupBWPND 11
M
Exponential
(M4) (constant
variance)
1.9
0.61
0.7264
243.14
-0.122
Dalsenter
et al.
(1999)
Daily Sperm
Production
M
Linear (constant
variance)
0.85
0.68
0.7639
345.39
-0.245
Dalsenter
et al.
(1999)
Relative
Testicular Weight
PND65
M
Linear (constant
variance)
1.17
0.91
0.1735
-286.66
1.100
BW = body weight.
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Hill Model with 0.95 Confidence Level
24
23
a)
to
1	22
a:
c
ro
a)
2
21
20
19 B
0	5	10	15	20	25	30
dose
09:00 04/27 2011
Figure D.l. Gilmore et al., 2006_Female Pup Body Weight PND H HillCV_RD10
Hill Model. (Version: 2.15; Date: 10/28/2009)
Input Data File: C:/l/Gilmore et al 2006 Female Pup body weight PND
ll_HillCV_RD10.(d)
Gnuplot Plotting File: C:/l/Gilmore et al 2006 Female Pup body
weight PND ll_HillCV_RD10.pit	~
Wed Apr 27 09:00:26 2011
add notes
The form of the response function is:
Y[dose] = intercept + v*doseAn/(kAn + doseAn)
Dependent variable = Mean
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
Hill
MDL BMD
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A constant variance model is fit
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-OOf
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha =	4.84843
rho =	0 Specified
intercept =	23.6
v =	-3.2
n =	1.78837
k =	3.14947
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Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho -n
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
alpha
intercept
alpha
1
-4 . 4e-008
5. le-008
-9.9e-009
intercept
-4 . 4e-008
1
-0.51
-0.33
v
5. le-008
-0.51
1
-0.53
-9. 9e-009
-0.33
-0.53
1
Confidence Interval
Variable	Estimate
Upper Conf. Limit
alpha	4.63307
5.98674
intercept	23.5988
24.4778
v	-3.5371
-2.01383
n	1
k	3.26121
8.39516
Parameter Estimates
Std. Err.
0. 690658
0.448458
0.777193
NA
2 . 61941
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
95.0% Wald
Lower Conf. Limit
3.27941
22 .7199
-5. 06037
-1. 87274
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Table of Data and Estimated Values of Interest
Dose
Res .
Obs Mean
Est Mean Obs Std Dev Est Std Dev Scaled
0
3.74
10. 8
29.8
23
23
23
21
23. 6
21.7
20.9
20.4
23. 6
21.7
20.9
20.4
1.73
2.21
2 . 59
2.2
2 . 15
2 . 15
2 . 15
2 . 15
0. 00258
-0. 0208
0. 0399
-0. 0227
Model Descriptions for likelihoods calculated
Model A1:	Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2:	Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3:	Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R:	Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
A1
A2
A3
fitted
R
Log(likelihood)
-113.993646
-112.152379
-113.993646
-113.994919
-126.468005
# Param's
5
8
5
4
2
AIC
237.987292
240.304758
237 . 987292
235.989837
256.936011
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
Test 2: Are Variances Homogeneous? (A1 vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df	p-value
Test 1	28.6313	6	<.0001
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Test 2
Test 3
Test 4
3. 68253
3. 68253
0. 00254481
3
3
1
0.2978
0.2978
0.9598
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect
0.05
Risk Type
Relative risk
Confidence level
0. 95
BMD
1.63249
BMDL
0.290268
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