United States
Environmental Protection
Agency
FINAL
ECA0-C1N-406
March, 1987
&EPA Research and
Development
DRINKING WATER CRITERIA DOCUMENT FOR
HEPTACHLOR, HEPTACHLOR EPOXIDE AND CHLORDANE
Prepared for
OFFICE OF DRINKING WATER
Prepared by
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH 45268

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DISCLAIMER
This document has been reviewed 1n accordance with the U.S. Environ-
mental Protection Agency's peer and administrative review policies and
approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11

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FOREWORD
Section 1412 (b)(3)(A) of the Safe Orlnklng Water Act, as amended In
1986, requires the Administrator of the Environmental Protection Agency to
publish maximum contaminant level goals (MCLGs) and promulgate National
Primary Orlnklng Water Regulations for each contaminant, which. In the
Judgment of the Administrator, may have an adverse effect on public health
and which Is known or anticipated to occur 1n public water systems. The
MCLG Is nonenforceable and Is set at a level at which no known or antici-
pated adverse health effects In humans occur and which allows for an
adequate margin of safety. Factors considered 1n setting the MCLG Include
health effects data and sources of exposure other than drinking water.
This document provides the health effects basis to be considered In
establishing the MCLG. To achieve this objective, data on pharmacokinetics,
human exposure, acute and chronic toxicity to animals and humans, epidemi-
ology and mechanisms of toxicity are evaluated. Specific emphasis 1s placed
on literature data providing dose-response Information. Thus, while the
literature search and evaluation performed In support of this document has
been comprehensive, only the reports considered most pertinent 1n the deri-
vation of the MCLG are cited 1n the document. The comprehensive literature
data base 1n support of this document Includes Information published up to
1984; however, more recent data may have been added during the review
process.
When adequate health effects data exist, Health Advisory values for less
than lifetime exposures (1-day, 10-day and longer-term, -10% of an
Individual's lifetime) are Included 1n this document. These values are not
used 1n setting the MCLG, but serve as Informal guidance to municipalities
and other organizations when emergency spills or contamination situations
occur.
Michael B. Cook
Director
Office of Drinking Water
111

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DOCUMENT DEVELOPMENT
W. Bruce Pelrano, Document Manager
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Helen Ball, Project Officer
Environmental Criteria and Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Authors
Sharon B. Wilbur
Dlpak K. Basu
Marljean B. Remington
Patrick Durkln
Syracuse Research Corporation
Syracuse, New York
(Contract 68-03-3112)
Yogendra M. Patal
(revised Chapter VIII)
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC
Finis L. Cavender
Brlon T. Cook
Norbert P. Page
D.E.B. Potter
Dynamac Corporation
Rockvllle, MD
(Carcinogen Assessment Group
contractor)
Scientific Reviewers
Charles H. RIs
V. James CogUano
Aparna M. Kopplkar
Dharm V. Singh
Carcinogen Assessment Group
U.S. Environmental Protection Agency
Washington, DC
Cindy Son1ch-Mul1 In
Michael L. Oourson
Environmental Criteria and
Assessment Office, Cincinnati
U.S. Environmental Protection Agency
George E. Whltmore
Consultant to Life Systems, Inc.
Cleveland, OH
Shane S. Que Hee
Oept. of Environmental Health
University of Cincinnati
Cincinnati, OH 45267
Chapter IV provided by the
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC
Editorial Reviewers
Erma Durden
Judith Olsen
Environmental Criteria and
Assessment Office, Cincinnati
U.S. Environmental Protection Agency
Document Preparation
Technical Support Services Staff: C. Cooper, P. Daunt, C. Fessler, K. Mann,
8. Zwayer, Environmental Criteria and Assessment Office, Cincinnati
tv

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TABLE OF CONTENTS
Page
I. SUMMARY		1-1
II. PHYSICAL AND CHEMICAL PROPERTIES		II-1
CHLOROANE		II-1
HEPTACHLOR		11 -4
HEPTACHLOR EPOXIDE		II-7
SUMMARY		11-10
III. TOXICOKINETICS		Ill-1
ANIMAL STUOIES		III-l
Chlordane			III-l
Heptachlor and Heptachlor Epoxide		111-11
HUMAN DATA		111-17
Chlordane		111-17
Heptachlor and Heptachlor Epoxide		111-21
SUMMARY		111-25
Chlordane		111-25
Heptachlor and Heptachlor Epoxide		111-27
IV. HUMAN EXPOSURE		IV-1
To be provided by the Office of Drinking Water
V. HEALTH EFFECTS IN ANIMALS 		V-l
GENERAL TOXICITY		V-l
Effects of Acute Exposure		V-l
Effects of Subchronlc and Chronic Exposures		V-l8
TARGET ORGAN TOXICITY 		V-52
Chlordane		V-52
Heptachlor and Heptachlor Epoxide		V-57
OTHER EFFECTS		V-59
Carcinogenicity		V-59
Mutagenicity 		V-92
Teratogenicity and Other Reproductive Effects		V-l02
SUMMARY			V-l 04
Chlordane		V-l 04
Heptachlor and Heptachlor Epoxide		V-l08
v

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TABLE Of CONTENTS (cont.)
Page
VI. HEALTH EFFECTS IN HUHANS		VI-1
CLINICAL CASE STUDIES		VI-1
CNS Effects Associated with Exposure to Chlordane
and Heptachlor		VI-1
Blood Dyscraslas Associated with Exposure to
Chlordane and Heptachlor			VI-5
Neuroblastoma Associated with Pre- and/or
Postnatal Exposure to Chlordane and Heptachlor ....	VI-Q
EPIDEMIOLOGICAL STUOIES		VI-11
HIGH RISK SU8P0PULATI0NS		VI-22
SUMMARY		VI-23
VII. MECHANISMS OF TOXICITY		VII-1
MECHANISM Of NEUROTOXICITY		VII-1
MECHANISMS OF EFFECTS ON ENDOCRINE AND REPRODUCTIVE
ORGANS			VII -4
ENZYME INDUCTION ANO RELATED MECHANISMS . 		VII-5
EFFECTS ON CELLULAR RESPIRATION . . . 		VII-7
SYNERGISM/ANTAGONISM		VII-8
MECHANISM OF CARCINOGENICITY		VII-10
SUMMARY 		VII-11
VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS 		VII1-1
INTRODUCTION		VIII-1
CHLORDANE 		VIII-6
Noncarclnogenlc Effects		VI11 -6
Quantification of Noncarclnogenlc Effects		VIII-11
Carcinogenic Effects		VI11-15
Quantification of Carcinogenic Effects 		VIII-20
HEPTACHLOR ANO HEPTACHLOR EPOXIDE 		VIII-29
Noncarclnogenlc Effects		VI11-29
Quantification of Noncarclnogenlc Effects		VIII-33
Carcinogenic Effects		VIII-4-0
Quantification of Carcinogenic Effects		VIII-44
EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS		VII1-56
Water		VIII-56
Food		VIII-57
A1r	•		VIII-58
v 1

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TABLE OF CONTENTS (cont.)
Page
SPECIAL CONSIDERATIONS		VIII-58
High Risk SubpopulatIons 		VIII-58
Multiple Pollutant Exposures 		VIII-59
IX. REFERENCES		IX-1
v 11

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LIST OF TABLES
No. Title	Page
II-1	Chemical	and Physical Properties of Chlordane 		II-3
II-2	Chemical	and Physical Properties of Heptachlor		11-5
11-3	Chemical	and Physical Properties of Heptachlor Epoxide. .	11-9
111-1 Metabolism of cls-Chlordane and trans-Chlordane in
vitro by Human and Rat Liver Microsomal Preparations:
The Data are Expressed as Percent of the Total
Starting Material	 111-20
111-2 Heptachlor Metabolism In vitro by Human and Rat Liver
Microsomal Preparations: The Data are Expressed as
Percent of Starting Material	 111-24
V-l Acute LD50S for Chlordane		V-2
V-2 Acute Toxicity of Chlordane 		V-5
V-3	Acute LD50S for Heptachlor and Heptachlor Epoxide ....	V-12
V-4	Acute Toxicity of Heptachlor and Heptachlor Epoxide . . .	V-15
V-5 Effects 1n Rats and Mice of Subchronlc and Chronic
Dietary Exposure to Chlordane 	 V-19
V-6 Design Suirvnary for NCI Bloassay of Chlordane 1n
Osborne-Mendel Rats	 V-28
V-7 Design Summary for NCI Bloassay of Chlordane In
B6C3FMice	 V-30
V-0 Effects of Subchronlc Exposure to Chlordane by Other
Routes	 V-34
V-9 Effects In Rats and Mice of Subchronlc and Chronic
Dietary Exposure to Heptachlor and Heptachlor Epoxide . . V-36
V-10 Design Summary for NCI Bloassay of Heptachlor In
Osborne-Wendel Rats	 V-48
V-11 Design Summary for NCI Bloassay of Heptachlor In
B6C3F] Mice	 V-50
V-12 Effects of Subchronlc Exposure to Heptachlor and
Heptachlor Epoxide by Other Routes	 V-53
V-13 Summary of Mouse Dietary CardnogencUty Tests for
Chlordane	 V-60
v111

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LIST OF TABLES (cont.)
No.	Title	Page
V-14 Summary of Rat Dietary Carcinogenicity Tests for
Chlordane	 V-61
V-15 Incidence of Liver Lesions and Tumors in CD-I Mice
Following Dietary Administration of Chlordane 	 V-65
V-16 Incidence of Hepatocellular Carcinoma in Mice
Following Chronic Dietary Administration of Chlordane . . V-66
V-17 Neoplastic and Toxic Lesions of the Liver In ICR Mice
fed Chlordane	 V-68
V-18 Liver Tumors and Nonneoplastic Lesions In Fischer 344
Rats Fed Chlordane	 V-73
V-19 Liver Lesions In Fischer 344 Rats Fed Chlordane for
26 or 52 Weeks	 V-75
V-20 Liver Neoplasms 1n Hale Fischer 344 Rats Fed Chlordane. . V-77
V-21 Summary of Mouse Dietary Carcinogenicity Tests for
Heptachlor and Heptachlor Epoxide 	 V-79
V-22 Summary of Rat Dietary Carcinogenicity Tests for
Heptachlor and Heptachlor Epoxide 		V-80
V-23 Incidences of Liver Lesions 1n C3H Mice Treated with
Heptachlor or Heptachlor Epoxide	 V-82
V-24 Incidences of Hepatocellular Carcinoma 1n C3H Mice
Treated with Heptachlor or Heptachlor Epoxide 	 V-83
V-25 Incidence of Hepatocellular Carcinoma in B6C3F1 Mice
Following Chronic Dietary Exposure to Heptachlor/
Chlordane Mixture 	 V-85
V-26 Incidence of Nodular Hyperplasia In CD-I Mice Exposed
to Heptachlor/Heptachlor Epoxide (25:75) Mixture		V-87
V-27 Incidence of Hepatic Carcinoma In CD-I Mice Following
Chronic Dietary Exposure to HeptachlorrHeptachlor
Epoxide (25:75) 	 V-88
V-28 Incidences of Neoplastic Nodules in Osborne-Mendel Rats
Following Chronic Dietary Exposure to Heptachlor	 V-90
V-29 Incidence of Hepatic Carcinoma and Neoplastic Nodules
1n CFN Rats Following Chronic Oletary Exposure to
Heptachlor Epoxide	 V-93
1 x

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LIST OF TA8LES (cont.)
No.	Title	Page
V-30 Mutagenicity Testing of Chlordane 	 V-94
V-31 Mutagenicity Testing of Heptachlor and Heptachlor
Epoxide	 V-97
VI-1 Summary of Cases with CNS Effects Associated with Exposure
to Chlordane or Heptachlor, Either Alone or 1n Combination
with Other Agents	 VI-2
VI-2 Summary of Cases of Blood Dyscraslas Associated with
Exposure to Chlordane or Heptachlor, Either Alone or
with Other Agents	 	 VI-6
VI-3 Cases of Neuroblastoma Associated with Exposure to
Chlordane and Heptachlor	 VI-9
VI11 -1 Summary of Data Used to Derive HAs and DUE L s for
Chlordane 	 VI11-16
VI11-2 Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas 1n Female nice 	 VIII-22
VI11-3 Cancer Data Sheet for OeMvatlon of Potency of Chlordane
from Hepatocellular Carcinomas 1n Male Mice	 VIII-23
VIII-4 Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas 1n Male Mice 	 VIII-24
VI11-5 Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas 1n Female Mice	 VI11-25
VI11-6 Cancer Data Sheet for Derivation of Potency of Chlordane
from Liver Adenomas and Carcinomas In Male Rats	 VIII-26
VIII-7 Human Potency Estimates by Chemical 	 VIII-27
VIII-8 Summary of Data Used to Derive HAs and DWELs for
Heptachlor/Heptachlor Epoxide 	 VIII-39
VIII-9 Cancer Data Sheet for Derivation of Potency of Heptachlor
frM Hepatocellular Carcinomas 1n Male Mice 	 VIII-45
VI11-10 Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Female Mice 	 VIII-46
VI11-11 Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Hale Mtce 	 VIII-47
x

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LIST OF TABLES (cont.)
No.	Title	Page
VIII-12 Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas 1n Female Mice 		VIII-48
VIII-13 Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas 1n Male Mice		VIII-49
VIII-14 Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas 1n Female Mice 		VIII-50
VIII-15 Cancer Data Sheet for Derivation of Potency of Heptachlor
Epoxide from Hepatic Carcinomas In Female Mice		VIII-SI
VIII-16 Cancer Data Sheet for Derivation of Potency of Heptachlor
Epoxide from Hepatic Carcinomas 1n Male Mice		VIII-52
VI11-17 Cancer Data Sheet for Derivation of Potency of Heptachlor
Epoxide from Hepatic Carcinomas In Female Rats		VI11-53
x 1

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LIST OF ABBREVIATIONS
ACTH
Adrenocorticotropic hormone
AOP
Adenosine diphosphate
AEL
Adverse effect level
cyclic AMP
Adenosine 3':51 -eye 11c phosphate
ATPase
Adenosine triphosphatase
CC14
Carbon tetrachloride
CI
Confidence Interval
CNS
Central nervous system
DUEL
OMnklng water equivalent level
EEG
Electroencephalogram
FEL
Frank effect level
GC-MS
Gas chromatography-mass spectrometry
GI
Gastrointestinal
GLC
Gas liquid chromatography
GOT
Glutamlne oxalacetlc transaminase
GPT
Glutamic pyruvic transaminase
HA
Health advisory
1.p.
Intraperitoneal
1 .v.
Intravenous
LOAEL
Lowest-observed-adverse-effect level
HW
Molecular weight
NAOH
Reduced nicotinamide-adenine dlnucleotlde
NAOPH
Reduced nicotinamide-adenine dlnucleotlde phosphate
NOAEL
No-observed-adverse-effeet level
NOEL
No-observed-effect level .

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LIST OF ABBREVIATIONS (cont.)
ppm	Parts per million
RfD	Reference dose
RNA	Ribonucleic acid
SHR	Standardized mortality ratio
TLC	Thin-layer chromatography
TWA	Time-weighted average
x 111

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I. SUMMARY
Chlordane (HW 409.76) and heptachlor {MW 373.32) are structurally
related chlorinated hydrocarbon Insecticides used to control termites and
pests on field crops. Heptachlor epoxide (MW 389.32) Is an oxidation
product of heptachlor. Technical grade chlordane consists of approximately
equal parts els- and trans- Isomers, along with heptachlor and other Impuri-
ties* and has a low water solubility. Technical grade heptachlor consists
of -73% heptachlor and 22% trans-chlordane, and also has a low water solu-
bility. Heptachlor epoxide 1s slightly water soluble.
Neither chlordane nor heptachlor epoxide undergoes significant chemical
reactions In drinking water; however, both may volatilize during aeration of
drinking water. Chlordane, heptachlor and heptachlor epoxide may sorb to
organic particulate matter and be removed from drinking water during filtra-
tion. Heptachlor may hydrolyze In drinking water and may undergo oxidation
with singlet oxygen during the water treatment process. Heptachlor will
probably volatilize faster than chlordane and heptachlor epoxide during
aeration.
The absorption of chlordane by the gastrointestinal tract of animals can
be Inferred from data on Its systemic toxicity and from excretion data.
Rats absorbed at least 2-8%, while rabbits absorbed at least 30% of orally
administered chlordane. Humans also absorbed chlordane after accidental
poisoning. Chlordane and/or metabolites are rapidly distributed to tissues
of animals, with highest levels detected In adipose tissue where 1t persists
as the metabolite oxychlordane. Oxychlordane has been detected 1n human
01760
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adipose tissue. At least 13 or 14 fecal metabolites and a urinary glucuro-
nic conjugate have been Isolated In rats. ffetabollsm Involves dehydrogena-
tlon, epoxldatlon (to oxychlordane)„ hydroxylatlon and dechlorination
reactions. Metabolism by human microsomes In. vitro is similar to metabolism
by rat microsomes 1_n vitro. The major route of excretion of chlordane and
metabolites In rats 1s via the feces with SOX of a single oral dose excreted
In 10 days. Rabbits eliminate more chlordane metabolites 1n the urine than
In the feces. In humans, the half-time for elimination from serum has been
measured at 21-86 days. Oxychlordane has been found In the milk of lactat-
1ng women.
Heptachlor Is absorbed from the gastrointestinal tract of rats; residues
appear in blood within an hour of oral dosing. Absorption of heptachlor by
humans can be Inferred from reports of heptachlor epoxide levels 1n adipose
tissue of the general population. Heptachlor epoxide, the major metabolite
of heptachlor, Is distributed to tissues of animals; the highest levels are
detected In adipose tissue where 1t persists. Heptachlor 1s epoxldlzed to
heptachlor epoxide that Is dechlorlnated and hydroxylated to a metabolite
eliminated 1n the feces. Other routes of metabolism Involve epoxldatlon of
other Intermediates. Rat liver microsomes form 4 times more heptachlor
epoxide than human microsomes; otherwise, In. vitro metabolism Is similar.
Rats eliminate >50% of administered heptachlor as fecal metabolites; <5X 1s
el1m1nated In urine.
The acute oral 10^ of chlordane (In a lipophilic vehicle) 1s -350
mg/kg bw In rats, 390 mg/kg bw In mice and 1720 «>g/kg bw 1n hamsters. Neo-
natal rats are less sensitive to chlordane toxicity than are adults.
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Symptoms of acute Intoxication Include CNS disturbances, such as Irritabil-
ity, tremors and convulsions. Sublethal doses result 1n microsomal enzyme
Induction and enhanced gluconeogenesls. Subchronlc dietary administration
of chlordane to rats and mice has resulted In changes In prostate homeosta-
sis and microsomal enzyme Induction. The no-effect level for enzyme induc-
tion 15 <5 mg/kg diet. In chronic dietary studies, a no-effect level of
chlordane In rats for histopathologic changes In liver, kidney, lungs,
pancreas, stomach, adrenals, thyroid, thymus, lymph, testes, ovaries, heart
and spleen; for food consumption; for growth rate; and for mortality was 5
mg/kg diet. At 10 mg/kg diet, mild liver changes (e.g., hepatocytomegaly)
were observed. At 150-300 mg/kg, progresslvely more severe histopathologic
damage to liver and other organs occurred.
The published literature on mutagenicity testing of chlordane and
heptachlor/heptachlor epoxide Is quite similar; Indeed, most studies report
results on both chemicals. Generally, the results have Indicated that these
chemicals are not mutagenic In bacteria or In manvnallan cells In culture and
do not Induce DNA repair as measured by unscheduled ONA synthesis In rodent
hepatocytes. While dominant lethal tests In mice have been negative for
both chemicals, the absence of direct cytogenetics tests In both germinal
and somatic cells precludes a conclusion of their potential for causing
chromosome aberrations.
Heptachlor was more toxic than chlordane to laboratory animals. The
acute oral L05Q of heptachlor Is -100 mg/kg bw for rats, 70 mg/kg bw for
mice and 105 mg/kg bw for hamsters. Neonatal rats were less sensitive than
adult rats to heptachlor administered intraperitoneal^. The acute LD5Q
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of heptachlor epoxide In rats Is -60 mg/kg bw. Symptoms of acute Intoxica-
tion of heptachlor and heptachlor epoxide Included tremors, convulsions,
paralysis and hypothermia. Sublethal dietary doses resulted In microsomal
enzyme induction; the no-effect level was <2 mg/kg diet. Moderate liver
damage In rats was accompanied by Increased serum levels of hepatic enzymes
at dietary levels of 7 or 12 mg/kg bw/day for 14 days; by 28 days tolerance
appeared to develop.
Subchronlc dietary exposure to heptachlor has been associated with
changes 1n rat prostate homeostasis and Induction of hepatic microsomal
enzymes. Chronic dietary exposure to heptachlor or heptachlor epoxide has
resulted In hepatocytomegaly, hyperplasia, hepatic vein thrombosis and
cirrhosis in mice.
For humans, clinical case studies of acute exposure to chlordane
describe CNS disturbances. An estimated dose of 0.14 mg/kg bw chlordane
resulted 1n convulsions and seizures In a child. Death occurred In a woman
who Ingested 104 mg chlordane/kg bw. Blood dyscraslas, such as aplastic
anemia, leukemia and pancytopenia, have been described In cases Involving
exposure to chlordane and heptachlor, either alone or 1n combination with
other pesticides. Neuroblastoma has been associated with pre- and/or post-
natal exposure to chlordane and heptachlor.
Based on the accumulated evidence, chlordane 1s a probable human
carcinogen, classified In Group B2 under the U.S. EPA's guidelines for
carcinogen risk assessment. Animal studies provide sufficient evidence for
carcinogenicity: chlordane Increased the Incidence of liver carcinomas In
C57B1/6N, CD-I and B6C3F1 mice; liver adenomas and hemangiomas In ICR mice;
01760
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03/26/87

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and liver adenomas In Fischer 344 rats. Epidemiologic studies provide
Inadequate evidence for carcinogenicity due to methodology and data limita-
tions .
Heptachlor/heptachlor epoxide 1s a probable human carcinogen, classified
In Group B2 under the U.S. EPA's guidelines for carcinogen risk assessment.
Animal studies provide sufficient evidence for carcinogenicity: heptachlor/
heptachlor epoxide Increased the Incidence of liver carcinomas in C3H, CD-I
and 66C3F1 mice and In CFN rats. Epidemiologic studies provide Inadequate
evidence for carcinogenicity due to methodology and data limitations.
Several studies on the mechanism of neurotoxicity Indicate that EEG
changes, acetylcholine content of brain, ammonia and glutamate content of
brain, and Inhibition of synaptosomal ATPases are Involved In CNS toxicity.
Other mechanisms studied Included enhanced gluconeogenesls and effects on
cellular respiration. Phenobarbltal and turpentine enhanced the toxicity of
chlordane and heptachlor In rats. Chlordane pretreatment enhanced the toxi-
city of CCl^. The content and quality of protein In the diet affects the
toxicity of chlordane and heptachlor. An eplgenetlc mechanism for carcino-
genicity has been suggested.
Quantitative risk assessment for chlordane was based on animal data
since human data were Insufficient. Satisfactory dose-response data was not
available for a derivation of a 1-day HA for chlordane. A 10-day HA for
chlordane for a 10 kg child Is 0.063 mg/l. A DWEL derived from chlordane
toxicity data Is 0.00? mg/l. The concentrations of chlordane In water
corresponding to an Increased upper limit 11 fet 1fi*e risk of cancer of 10"4,
1Q~S and 10"* are 2.7, 0.27 and 0.027 Mg/I# respectively.
01760	1-5	03/26/87

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Quantitative risk assessment for heptachlor and heptachlor epoxide was
based on animal toxicity- data, since human data were Insufficient. A 1-day
HA for heptachlor for a 10 kg child 1s 10 pg/i. Because there was a
paucity of data to derive a 10-day HA and because there was some Indication
that rats develop tolerance to heptachlor, the value for a 1-day HA was
recommended to serve as the 10-day HA. as well. A DWEL derived from
heptachlor toxicity data 1s 20 yg/l. A DWEL derived from heptachlor
epoxide toxicity data 1s 0.4 yg/l. The concentrations of heptachlor in
drinking water corresponding to Increased lifetime risk of cancer of 10**,
10~5 and 10"* are 0.76, 0.076 and 0.0076 yg/l, respectively. The
concentrations of heptachlor epoxide In drinking water corresponding to
Increased lifetime risk of cancer of 10"4, 10~5 and 10"* are 0.38,
0.038 and 0.0038 yg/t, respectively.
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II. PHYSICAL ANO CHEMICAL PROPERTIES
Chlordane
CI
CI
CI
CI
CI
Molecular formula: C^H^Clg
Molecular weight: 409.76
RTECS number: P8 98000
Chemical Abstracts Service number; 57-74-9 and 12789-03-6
Chlordane Is the conmon name for 1,2,4,5,6,7,8,8-octachlor-
2f3,3a,4,7,7a-hexahydro-4,7-methano-lH-1ndene. This chemical Is also known
as dlchlorodene; 1,2,4,5,6,7,8,8-octachloro-4,7-methano-3a,4.7,7a-tetra-
hydrolndane; and 1.2,4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-
methanlndan, among others. Some trade names of chlordane Include Octachlor,
Velslcol 1068, Toxlchlor and Dowchlor (IARC, 1979a).
Chlordane 1s a contact Insecticide (8CPC, 1977) and has been used In the
control of cutworms, ants, root weevils, rose beetles, grasshoppers, grubs
and termites (Delchmann, 1981). Technical grade chlordane consists of 24%
transchlordane, 21.5* chlordene Isomers, 19% cls-chlordane (CAS No.
5103-74-2; RTECS No. PC01750), 10% heptachlor, 7% nonachlor and 18.5% other
miscellaneous Impurities (IARC, 1979a). HCS-3260, a high purity formulation
(98*%) contains 3:1 cls/trans chlordanes.
01770	II-l	03/26/87

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The chemical aruJ physical properties of chlordane, a viscous amber
liquid (IARCt 1979a)t are given In Table II-l.
The fate of chlordane 1n aquatic media cannot be assessed with
certainty. Although there Is evidence that chlordane may undergo sensitized
(acetone, etc.) photolysis In aquatic media (Callahan et al., 1979), the
direct photolysis of this compound 1n ambient aquatic media without any
sensitizer appears to be an insignificant process (Verschueren, 1983; Nabey
et al., 1981). From the oxidation rate constant values given by Mabey et
al. (1981), the two likely oxidation processes 1n aquatic media, Involving
singlet oxygen and peroxy/radlcal, do not appear to be significant processes
for chlordane. Similarly, both hydrolysis and blodegradatlon of this
compound In ambient aquatic media are not likely to be significant fate-
determining processes (Mabey et al., 1981; Callahan et al., 1979; Tabak et
al., 1981). The three likely processes that may determine the fate of this
compound In aquatic media are evaporation, sorption and bloaccumulatlon.
The evaporative half-life of this compound from 1 m depth of water was
estimated to be 28-33 hours (Atlas et al., 1982). Thus, although the
evaporation from aquatic media may not be rapid, 1t could be significant In
the absence of any other faster processes.
From the sediment-water sorption coefficient value (1.4x10"*) given by
Mabey et al. (1981), chlordane Is expected to be significantly sorbed from
the aquatic phase to suspended particles and sediment of high organic con-
tent In water. Therefore, sorption may play a significant role In determin-
ing the aquatic fate of chlordane (Callahan et al., 1979). In the treatment
of wastewater, a 2.1 mg carbon/t water was required to reduce the pollu-
tant concentration from 0.1 to 0.01 mq/l (U.S. EPA, 1981).
01770	II-2	03/26/87

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TABLE 11-1
Chemical and Physical Properties of Chlordane
Property
Value or Comment
Reference
Boiling point
175°C at 2 mm Hg
Callahan et al., 1979
Melting point
106-107'C (ds-lsomer)
104-105'C (trans-lsomer)
BCPC, 1977
Specific gravity
1.59-1.63 at 25°C
Delchmann, 1981
Vapor pressure
1 x 10"® mm Hg at 25aC
IARC, 1979a
Solubility .
9 yg/l at 25*C In water
for technical grade and
56 yg/t In water for
c1s:trans (75:25)
chlordane; mlsclble with
allphat1c and aromatic
hydrocarbon solvents
Verschueren, 1983;
Delchmann, 1981
Evaporation
half-life {t-j/2)
from water
28 hours (trans-lsomer)
from 1 meter depth;
33 hours (c1s-1somer)
from 1 meter depth
Atlas et al., 1982
Log (octanol/water
partition coefficient)
3.32
Rao and Davidson, 1982
Air conversion factor
1 mg/t * 59.7 ppm and
1 ppm = 16.76 mg/ra*
at 25*C; 760 mm Hg
Delchmann, 1981
01770
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Heptachlor
CI
CC1
CI
Molecular formula:
Molecular weight: 373.32
RT6CS number: PC 07000
Chemical Abstracts Service number: 76-44-8
Heptachlor 1s the conmon name for 1l4,5»6,7t8,8-heptachlor-3a.4,7,7a-
tetrahydro-4,7-niethano-lH-1ndene. This chemical 1s also known as 3-chloro-
chlordene and 3,4t5,6,7,8l8a-heptachlorod1cyclopentad1ene. Some trade names
of heptachlor include Velslcol 104, Orlnox, Heptagran, Rhodlachlor and E3314
(IARC, 1979b).
Heptachlor is a contact Insecticide with some fumlgant action {8CPC,
1977) used to control termites and Insects that attack field crops (Delch-
mann, 1981). Technical heptachlor consists of -73* heptachlor, 22% trans-
chlordane and 5% nonachlor (Oelchmann, 1981).
The chemical and physical properties of 99% pure heptachlor (unless
otherwise stated), a white crystalline solid (IARC, 1979b), are given In
Table II-2.
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03/12/84

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TABLE II-2
Chemical and Physical Properties of Heptachlor
Property
Value or Description
Reference
Bo 111ng point
135-145'C at 1-1.5 mm Hg
IARC, 1979b
Melting point
93°C
IARC, 1979b
Specific gravity
1.57-1.59 (unknown purity)
Delchmann, 1981
Vapor pressure
3 x 10"* mm Hg at 25*C
IARC, 1979b
Absorption
spectrometry
\nax 236 nm
(e] . 1611), 309 nm
(E - 270), 328 nm

-------
The fate of heptachlor 1n aquatic media has been studied by several
Investigators. This compound may undergo significant photolysis 1n ambient
aquatic media (Callahan et al., 1979). Verschueren (1983) reported that
sunlight and artificial fluorescent light Irradiation of 10 pg/i of this
compound In a river water placed Inside a sealed glass Jar produced 75%
decomposition In 1 week and complete decomposition 1n 2 weeks. From the
estimated oxidation rate constant values given by Mabey et al. (1981), the
singlet oxygen reaction with heptachlor 1n aquatic media may be signifi-
cant. Based on an estimated rate constant of 3x10~LO H~x hr-1 (Habey
et al., 1981), and a concentration of 10"12 H for singlet oxygen in
surface water (H111 et al., 1982), the half-life of heptachlor for this
reaction 1s estimated to be *1 day.
The hydrolysis of heptachlor In aquatic media Is also an important
process. It has been estimated that the hydrolytlc half-life of heptachlor
Is In the range of 1-3 days (Callahan et al., 1979; Habey et al., 1981).
The volatilization half-Hfe of heptachlor from aquatic media Is estimated
to be In the range of 2-10 days from pond, river and lake water. This
estimate Is based on the re-aeration rate ratio of 0.355 given by Mabey et
al. (1981) and oxygen re-aerat1on rate of 0.19 day"1, 0.96 day*1 and
0.24 day'1 for pond, river and lake water, respectively as given by Mil
et al. (1982). Although the evaporative half-life of heptachlor 1n aquatic
media appears to be longer than that of chlordane (2-10 days vs. 28-33
hours) from this estimate, Huang (1970) reported that the evaporation rate
of heptachlor from aquatic media Is faster than chlordane.
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08/09/84

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From the sediment-water sorption coefficient value of 1.2xl0~4 given
by Mabey et al. (1981), heptachlor may be significantly sorbed onto suspend-
ed particles and sediments of high organic content present In aquatic
media. Therefore, sorption may play a significant role In determining the
fate of heptachlor 1n aquatic media (Callahan et al., 1979). In the treat-
ment of wastewater, 5.9 mg carbon/t water was required to reduce the pol-
lutant concentration from 0.1 to 0.01 mg/t (U.S. EPA, 1981). The blode-
gradatlon rate of heptachlor 1n aquatic media 1s slower than the rate of
hydrolysis (Mabey et al.t 1981; Callahan et al., 1979).
Heptachlor Epoxide
CI
CC1
CI
CI
Molecular formula: C^H^C^O
Molecular weight: 389.32
RTECS number: PB 94500
Chemical Abstracts Service number: 1024-57-3
Heptachlor epoxide 1s the common name for 2,3,4,5,6,7.7-heptachloro-
1a,1b,5,5a,6,6a-hexahydro-2,5-methano-2H-1ndene(l,2-b)oxIrene. This chemi-
cal Is also known as epoxyheptachlor; Velslcol 53-CS-17; ENT 25, 584;
1,4,5,6,7,8,8-heptachloro-2,3-epoxy-3a,4,7,7a-tetrahydro-4,7-methano1ndan.
Heptachlor epoxide 1s not conmerclal1y available In the United States, but
1t 1s an oxidation product of heptachlor (IARC, 1979b; Verschueren, 1983).
01770
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02/18/87

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The chemical and physical properties of heptachlor epoxide are given In
Table II-3.
The fate of heptachlor epoxide In aquatic media has not been comprehen-
sively studied. From the Information available In the reviewed literature,
photolysis, oxidation or hydrolysis are not expected to be a significant
fate-determ1n1ng process for this chemical 1n aquatic media (Mabey et al.,
1981; Callahan et al., 1979; Verschueren, 1983). from the estimated bio-
transformation rate constant (3x10"* ml cell"1 hr"1) given by Mabey
et al. (1981), and the estimated microbial population in surface water
(5xl0~* cell mi*1) given by Burns et al. (1981), the blodegradatlon of
this compound does not appear to be a significant process.
The two processes that may determine the fate of heptachlor epoxide 1n
aquatic media are volatilization and sorption. Huang (1970) reported that
the rate of volatilization of heptachlor epoxide from aquatic media Is much
slower compared with heptachlor. However, tt Is difficult to make a reason-
able estimate of the evaporative half-life for this compound because of the
lack of appropriate data. The sorption of this compound on suspended
particulate matter or sediment of high organic content may be a moderately
significant process (Mabey et al., 1981; Callahan et al., 1979). In the
treatment of wastewater, a 2.2 mg/l carbon dose was required to reduce the
concentration of this compound from 0.1 to 0.01 mg/l (U.S. EPA, 1981).
01770
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02/18/87

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TABLE II-3
Chemical and Physical Properties of Heptachlor Epoxide
Property
Value or Description
Reference

Melting point
160-161.5°C {99.5% pure)
I ARC, 1979b

Vapor pressure
(estimated)
3 x 10*« mm Hg at 25°C
Mabey et al. t
1981
Solubility
0.35 mg/l at 25'C
Mabey et al.,
1981
Log octanol/water*
partition coefficient
5.40
4.43
2.65
Mackay, 1982
Geyer et al.,
Mabey et al.,
1982
1981
Conversion factor
1 ppm = 15.9 mg/ma
at 25°C and 760 mm of Hg


•The difference In these calculated values Is due to the different methods
used for estimating the partition coefficient.
01770
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08/09/84

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Sunyiar v
Chlordane and heptachlor are structurally related chlorinated hydro-
carbon Insecticides that have been and/or are being used to control pests on
field crops and termites. Heptachlor epoxide Is a degradation product of
heptachlor. Technical grade chlordane Is a yellow-colored liquid consisting
of approximately equal parts c1s- and trans-chlordane Isomers, along with
chlordane Isomers, heptachlor, nanachlor and their impurities. It bolls at
175°C at 2 mm Hg (Callahan et al., 1979), melts at 104-107"C (BCPC, 1977 ),
has a vapor pressure of lxlO*5 mm Hg at 25°C (I ARC * 1979a) and has a low
water solubility but Is soluble 1n most hydrocarbon solvents (Delchrnann,
1981 J. Chlordane has a log octanol/water partition coefficient of 3.32 (Rao
and Davidson, 1982).
Technical grade heptachlor Is approximately 73% heptachlor and 22%
transchlordane. The boiling point of the pure chemical (a white crystalline
solid) Is 135-145°C at 1-1.15 mm Hg; Its melting point Is 93°C and Its vapor
pressure Is 3x10** mm Hg at 25°C {I ARC. 1979b). Like chlordane, 1t has a
low water solubility but Is soluble In organic solvents. Literature values
for log octanol/water partition coefficient were reported as 3.87 (Rao and
Davidson, 1982) and 5.44 (Mackay, 1982) For heptachlor.
The melting point of pure (99.5%) heptachlor epoxide Is 160-161.5®C
(IARC, 1979b) and has an estimated vapor pressure of 3x10~4 mm Hg at 25°C
(Mabey et al., 1981). It is slightly soluble In water (Mabey et al., 1981)
and has a log (octanol/water partition coefficient) In the range of
2.65-5.40 (Mackay, 1982; Geyer et al., 1982; Mabey et al., 1981).
01770
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02/18/87

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Neither chlordane nor heptachlor epoxide will undergo significant chemi-
cal reactions In drinking water (Callahan et al., 1979; Habey et al., 1981).
Volatilization of both chlordane and heptachlor epoxide 1s possible during
aeration of drinking water; however, the rate of volatilization is faster
for chlordane than heptachlor epoxide (Huang, 1970). Both compounds may be
sorbed onto organic particulate matter and may be removed during the
filtration processes. The rate of sorption with chlordane Is higher than
heptachlor epoxide (Habey et al., 1981; Callahan et al.# 1979). Heptachlor.
on the other hand, may hydrolyze In drinking water (Habey et al., 1981;
Callahan et al., 1979) and may undergo oxidation with singlet oxygen (Habey
et al., 1981) during the water treatment process. Volatilization of hepta-
chlor Is possible during aeration of drinking water at a rate faster than
that of either chlordane or heptachlor epoxide (Huang, 1970). Heptachlor
may be sorbed onto organic particulate matter (Habey et al., 1981; Callahan
et al., 1979) and may be removed during filtration of drinking water.
01770
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08/09/8A

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III. TOXICOKINETICS
Animal Studies
Chlordane.
Absorption -- Quantitative data on the absorption of chlordane from
the gastrointestinal tract of animals are not available; however, the
systemic toxicity of the Insecticide to laboratory animals following oral
exposure (Chapter V) Indicates that some absorption does take place. That
chlordane 1s absorbed can also be Inferred from excretion data. As dis-
cussed 1n Chapter II, chlordane consists of a mixture of components, Includ-
ing the c1s- and trans-1somers. A high purity (98+%) formulation of the
Insecticide, designated HCS-3260, contains els- and trans-chlordane 1n a 3:1
ratio. Sprague-Dawley rats (one of each sex) received a single dose of
0.05, 0.2 or 1.0 mg 14C-HCS-3260/kg bw by gavage, or a single oral dose of
each 14C-labeled Isomer at 0.2 rug/kg bw (Barnett and Dorough, 1974).
Elimination of radioactivity 1n the urine over 7 days was 6* for females and
2% for males following l4C-HCS-3260 treatment. Following treatment with
c1s- and trans-ch1ordane-"C, female rats eliminated 8.5 and 5% of the
administered radioactivity, respectively. These results Indicate that at
least 2-8.5% of the administered chlordane dose was absorbed by the gastro-
intestinal tract of rats. In contrast, a male rabbit that was given
14C-HCS-3260 1n a dietary concentration of 25 ppm (25 mg/kg diet) for 2
days excreted 33% of the radioactivity 1n the urine and 21% in the feces 24
hours after termination of the treatment (Barnett and Dorough, 1974).
Pulmonary absorption of an unspecified amount of 14C-chlordane (11,500
dpm/wg) in 20 ethanol administered as an aerosol Intratracheally to
female Sprague-Oawley rats was observed by Nye and Dorough (1976). No
01780
r i c-i
08/09/84

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Intact "C-chlordane was detected In exhaled air. A peak blood concentra-
tion of radioactivity of -4% of the applied dose was reached 1n <5 minutes.
Ambrose et al. (1953b) reported 1n an abstract that dermal application
of 50 mg chlordane/kg bw was more toxic to rats when the chemical was
applied In cottonseed oil rather than ethyl alcohol, Indicating greater
absorption with the lipophilic vehicle.
Distribution — The tissue distribution of l4C-HCS-3260, ds- or
trans-ch1ordane-14C and the metabolite, oxychlordane, In male and female
rats following treatment with single oral doses was compared by Barnett and
Oorough ( 1974). At one day following doses of 0.05-2.0 mg/kg bw of the
respective compounds, the concentrations of radioactive equivalents In
brain, muscle, liver and kidney were generally low (0.00-0.08 ppm), while
the concentrations of radioactivity in fat were somewhat higher (average for
all treatments -0.47 ppm). Male and female rats treated with 0.1 mg
14C-HCS-3260/kg bw had higher tissue residue levels of radioactive
equivalents 1n liver (0.50 ppm}, kidney (0.26 ppm) and fat (3.71 ppm for
females, 2.58 ppm for males). In general, female rats accumulated greater
concentrations of radioactivity 1n fat than male rats after treatment with
any preparation. At 7 days after dosing with 1.0 mg l4C-HCS-3260/kg bw,
radioactivity 1n all tissues declined; radioactivity In fat declined to 2.0
ppm for females and 1.19 ppm for males. Slightly more radioactivity was
present 1n rat tissues following oral doses of trans- than ds-chlor-
dane-14C,
01780
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08/09/84

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When 1*C-HCS-3260 was administered to male rats In the diet at 5 ppm
(S rug/kg diet) for 56 days, the tissue distribution of radioactive equiva-
lents was 0.42, 0.91, 0.55, 0.68 and 14.73 ppm for muscle, brain, kidney,
liver and fat, respectively (Barnett and Oorough, 1974). After discontinu-
ing treatment for 28 and 56 days, the concentration of radioactivity 1n fat
declined to 3.67 and 2.49 ppm, respectively. Radioactivity was still
detected In other tissues 56 days after treatment was terminated. Greater
accumulation of radioactivity 1n all tissues resulting from trans- rather
than from c1s-chlordane-14C occurred In female rats treated with 25 ppm
(25 mg/kg diet). Analysis of the nature of the radioactivity revealed that
-30-60% of the radiocarbon was associated with oxychlordane.
Ambrose et al. (1953b) found that the perirenal fat of male rats con-
tained 43, 41 and 81 ppm of chlordane residues following feeding for 5, 148
and 407 days, respectively, of a diet containing 320 ppm (320 mg/kg diet).
The fat of female rats contained approximately twice the values for males.
Residues of parent Isomers and oxychlordane In adipose tissue from male
and female Holtzmann rats maintained on diets containing 50-200 ppm (50-200
mg/kg diet) c1s- or trans-chlordane, or 100 ppm (100 mg/kg diet) of fixed
ratios of the Isomers from 9:1 to 1:9 trans:cls ratios, or 50 ppm (50 mg/kg
diet) technical chlordane for 15 days were determined by Street and Blau
(1972). Adipose tissue of female rats fed trans-chlordane at 50, 100 and
200 mg/kg diet contained -6, 10 and 23 wg/g lipid of trans-chlordane,
respectively. Much greater concentrations of oxychlordane than trans-chlor-
dane were stored 1n adipose tissue by females (-104, 202 and 471 ug/g
lipid) and males (-5, 15 and 22 vq/q lipid) after being fed trans-chlor-
01780
III-3
03/26/87

-------
dane. The feeding of ds-chlordane likewise resulted In greater fat storage
of the metabolite than of the parent Isomer In both male and female rats;
however, the ratio oxychlordane:Isomer was reduced. Female rats fed 50 ppm
technical grade chlordane (50 mg/kg diet) stored -7 times more oxychlordane
than the parent compound In adipose tissue. The results of feeding ratios
of c1s- and trans-chlordane Indicated that oxychlordane accumulation was
additive for each Isomer.
Polen et al. (1971) also detected oxychlordane In the fat depots of
rats, dogs and pigs maintained on diets containing Individual Isomers or
technical chlordane. Rats received dietary concentrations of els-, trans-
or technical grade chlordane of 0-150 ppm (0-150 mg/kg diet) for 1 year;
dogs received technical grade chlordane at 3 or 30 ppm (3 or 30 mg/kg diet)
for 2 years; pigs were fed diets containing the c 1 s- or trans-lsomer at 300
ppm (300 mg/kg diet) for 90 days. The respective levels of oxychlordane In
the fat for rats, dogs and pigs were 0.2-150, 3-24 and 36-90 ppm.
Rabbits received trans-chlordane-14C dally fier os In doses of 14.3
mg/rabb1t/day for 10 weeks (Poonawalla and Korte, 1971). Two weeks after
treatment was discontinued, low levels of radioactivity were detected In
kidney (0.05%), liver (0.52%), heart (0.09%), lung (0.04%), spleen (0.03%),
testes (0.03%) and brain (0.04%). Higher levels were found In adipose
tissue (2.59% In abdominal fat, 1.53% in subcutaneous fat) and muscle
(5.60%). Barnett and Oorough (1974) found that the tissues of a rabbit fed
14C-HCS-3260 (25 mg/kg diet for 2 days) contained 14C-oxychlordane and
l4C-d1chlorochlordene.
01700
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03/26/07

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The distribution of radioactivity In the tissues of rabbits administered
c1s- or trans-chlordane-14C orally 1n four doses, one given every 4 days,
of 100 mg )n a capsule per rabbit was as follows: fat > kidney > muscle >
liver > brain (c1s-); kidney > fat > liver > muscle > brain (trans-) (Balba
and Saha, 1978). The majority of the radioactivity 1n the tissues was
associated with oxychlordane, regardless of which Isomer was administered.
The mobilization of chlordane or Its metabolites from adipose tissue of
rats after food deprivation was studied by Ingle (1952). Chlordane was
removed from the diet after 80 week* maintenance of 16 rats on a diet
containing chlordane at 150 ppm (150 mg/kg diet). At weekly Intervals, two
male and two female rats were fasted for 48 hours and observed for symptoms
of toxicity. After 4 weeks, symptoms such as tremors and hyperactivity were
no longer observed.
The distribution of radioactivity (expressed as % of administered radio-
activity/tissue) 1n selected tissues of rats following the previously
described Intratracheal administration of ^C-chlordane (Nye and Oorough,
1976) was 23.9% 1n lungs, 19.6% 1n liver, 0.3% 1n kidney and 0.1% 1n the
bladder and Its contents.
Metabolise — The metabolism of chlordane has been fairly well studied
1n rats and rabbits Vn vivo and In vitro. Oxychlordane was a metabolite of
both els- and trans-chlordane (Street and Blau, 1972). A metabolic pathway,
based upon In. vitro studies with rat liver homogenates, was proposed In
01780
111-5
08/09/84

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which each Isomer \s dehydrogenated to 1,2-d1chlorochlordene with subse-
quent epoxldation to oxychlordane. In these experiments, trans-chlordane
was converted to oxychlordane at a 7-fold greater rate than was the cls-
Isomer.
Barnett and Oorough (1974) Isolated seven radioactive metabolites (In
addition to the respective unchanged parent compounds) In the feces of rats
that had been administered c1s- or trans-chlordane-1*C or the 3:1 mixture
of c 1 s- and trans-chlordane, designated as 14C-HCS-3260, either as single
oral doses (0.2 mg/kg bw) or by continuous feeding (5 mg/kg diet of
14C-HCS-3260 for 56 days or 25 mg/kg diet els- or trans-chlordane-14C
for 14 days). These metabolites, analyzed by TLC and GLCt were tentatively
Identified as hydroxychlordane; chlordene chlorohydrln; monochloro and
dlhydroxy derivatives of chlordane; ds- and/or trans-dlhydroxychlordane
derivatives; a trlhydroxylated chlordene; and a conjugated form of a
hydroxylated chlordane metabolite. No oxychlordane or dlchlorochlordene was
detected 1n feces; however, oral administration of oxychlordane resulted In
fecal excretion of unchanged oxychlordane. The nature of urinary metabo-
lites was essentially the same as fecal metabolites In rats fed HCS-3260 in
dietary concentrations of 25 mg/kg diet: oxychlordane was also present. The
24-hour feces of a rabbit fed l4C-HCS-3260 at 25 mg/kg diet for 2 days
contained the same fecal metabolites found In the rat, although the amounts
of unchanged Isomers were greater. The urine of the rabbit contained a
greater percentage of the conjugated hydroxylated metabolites than did the
urine of rats.
01780
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03/26/87

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In the previously described study 1n rabbits, 8alba and Saha (1970)
Identified the following urinary metabolites of c1s-chlordane: l-hydroxy-2-
chlorochlordene, trans-chlordene chlorohydrln and 1-hydroxychlordene.
Urinary metabolites 1n rabbits of trans-chlordane were l-hydroxy-2-chloro-
chlordene, 1,2-dichlorochlordene, trans-chlordene chlorohydrln and
3-hydroxychlordane.
The metabolism of ds- and trans-ch1ordane-1#C was also studied in
rats 1_n vivo and in v1 tro by Tashlro and Matsumura ( 1977). Male rats main-
tained on diets containing c1s- or trans-chlordane-14C In a concentration
of 100 ppra (100 mg/kg diet) for 4 weeks excreted 13 metabolites from cis-
and 14 metabolites from trans-chlordane 1n the feces. The fecal metabolites
of both Isomers, Identified by TLC and*.GLC, included heptachlor, 1,2-d1-
chlorochlordene, oxychlordane, l-hydroxy-2-chlorochlordene, 1-hydroxy-2-
chloro-2,3-epoxychlordene, chlordene chlorohydrln, monohydroxy dlhydrochlor-
dene, 1,2-d1hydroxychlordene and trihydroxydihydrochlordene. These metabo-
lites were found In different proportions, depending upon the administered
Isomer. The only urinary metabolite identified from both c1s- and trans-
chlordane was a glucuronlde conjugate of 1-hydroxyd1hydrochlordene. The in.
v1 tro Incubation of the Isomers with rat liver microsomes and cofactors
resulted in the same metabolites qualitatively.
Based upon these In. v1 vo and In, v1 tro results, Tashlro and Hatsumura
(1977) concluded that chlordane 1s dehydrogenated to dlchlorochlordene,
which Is epoxldlzed at carbons 2 and 3 and hydroxylated at carbon 1 (Route
A). An additional route whereby chlordane 1s hydroxylated at carbon 1 to
yield 1-hydroxydlhydrochlordene was also proposed (Route 8). These pathways
are Illustrated 1n Figure 111-1.
01700	III-7	00/09/84

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-------
Brlmfleld et al. (1978) also studied the jn, vitro metabolism of the pure
els- and trans-1somers of chlordane by microsomal preparations from Sprague-
Dawley rats fed the Individual Isomers In dietary concentrations of 100 ppn
(100 mg/kg diet) for 9 days. Incubation of cls-chlordane-lnduced enzymes
with cls-chlordane resulted in the following metabolites, Identified by
GC-MS: cls-chlordane, dlchlorochlordene, oxychlordane, dlhydroheptachlor,
hydrochlordene and 1 -chloro-2-hydroxyd1hydrochlordene. Similar products
from trans-chlordene were Identified; however, heptachlor rather than
dlhydroheptachlor was formed, as was hydroxychlordane. A metabolic pathway,
based upon reductive dechlorination via dlhydroheptachlor to dlhydrochlor-
dene, was proposed. According to this scheme, these molecules can also be
hydrolyzed, desaturated and epoxldlzed. In the Brlmfleld et al. (1978)
proposal, oxychlordane Is an endpolnt, whereas, 1n the scheme proposed by
Tashlro and Matsumura (1977), oxychlordane undergoes further hydroxylatlon
and reduction. Many of the proposed Intermediates In the Brlmfleld scheme
have not actually been found In this or other studies (Brlmfleld et al.,
1978).
Excretion — In the previously described experiments of Barnett and
Oorough (1974), >90% of the administered radioactivity was excreted over 7
days by rats that were given single oral doses (0.2 mg/kg diet) of c1s- or
trans-chlordane-14C or 1AC-HCS-3260. Females excreted 5-6% of the
radioactivity In the urine; males excreted 2-3%. At higher doses (0.5 or
1.0 mg/kg diet) of HCS-3260, the pattern of elimination was essentially the
same. When 1*C-HCS-3260 was fed in the diet for 56 days, fecal elimina-
tion as measured by radioactivity was 70% for the 1 mg/kg diet level, 75%
01780
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for the 5 mg/kg diet level and 80% for the 25 mg/kg diet level. Indicating
possible decreased absorption: the Investigators made no mention of possible
biliary excretion. Elimination of cls-chlordane (75%) was greater than
trans-chlordane (65%) following 14 days of dietary levels of the Isomers at
25 mg/kg diet.
Tashlro and Natsumura (1977) reported similar results for elimination
from rats treated with single oral doses of els- (5.4 mg/kg bw) and trans-
ch1ordane-A*C (9.7 mg/kg bw) 1n corn oil. The total 7-day elimination of
radioactivity from els- and trans-chlordane was 86 and 66% of the adminis-
tered dose, respectively. The 24-hour total excretion was 59% for els- and
27% for trans-chlordane.
Rabbits excreted 18% of an orally administered single dose of 200 mg/kg
bw of chlordane In the urine collected over 16 days when organic chlorine
content was measured (Stohlman et al., 1950). The peak urinary elimination
of organic chlorine occurred within 2 days and amounted to 9% of the dose.
Poonawalla and Korte (1971) observed appreciable urinary excretion of
radioactivity by rabbits that received 14.3 mg/rabblt of trans-chlordane-
14C dally for 10 weeks. At the end of this period, -70% of the dally dose
had been eliminated: 22.7% was excreted In the feces, 30% of which was
unchanged trans-chlordane; and 47% was excreted as urinary metabolites. In
agreement, with these results, excretion of radioactivity by one rabbit 24
hours after termination of feeding 25 ppm (25 mg/kg diet) of HCS-3260-14C
for 2 days was 21% In feces and 33% In urine (Barnett and Dorough, 1974).
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Balba and Saha (1978) also observed appreciable urinary excretion of the
1*C-label from els- or trans-chlordane-14C by rabbits that were treated
with either Isomer orally at a dose of 100 mq/rabblt In a capsule every 4
days up to 400 mg/rabb1t; however, In this study, urinary excretion did not
exceed fecal excretion. For the cls-lsomer, 48.5% and 20.4% of the radio-
activity were eliminated In the feces and urine, respectively. For the
trans-1somer, fecal and urinary excretion were 46.1% and 35.8%, respectively.
When an unspecified amount of chlordane-14C In 20 ul of ethanol was
administered Intratracheally to female Sprague-Oawley rats, elimination of
radioactivity was primarily In the feces (Nye and Oorough, 1976). After a
lag period of 2 days during which <20% of the dose was eliminated, fecal
excretion rose to -50% by day 4 and 56% by day 6. Urinary excretion over 6
days amounted to 12% of the dose. No radioactivity was detected In the
expired air of these animals.
Heptachlor and Heptachlor Epoxide.
Absorption — Limited Information on the absorption of heptachlor fol-
lowing Ingestion by animals was available. A Russian study (Mlzyukova and
Kurchatov, 1970) has reported that heptachlor administered Intragastrlcal1y
In a single oral dose of 120 mg/kg bw to rats was detected In blood within
0.5-1 hour of administration.
The absorption of heptachlor following Inhalation exposure also has not
been well studied. From July 1 , 1972 to October 4, 1972, Arthur et al.
(1975) placed 10 rabbits of each sex 1n open-air cages so that they were
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exposed to the ambient air of Stonevllle, Mississippi, an area where Insec-
ticides had been heavily used. Control groups of male and female rabbits
(10 each) were housed 1n a room at Mississippi State University, a low
pesticide use area. The average air levels of heptachlor epoxide (hepta-
chlor was either not measured or not detected) 1n Stonevllle air was 1.86
5
ng/ma; the air at Mississippi State was not sampled. Heptachlor epoxide
residue levels 1n adipose tissue of the test rabbits was 0.039 ppm compared
with 0.016 ppm In controls (p<0.001). The average respiratory Intake of
heptachlor epoxide was calculated ' as 0.002 yg/day for rabbits In the
Stonevl11e area.
Distribution and Retention — An abstract of a Russian study (Mlzyu-
kova and Kurchatov, 1970), reported that following a single Intragastric
dose of 120 mg/rat heptachlor to female rats, heptachlor was detected In
blood, liver, kidney and adipose tissue within 1 hour. After 4 hours,
heptachlor epoxide, a metabolite of heptachlor, was detected 1n blood, liver
and fat and persisted 1n the adipose tissue for 3-6 months.
Radomskl and Oavldow (1953) studied the tissue distribution of hepta-
chlor epoxide In rats and dogs following oral administration of heptachlor.
Nine rats of either sex received dietary concentrations of heptachlor at
30-35 pp« (30-35 mg/kg diet) for 2 months. Heptachlor epoxide levels 1n the
fat of six female rats averaged 384 yg/g tissue, while fat In male rats
contained an average of only 43 yg/g tissue. Much lower levels of hepta-
chlor epoxide were detected In liver (0.4-33 yg/g), kidney (0-21 yg/g)
and muscle (0-27 vg/g). None was detected Ir-1 brain tissue. A similar
pattern of distribution was observed In three female dogs that received 1 mg
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heptachlor/kg bw 1n capsules dally for 12-18 months. Fat contained an
average heptachlor epoxide concentration of 636 wg/g; liver, 36 wg/g;
kidney, 7.6 wg/g; and muscle, 13 »g/g. Again, no heptachlor epoxide was
detected 1n the brain. Small amounts of unchanged heptachlor were detected
In the fat of dogs when higher levels of the Insecticide were administered.
Administration of 1 mg heptachlor/kg bw to four dogs resulted 1n no detect-
able heptachlor 1n fat after 26 weeks, while three of eight dogs, surviving
6-18 weeks, dosed with 3 mg/kg bw dally had <1 yg heptachlor/g fat tissue.
A dally dose of 5 mg/kg bw resulted in an average concentration of hepta-
chlor 1n fat of 6.5 wg/g. This dose was fatal to the dogs within 2-11
weeks.
Radomskl and Davldow (1953) also determined the rate of accumulation and
disappearance of heptachlor epoxide 1n rats (three rats/sex/group) fed a
diet containing 30 ppm (30 mg/kg diet) heptachlor. One group was sacrificed
each week for 12 weeks. Female rats accumulated heptachlor epoxide to a
maximum level of -225 wg/g fat 1n 8 weeks compared with only 50 wg/g fat
In males. When the test diet was discontinued, heptachlor epoxide was no
longer detected at 8 weeks for female fat tissue and 6 weeks for male fat
tissue. Further tests established that the maximum dietary level at which
fat storage did not occur was 0.3 mg/kg diet for male rats and 0.1 mg/kg
diet for female rats.
Although Radomskl and Davldow (1953) failed to detect heptachlor epoxide
in rat and dog brain tissue after heptachlor treatment, Yamaguchl et al.
(1980), using a GC method, detected 3.15 ppm heptachlor epoxide In brain
tissue of rats 5 hours after an Intraperitoneal Injection of 200 mg hepta-
chlor/kg bw.
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Metabolism — Davldow and Radomskl (1953) first Identified heptachlor
epoxide in the adipose tissue of dogs that had been treated with dally oral
doses of 1-3 mg heptachlor/kg bw 1n corn oil. They concluded that the
metabolite arose by the epoxldlzatlon of heptachlor. Subsequently, these
Investigators (Radomskl and Davldow, 1953) also Isolated heptachlor epoxide
from the adipose tissue of male and female rats maintained on diets contain-
ing 30-35 ppm (30-35 mg/kg diet) heptachlor.
In an abstract of a Russian study, Ermakov (1977) reported that hepta-
chlor, administered to rats and rabbits In single oral doses of 28-50 mg/kg
bw, was metabolized by reactions Involving hydrolysis, hydroxylatlon,
epoxldatlon, dehydrogenatlon and molecular rearrangement, but details as to
Intermediate structures were not given 1n the abstract.
The most extensive Investigation of the metabolic fate of heptachlor In
rats was performed by Tashlro and Matsumura (1970) U\ v1 vo and J_n vitro.
Over a 10-day period following a single oral unspecified dose of
A4C-heptachlor (position of label not specified) In corn oil, rats
excreted >50% of the administered radioactivity in feces, and <5% In urine.
The relative abundance of fecal metabolites, expressed as percent of
14C-compounds, were as follows: unchanged heptachlor, 26.2%; heptachlor
epoxide, 13.1%; 1-hydroxychlordene, 19.5%; 1-hydroxy-2,3-epoxychlordene,
17.5%; 1,2-d1hydroxyd1hydrochlordene, 3.5%; and two other unnamed metabo-
lites, one of which accounted for 19% of the radioactivity, the other for
<0.1%. The latter metabolite was designated as H-2 by these authors, and
Its structure is Identical to that proposed by Matsumura and Nelson (1971)
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for what they called the fecal metabolite of heptachlor epoxide. The In
vitro metabolism of 14C-heptachlor by rat liver microsomal preparations
resulted In the following relative abundance of metabolites, expressed as %
of the total 14C-compounds: heptachlor, 4.4%; heptachlor epoxide, 85.8%;
1-hydroxychlordene, 2.9%; 1-hydroxyepoxychlordene, 3.0%; 1,2-d1hydroxyd1hy-
drochlordene, 0.9%; and an unknown metabolite, 3.0% (Tashlro and Matsumura,
1978). No H-2 fecal metabolite was detected. The Identity of the metabo-
lites was confirmed by TLC analysis and comparison with authentic standards
of fecal metabolites from rats fed diets containing 100 ppm (100 rag/kg diet)
heptachlor for 4 weeks. A pathway of heptachlor metabolism by rats was pro-
posed and Is Illustrated 1n Figure II1-2.
Hatsumura and Nelson (1971) administered heptachlor epoxide to four rats
1n dietary concentrations of 10 ppm (10 mg/kg diet) for 30 days. The
authors estimated that each rat consumed 5 rog of heptachlor epoxide over the
test period and excreted 950 »g of a fecal metabolite (see Figure III-2)
and 66 ug of heptachlor epoxide In the feces.
Brooks et al. (1968, 1970) Investigated the .In vitro metabolism of
heptachlor epoxide by pig liver microsomes. The product, formed upon
Incubation at 45*C for 60 hours, was Identified as heptachlor epoxide dlol:
CI
CI cx
Incubation of heptachlor epoxide with rabbit microsomes also resulted In the
formation of heptachlor epoxide dlol as well as another unidentified product.
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CI CI
Hepcachlor Epoxide
un-named fecal metabolite
H-2
CI OH
1-Hydro*ychlordene
CI OH
l-hydroxy-2,3-epoxychlordene
CI OH
1,2-dihydroxydlhydrochlordene
FIGURE 111-2
Proposed Metabolic Pathway of Heptachlor In Rats
Source: Tashlro and Matsumura, 1978
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Excretion — Tashlro and Matsumura (1978) reported that rats excreted
>50% of the administered single oral unspecified dose of radioactivity from
l4C-heptachlor In the feces over 10 days. Urinary excretion of radio-
activity accounted for <5% of the administered amount.
In an abstract of a Russian study (Ermakov, 1977), It was reported that
16-40% of the orally administered dose of heptachlor (28-50 mg/kg bw) to
rats and rabbits was excreted unchanged In the feces and that heptachlor
epoxide was excreted over at least a 12-month period. Further details were
not given In the abstract. As reported In an abstract of another Russian
study, Mlzyukova and Kurchatov (1970) showed that heptachlor and Its metabo-
lites were excreted mainly In the feces of rats within 5 days of an Intra-
gastric dose of 120 mg/kg bw.
Each of four male albino rats excreted an average of 950 wg of the
fecal metabolite (see Figure III-2) of 99% pure heptachlor epoxide during 30
days of maintenance on a diet containing 10 ppm (10 mg/kg diet) heptachlor
epoxide (each rat consumed -5 mg over 30 days) (Natsumura and Nelson, 1971).
Approximately 66 yg of unchanged heptachlor epoxide was also excreted In
the feces.
Huwan Data
Chlordane.
Absorption — A 20-month-old boy (12.7 kg) drank an unknown quantity
of 74% technical grade chlordane from a bottle (Curley and Garrettson,
1969). He vomited 45 minutes later and convulsions began shortly there-
after. He was admitted to the hospital 75 minutes after ingestion of
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the pesticide. The concentration of chlordane In a blood sample taken 2.75
hours after Ingestion was 2.71 mg/i. The concentration In fat was 3.12
mg/kg bw. A 4-year-old girl (11 kg) drank an unknown amount of a 45%
chlordane formulation (Aldrlch and Holmes, 1969). She was admitted to the
hospital 3 hours later, and gastric lavage was performed. The first serum
sample taken (time not specified) contained 3.4 ppm (mg/kg serum), from
which the Investigators calculated an absorbed quantity of at least 1.62 mg.
Distribution and Retention — Information on the distribution of
chlordane to tissues following chlordane Ingestion 1s available from a case
report of accidental poisoning and from autopsy samples.
In the previously described case of the 20-month-old boy who drank an
unknown quantity of 74% chlordane, Curley and Garrettson (1969), assuming
that fat comprised 15% of the body weight, calculated that 5.9 mg chlordane
was In the fat 3 hours after Ingestion; 36 mg chlordane was present on the
next day, and 65.0 mg chlordane was In fat 8 days after Ingestion. Three
months after Ingestion, the concentration of chlordane 1n fat was 25.53
mg/kg. At this time the ratio of chlordane In fat to serum was 1470:1. No
fat samples were collected by Aldrlch and Holmes (1969) from the 4-year-old
girl who Ingested chlordane.
Biros and Enos (1973) reported that of 27 adipose tissue specimens ob-
tained at .autopsies from people of the general population, 21 were positive
for oxychlordane, a metabolite of chlordane, with a mean concentration of
0.14*0.09 ppm and a range of 0.03-0.40 ppm. The mean adipose tissue levels
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of oxychlordane 1n 10 autopsy samples from the Oade County, Florida, white
female population 1n 1978 was 0.193 ppm, with a range of 0.07-0.49 ppm
(Barquet et al.# 1981). CIs- and trans-chlordane levels were below the
limits of 6C-electron capture detection {<0.10 ppm). Sovocool and Lewis
(1975) also detected oxychlordane In human adipose tissue obtained at
autopsy from several hospitals.
Metabolism — The J_n vitro metabolism of els- and trans-chlordane-
1JC by rat and human liver microsomal preparations was compared by Tashlro
and Hatsumura (1978). Their data are presented In Table III-l. Examination
of the table reveals very little Interspecies difference between the rat and
human In vitro metabolism of the two Isomers.
Excretion — In the case of accidental poisoning of a 4-year-old, 11
kg girl by Ingestion of an unknown quantity of a 45% chlordane formulation
(Aldrlch and Holmes, 1969), a nonlinear decline of serum chlordane concen-
tration from 3.4 to 0.138 mg/kg serum 1n the first 3 days and 0.03 mg/kg
serum by 130 days was observed. During the last 90 days of this period, the
concentration of chlordane In serum declined at a rate of 0.036 mg/kg day,
and a biological half-life was estimated as 88 days. The chlordane concen-
tration In urine declined from 1.93 to 0.05 ppm over the first 3 days after
Ingestion; however, 35 days later the urinary level was 0.13 ppm. The
Investigators speculated that the rise may have resulted from release of
stored chlordane. On days 2 and 3 after Ingestion, fecal chlordane levels
were 719 and 105 ppm, respectively. No chlordane was detected In the feces
1 or 2 months later.
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TABLE III-l
Metabolism of c1s-Chlordane and trans-Chlordane l£ v1tro by Human
and Rat Liver Microsomal Preparations: The Oata Are
Expressed as Percent of the Total Starting Material4*15
Starting Material
and Metabolites
c1s-
Chlordane
trans-
-Chlordane
Humanc
Rat
Human*1
Rat
1,2-Cl-chlordene
0.5
2.1
0.4
NA
Oxychlordane
1.0
5.2
0.4
2.3
ds-Chlordane
83.1
85.5
NA
NA
trans-Chlordane
NA
NA
85.25
86.4
C-4 (T-4)*
0.5
0.6
1.0
0.8
C-5 (T-5)f
0.1
0.1
0.2
0.1
H«9
0.6
NA
2.5
NA
Chlordene chlorohydrln
5.7
3.8
4.5
7.9
C-10 (T-10)h
2.9
0.9
0.8
1.3
1(2-0H-chlordene
0.4
0.3
0.4
0.3
C-12 (T-12).1
0.6
0.2
0.5
0.0
C-13 (T-13)J
4.6
1.3
3.5
0.8
aSource: Tashlro and Matsumura, 1978
^Microsomal fraction prepared by centrlfuglng human liver tissue homogen-
ate at 20r000 x g for 20 minutes. Incubation mixture made up to 5 im
with Incubation at 37*C 1n shaking Incubator for 2 hours.
^Patient A only
^Average of two patients (A and B). The amounts of trans-chlordane
remaining were 86.b% for A and 83.9% for B.
eC-4, T-4 - l-hydroxy-2-chlorochlordene derived from c1s- or trans-
chlordane
^C-5, T-5 « l-hydroxy-2-chloro-2.3-epoxychlordene
^Unknown metabolite found only 1n human experiments
hC-10( T-10 » monohydroxylated dlhydrochlordene
1C-12, T-12 » trlhydroxydlhydrochlordene
3C-13, T-13 « beta-g1ucuron1de-l-hydroxyd1hydroch*i>rdene
NA « Not applicable
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In th0 case of the 20-month-old boy who ingested an unknown quantity of
74% chlordane, Curley and Garrettson (1969) estimated the half-life of
chlordane In serum to be 21 days. The concentration of chlordane In the
24-hour urine was 0.309 mg/i, corresponding to 48.8 vq chlordane.
Further urine samples were not collected.
In a survey conducted 1n Arkansas and Mississippi In 1973 and 1974,
Strassman and Kutz (1977) reported that of 57 human milk samples, 45.6% were
positive (quantifiable) for oxychlordane residues, while 54.4% contained
unquantlflable traces. The mean level of oxychlordane residues in positive
samples was 0.012 ppm. Thus, a route of chlordane or oxychlordane excretion
1n human females Is lactation. The Investigators stated that, to their
knowledge, this was the first report of oxychlordane occurrence In human
ml Ik.
Heptachlor and Hepachlor Epoxide.
Absorption — No Information on the absorption of heptachlor or hepta-
chlor epoxide by humans was available. That these Insecticides are absorbed
can be Inferred from the many reports of adipose tissue levels of heptachlor
epoxide, although the route cannot be readily ascertained.
Distribution — Klemmer et al. (1977) reported that heptachlor epoxide
was detected In tissue samples from 77 autopsies performed from 1966-1968 1n
a range of concentrations of 1-32 ppb whole tissue. The highest concentra-
tions occurred 1n bone marrow and liver.
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Heptachlor epoxide has been detected In human adipose tissue In a large
number of surveys conducted 1n Great Britain (Abbott et al.t 1972; 1981),
Brazil (Uassermann et al.t 1972), Japan (Curley et al., 1973), Israel (Was-
sermann et al., 1974), Texas (Burns, 1974), Louisiana (Greer et al., 1900)
and the continental United States (Kutz et al., 1979, Sovocool and Lewis,
1975).
Several studies have reported transplacental transfer of heptachlor or
heptachlor epoxide In humans. Curley et al. (1969) detected heptachlor
epoxide 1n adipose tissue, brain, adrenals, lungs, heart, liver, kidney and
spleen of 10 stillborn babies and 2 babies who died soon after birth and In
27 of 30 samples of cord blood from healthy neonates. All concentrations
fell within the range of levels detected In the general adult population.
Others have also reported that heptachlor epoxide levels In tissues of
stillborn and soon dead Infants correspond to concentrations found In adults
(Zavon et al., 1969; Wassermann et al., 1972, 1974). A comparison of hepta-
chlor and heptachlor epoxide residues In 53 maternal blood and 54 placental
tissue samples revealed that, In general, high levels of the compounds 1n
maternal blood were reflected as high levels In placentae and vice versa
(Selby et al., 1969). The ratio of the concentration of heptachlor epoxide
In placentae to the concentration In maternal blood was 5.8:1, based upon
geometric means. For total equivalents of heptachlor, defined as the sum of
the values for heptachlor and heptachlor epoxide, the ratio was 8.8:1.
Pollshuk et al. (1977a) reported mean levels of heptachlor epoxide 1n the
extracted lipid of tissues from 24 women during labor as follows: adipose
tissue, 0.2856 ppm; maternal blood, 0.2798 ppm;, fetal blood, 0.9959 ppm;
uterine muscle, 0.4895 ppm; placenta, 0.5000 ppm; and amniotic fluid
0.6730 ppm.
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Hetabollsm — The results of a comparison of the v1 tro metabolism
of heptachlor by human and rat liver microsomal preparations (Tashlro and
Matsumura, 1978) are presented 1n Table III-2. A major difference between
the species Is that 4 times more heptachlor epoxide was formed 1n the rat
system than In the human system. The investigators pointed out that since
heptachlor epoxide Is a stable product not Involved In the major metabolic
pathway of heptachlor (see Figure III-2), the relative ease of Its formation
may Influence the relative degree to which heptachlor and heptachlor epoxide
are stored by adipose tissue.
Excretion — Information regarding the urinary and fecal excretion of
heptachlor and heptachlor epoxide by humans was not available. Excretion of
heptachlor epoxide In the milk of lactatlng mothers, however, has been
reported by several Investigators. Kroger (1972) detected an average of
0.16 ppm heptachlor epoxide 1n 53 human milk samples. A mean concentration
of 0.003 mg/kg of heptachlor epoxide In milk was reported by RUcey et al.
(1972) 1n 147 human milk samples collected In Canada In 1967-1968. Hepta-
chlor epoxide. In trace amounts up to concentrations of 5 ppb, was detected
in 10 of 40 milk samples collected from women In Colorado 1n 1971 -1972
{Savage et al., 1973). The mean concentration of heptachlor epoxide 1n 18
of 50 human m11k samples collected In Norway was 1.57 ppb (Bakken and Selp,
1976). In Israel 1n 1975, a mean concentration of 0.0091 ppm was detected
In whole milk collected from 29 women 2*4 days after delivery (Pollshuk et
al., 1977bJ. Of 51 human milk samples collected In St. Louis, M0, In 1973,
12 were positive for heptachlor epoxide (mean concentration * 0.0027 ppm)
and 3 were positive for heptachlor (mean concentration » 0.019 ppm) (Jonsson
et al., 1977). Strassman and Kutz (1977) detected heptachlor epoxide In
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TABLE III-2
Heptachlor Metabolism In, v1tro by Human and Rat Liver Microsomal
Preparations: The Data Are Expressed as Percent of Starting Material*
Starting Material and
Metabolites
Human
Rat
Heptachlor
68.6
4.4
H-l (heptachlor epoxide)
20.4
85.8
H-2
<0.1
—
H-3 (1-OH-chlordene)
4.8
2.9
H-4 (1-OH-chlordene epoxide)
5.0
3.0
H-5 (1,2-OH-chlordene)
0.1
0.9
H-6
1.0
3.0
•Source: TasMro and Matsumura, 1978
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4
35.IX of 57 human milk samples from Arkansas and Mississippi 1n 1973-1974 1n
a mean concentration of 0.004 ppm. All milk samples from 50 lactatlng women
In Hawaii collected from 1979-1980 contained heptachlor epoxide In a mean
concentration of -34 ppb (Takahashl et al.f 1901). Lactation may, there-
fore, represent an Important route of excretion of heptachlor epoxide In
nursing mothers and reduce the body burden In this human subpopulatlon. On
the other hand, breast feeding results In an additional source of pesticide
exposure for Infants.
Summary
Chlordane. The absorption of chlordane by the gastrointestinal tract
of animals can be Inferred from data on Its systemic toxicity and from
excretion data following Ingestion. At least 2-8% of orally administered
14C-chlordane was absorbed by rats and at least 30% was absorbed by
rabbits, as evidenced by the percent radioactivity recovered In urine
(Barnett and Dorough, 1974). Chlordane was absorbed by the pulmonary route
by rats (Nye and Dorough, 1976). Hore chlordane in lipophilic vehicle than
In ethanol was absorbed by rats dermally (Ambrose et al., 1953b). Informa-
tion on the absorption of chlordane by humans following Ingestion Is avail-
able from case studies of accidental poisoning of children (Curley and
Garrettson, 1969; Aldrlch and Holmes, 1969).
Once absorbed, chlordane and/or Us metabolites are rapidly distributed
throughout-the body with highest levels detected In adipose tissue (Barnett
and Dorough, 1974). Radioactivity in all tissues declined when treatment
was discontinued, but detectable levels were still present In all tissues of
rats 56 days after termination of feeding 14C-chlordane for 56 days.
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Female rats accumulated greater concentrations of chlordane or metabolites
than did male rats (Barnett and Oorough, 1974; Ambrose et al.f 1953b; Street
and Blau, 1972). Greater accumulation of residues by rats from trans- than
from cls-chlordane was observed (Barnett and Oorough, 1974; Street and Blau,
1972). The major form of chlordane stored in tissues has been Identified as
oxychlordane (Barnett and Oorough, 1974; Street and B1au, 1972; Polen et
al., 1971). Similar results have been observed In dogs, pigs (Polen et al.,
1971) and rabbits (Poonawalla and Korte, 1971; Barnett and Oorough, 1974)
and In rats following pulmonary absorption (Nye and Oorough, 1976). Oxy-
chlordane has been detected In adipose tissue of the general human popula-
tion obtained at autopsy (Biros and Enos, 1973; Barquet et al., 1981; Sovo-
cool and Lewis, 1975). Chlordane was stored for at least 3 months 1n the
fat of one child who drank a formulation of the Insecticide (Curley and
Garrettson, 1969).
Oxychlordane Is a metabolite of both els- and trans-chlordane In rats U\
vivo and .In vitro, although more oxychlordane Is formed from trans-chlordane
(Street and Blau, 1972; Barnett and Oorough, 1974; Tashlro and Matsumura,
1977; Brlmfleld et al., 1978). At least 13 or 14 fecal metabolites have
been Isolated, many of which have been Identified; they are Identical,
whether formed from ds- or trans-chlordane. A glucuronlde conjugate was
found In the urine (Tashlro and Matsumura, 1977). A metabolic scheme
Involving dehydrogenatlon, epoxldatlon, hydroxylatlon and dechlorination
reactions was presented. Metabolism of chlordane hi vitro by human liver
microsomal preparations was found to be very similar to rat microsomal .In
vitro metabolism (Tashlro and Matsumura, 1978).
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The major route of elimination of chlordane and Its metabolites by rats
following Ingestion 1s by the feces, amounting to as much as 88% of the
administered dose (Barnett and Dorough, 1974). The cls-lsomer was excreted
to a greater extent than was the trans-1somer (Barnett and Dorough, 1974;
Tashlro and Hatsumura, 1977). Since more oxychlordane, which Is stored In
adipose tissue, 1s formed from trans- than from cls-chlordane, the greater
elimination of the ds-1somer Is not surprising. Female rats excreted a
greater percentage of metabolites In urine than did males (Barnett and
Dorough, 1974). Urinary excretion of metabolites by rabbits was much great-
er than by rats, and In some cases exceeded fecal excretion of metabolites
by rabbits (Poonawalla and Korte, 1971; Barnett and Dorough, 1974; 8a1ba and
Saha, 1978). Elimination of chlordane or metabolites by the pulmonary route
was not observed 1n rats (Nye and Dorough, 1976). Excretion of chlordane by
a 4-year-old girl was nonlinear, with a calculated half-life In serum of 88
days (Aldrlch and Holmes, 1969). Urinary excretion declined for 3 days
after Ingestion, but rose at 35 days possibly as a result of a release of
stored chlordane. A half-life of chlordane In serum of 21 days was calcu-
lated for a 2-year-old boy who drank chlordane I Cur ley and Garrettson,
1969). Oxychlordane has been found In the milk of lactatlng women (Strass-
man and Kutz, 1977).
Heptachlor and Heotachlor Epoxide. Heptachlor was absorbed from the
gastrointestinal tract of rats following Intragastric administration, as
evidenced by the appearance of residues in blood within 1 hour of oral
dosing (Mlzyukova and Kurchatov, 1970), and by the pulmonary route by
rabbits that breathed ambient air In a heavy pesticide use area, as
evidenced by increased levels of heptachlor epoxide 1n adipose tissue over
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03/26/87

-------
control levels {Arthur et al., 1975). Absorption of heptachlor by humans
can be Inferred from many reports of levels of heptachlor epoxide in adipose
tissue obtained In autopsy and biopsy samples from the general population
(Klemmer et al., 1977; Abbott et al., 1972; 1981; Wassermann et al.# 1972;
1974; Curley et al., 1973; Burns, 1974; Greer et al., 1980; Kutz et al.,
1979). Heptachlor epoxide has been detected In all monitored tissues of
deceased Infants, and 1n cord blood of healthy neonates at levels corre-
sponding to levels In the general adult population (Curley et al., 1969;
Zavon et al., 1969; Wassermann et al., 1972, 1974). Following absorption of
heptachlor. Its metabolite, heptachlor epoxide. Is distributed throughout
the body of rats and dogs (Mzyukova and Kurchatov, 1970; Radomskl and
Davldow, 1953). Female rats accumulated 5-10 times as much heptachlor
epoxide 1n adipose tissue as did males following continuous feeding of
heptachlor for 8 weeks. Heptachlor epoxide persisted In fat of female rats
for 8 weeks and male rats for 6 weeks after treatment was terminated. The
maximum dietary level at which fat storage could not be detected was 0.3
mg/kg diet for male rats and 0.1 mg/kg diet for female rats. Heptachlor was
metabolized by rats tn v1 vo and in vitro to heptachlor epoxide, which was
dechlorInated and hydroxylated to a fecal metabolite (Tashlro and Matsumura,
1978). Heptachlor was also metabolized to 1-hydroxychlordene, which was
epoxldlzed to 1-hydroxy-2,3-epoxychlordene; 1-hydroxy-2,3-epoxychlordene was
hydrolysed to 1,2-dlhydroxydlhydrochlordene. As much as 4 times more hepta-
chlor epoxide was formed \n vitro by rat microsomes than by human micro-
somes; otherwise, metabolism was similar (Tashlro and Matsumura, 1978).
01780
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02/15/87

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Like chlordane, the major route of heptachlor elimination by rats Is by
the feces, amounting up to 50% over 10 days of the administered oral dose
(Tashlro and Matsumura, 1978). Urinary excretion of metabolites amounted to
<5% of the dose. The only Information available on human excretion of
heptachlor are reports of heptachlor epoxide detected In milk of lacta11ng
women {Kroger, 1972; RUcey et al., 1972; Savage et al., 1973; Bakken and
Se1p, 1976; Pollshuk et al., 1977b; Jonsson et al., 1977; Strassman and
Kutz, 1977; Takahashl et al., 1981). This excretion reduced the body burden
of heptachlor epoxide In these women but Increases the exposure of breast-
fed Infants.
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03/26/87

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IV. SOURCE OP HUMAN EXPOSURE - CHLORDANE, HEPTACHLOR AND
HEPTACHLOR EPOXIDE
This chapter summarizes available data on use, environmen
fate, and occurrence of chlordane, heptachior and heptachlor
epoxide and characterizes their potential for drinking water
exposure. A more extensive discussion of the available
information on these compounds is presented in the draft
document entitled "Occurrence of Pesticides in Drinking Water,
Food and Air" (USEPA 1987).
Humans may be exposed to chlordane and heptachlor from a
variety of sources, including drinking water, food, ambient
and possibly indoor air. Heptachlor epoxide is a degradation
product of heptachlor. Humans may be exposed to heptachlor
epoxide from water, food and air. This analysis of human
exposure is limited to drinking water, food and air. Some
~
individuals {pesticide applicators) will receive additional
exposure to chlordane and heptachlor from their occupations.
Chlordane is not restricted to licensed applicators and is
available to the public. Additional exposure can occur from
consumer use. Individual exposure to chlordane, heptachlor
and heptachlor epoxide will vary widely based on factors
such as where a person lives and travels, and what a person
eats. Intake of chlordane, heptachlor and heptachlor epoxide
will be affected by age, weight and lifestyle.

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- V —
The Exposure Estimates section of this chapter presents
available information on the range of human exposure to and
intake of chlordane, heptachlor and heptachlor epoxide from
drinking water, food and air. It is not possible to provide
an estimate of the number of individuals experiencing specific
exposures from these three sources. However, this section
provides insight into the relative contributions of the three
sources.
A. 'Jse/Environmental Fate
Chlordane and heptachlor are broad spectrum insecticides
which, until the mid-1970's, were widely used as pre-emergent
insecticides on many crops. During the mid-1970's, concern
over the possible health risks of these two chemicals led to
a ban of all their uses other than subsurface ground injection
for the control of termites. Heptachlor is often used in
conjunction with chlordane for this purpose (USEPA 1983).
Heptachlor also retains some additional use on certain crops
in Hawaii (Kuch, 1986). In the past, heptachlor has occurred
as an impurity in commercial chlordane (USEPA 1980).
Once released into the environment, chlordane is very
resistant to chemical and biological degradation. It is
highly immobile, and does not leach readily from soil. The
average half-life of chlordane in soil is 3.3 years, but some
residues have been reported to persist for up to 14 years at

-------
: v-3
detectable levels. The major mechanism for removing chlordar.e
from soil is volatilization (Lyman et al., 1982; Rao and
Davidson, 1980). Despite its lack of mobility in the soil
column, chlordane's lengthy persistence in the soil may resjlt
in a long-term, low-level source of ground water contamination
(USEPA 1987).
Chlordane is not removed, from natural waters by hydrolysis
or biodegradation (Eichelberger and Lichtenberg, 1971, as
cited in Callahan et al,, 1979). However, chlordane is
removed from surface water by adsorption to bottom sediments
and by volatilization into the atmosphere. Based on chlordane's
physical and chemical properties, it is expected to adsorb to
suspended sediments and particulates in surface waters (Lyman
et al., 1982 ).
Heptachlor has environmental fate properties similar to
~
chlordane except that heptachlor is susceptible to hydrolysis.
Eichelberger and Lichtenberg (1971, as cited in Callahan et al.,
1979), reported that heptachlor can undergo hydrolysis to
form 1-hydroxychlordene. This product is then susceptible to
biotransformation under either aerobic or anaerobic conditions
to produce heptachlor epoxide and other compounds (Miles et al.,
1969 and 1971, both cited by Callahan et al., 1979). The
mean half-life of heptachlor in soils under aerobic conditions
may range from 63 days, as observed in the laboratory, to 426
days, as observed under field conditions (Rao and Davidson, 1980).

-------
:v-4
Heptachlor epoxide, however, is resistant to hydrolysis and
biodegradation, and is very persistent in soils (Callahan
et al., 1979).
As with chlordane, both heptachlor and heptachlor epoxide
bind strongly to the soil and sediments. 3oth compounds are
therefore resistant to leaching and transport into ground
water (USEPA 1987).
In surface waters, heptachlor is susceptible to hydrolysis
to 1-hydroxychlordene with an estimated half-life of
approximately 1 to 3 days (Demayo, 1972 and Eichelberger and
Lichtenberg, 1971, both cited by Callahan et al., 1979). The
compound 1-hydroxychlordene degrades in water to form heptachlor
epoxide. 3oth heptachlor and heptachlor epoxide will tend to
adsorb onto suspended particulate matter and partition to
bottom sediments (Mabey et al, 1981). Under certain conditions,
«»
the two compounds may also volatilize.
B. Occurrence
Drinking Water
No national surveys of chlordane, heptachlor and heptacnlor
epoxide are available to determine the extent of occurrence
of these chemicals in public water supplies. A few limited
studies have reported detectable levels of the compounds

-------
(CJSEPA, 1987).
rarely occurred
the levels were
These surveys
at detectable
almost always
determined that the compounds
levels and when they did occur,
less than 0.1 ug/1.
It is not clear how to relate the results of the above
surveys to the current levels of these compounds in drinking
water. The surveys generally were designed to detect
contaminants resulting from agricultural uses, which were
later banned or greatly restricted. EPA has no data on the
degree of contamination of drinking water supplies which
occurs as a result of current uses of these compounds.
However, based on the manner of application (subsoil injection
and the ability of the compounds to bind to soils, contaminati
would be expected to be limited to ground water supplies.
Levels of these compounds, where they occur, are expected to
be low.
Diet
Information and estimates on the dietary intake of
chlordane, heptachlor and heptachlor epoxide were obtained
from the Pood and Drug Administration's Total Diet Studies,
which are commonly referred to as "Market Basket Surveys'1
{see Table 1). The dietary intake of heptachlor by 25-30
year old adults is significantly lower than the intake of
heptachlor epoxide (FDA, 1986), reflecting the conversion of
the compound in the environment.

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Table 1
Estimated Daily Intake Levels for 25-30 Year Old Males
Estimated
Compound	Adult Intake
Chlordane	0.048 ug/day
Heptachlor	0.007 ug/day
Heptachlor epoxide	0.134 ug/day
The origins of these dietary levels are unclear. There
are currently no agricultural uses of chlordane, and heptachlor
has only limited agricultural uses. The current dietary
levels could be the result of past uses of these compounds.
Due to the restricted use of the chemicalsf future levels are
expected to decline.
Air
No monitoring data are available on the current levels
of chlordane, heptachlor and heptachlor epoxide in ambient
air. In 1976, a survey of air levels in high use areas
reported finding both chlordane and heptachlor in a large
percentage of samples taken. The levels found were less than
0.1 ug/m3. This survey reflects the then widespread agricultural
use of chlordane and heptachlor. Because these uses have
been discontinued and the current uses have a low potential
for volatilization (subsurface injection), the levels reported

-------
for the survey are not indicative of current levels of
occurrence, Based on the current uses, levels of chlordane,
heptachlor and heptachlor epoxide in air are expected to be
negligible.
EPA is concerned over the potential occurrences of
these compounds in indoor air. Based on the recent experience
with radon and other volatile ground water contaminants, EPA
suspects that the current practices of subsurface injection
for chlordane and heptachlor can result in the contamination
of indoor air. No data are available on actual levels.
C. Exposure Estimates
The information that is currently available on the
occurrence of chlordane, heptachlor and heptachlor epoxide in
the environment and the potential for exposure are insufficient
to determine the national distribution of intake by any of
the three routes, for any of the compounds. (Table 2). The
data reported for drinking water and air exposure were derived
from studies conducted during the early to mid-1970's (prior
to restrictions) and data for food exposure were collected
during the early to mid-1980's? therefore, it is likely that
current inhalation and drinking water exposure levels will
oe much less than the reported levels and that current dietary
exposure levels will be similar to the reported levels.
Although the exposure to heptachlor is expected to be equally

-------
: v- a
minimal from all'three sources, diet is likely to be the
greatest source of exposure to heptachlor epoxide.
Because the ranges of potential intake levels from
drinking water and air exposure are so wide and varied and
because the estimated intake levels provided for food are
single values and were collected under different usage condi-
tions it is not possible at this time to generate an accurate
estimate of the total exposure. Additional data are needed
for estimating the total combined intake from each media.
Table 2
Exposure Estimates
Source
Reported
Exposure Levels
Estimated
Adult Intake
CHLORDANE
Drinking Water
Diet
Air
<0.1 ug/1
<0.2 ug/day
0.048 ug/day
HEPTACHLOR
Drinking Water
Diet
Air
<0.1 ug/1
?
<0.2 ug/day
0.0007 ug/day
HEPTACHLOR EPOXIDE
Drinking Water
Diet
Air
<0.1 ug/1
?
<0.2 ug/day
0.184 ug/day
~Ambient air levels of the compounds are believed to be very
low. Indoor air levels while unknown are expected to be
higher.

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rv-9
D. References
Callahan, M.A., M.W. Slimak, N.W. Gabel, et al. 1979. water-
related environmental fate of 129 priority pollutants.
Final report. Prepared by Versar, Inc., Springfield,
VA, and SRI International, Menlo Park, CA, for"Monitoring
and Data Support Division, U.S. Environmental Protection
Agency, Washington, DC~ EPA-440/4-79-029a.
Demayo, A. 1972. Gas chromatographic determination of the
rate constant for hydrolysis of heptachlor. Bull.
Environ. Contamin. and Toxicol. 8(4):234-237. Cited in
Callahan et al. 1979.
Eichelberger, J.w. and J.J. Lichtenberg. 1971. Persistence
of pesticides in river water. Environ. Sci. and Technol.
5(6):541-544. Cited in Callahan et al. 1979.
FDA. 1986. Food and Drug Administration. Memorandum from
E. Gunderson, Division of Contaminants Chemistry, Center
for Food Safety and Applied Nutrition, Washington, DC to
Dr. Paul S. Price, Office of Drinking Water, U.S. Environ-
mental Protection Agency, Washington, DC. Nov. 6, 1986.
Kuch, P. 1986. Letter to Frederic A. Zafran, Oct. 28, 1986.
SAIC, McLean, VA.
Lyman, W.J,, W.F. Reehl and D.H. Rosenblatt. 1982. Handbook
of chemical property estimation methods. Environmental
behavior of organic compounds. New York: McGraw-Hill.
Mabey, W.R., J.H. Smith, R.T. Podall et al. 1981. Aquatic
fate process data for organic priority pollutants. Final
draft report. Prepared by SRI International, Menlo
Park, CA, for Monitoring Support Division, U.S. Environ-
mental Protection Agency, Washington, DC. EPA4 40/4-81-014.
Miles, J.R.w., C.M. Tu and C.R. Harris. 1969. Metabolism of
heptachlor and its degradation products by soil micro-
organisms. J. Econ. Entomol. 62( 6 ) :1334-1338. Cited in
Callahan et al. 1979.
Miles, J.R.W., C.M. Tu and C.R. Harris. 1971. Degradation
of -heptachlor epoxide and heptachlor by a mixed culture
of soil microorganisms. J. Econ. Entomol. 64(4):839—041.
Cited in Callahan et al. 1979.
Rao, P.S.C. and J.M. Davidson. 1980. Estimation of pesticide
retention and transformation parameters required in non-
point source pollution models. In: M.R. Overcash and
J.M. Davidson (eds.). Environmental impact of non-point
source pollution. Ann Arbor, MI: Ann Arbor Science
Publishers. pp. 23-67.

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iv-io
USEPA. 1980. U.S. Environmental Protection Agency.
Ambient water quality criteria for chlordane. Washington,
DC: Office of water Regulations and Standards, U.S.
Environmental Protection Agency. EPA-440/5-80-027.
USEPA. 1983. U.S. Environmental Protection Agency.
Analysis of the risks and benefits of seven chemical
jsed for subterranean termite control. wasnington, DC:
Office of Pesticide Programs, U.S. Environmental Protection
Agency. EPA-540/9-83-005.
USEPA. 1987. U.S. Environmental Protection Agency.
Occurrence of pesticides in drinking water, food and
air. Prepared by SAIC, McLean, VA, for Office of Drin.Kir.g
Water, U.S. Environmental Protection Agency, Washington,
DC.
~

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V. HEALTH EFFECTS IN ANIMALS
General Toxicity
Effects of Acute Exposure. In the discussion of the health effects of
acute exposure of animals to chlordane, heptachlor and heptachlor epoxide,
durations of exposure from a single dose up to 15 dally doses will be
considered acute.
Chlordane « Values for the acute oral LD^ of chlordane In rats of
various strains ranged from 83 mg/kg bw for pure cls-chlordane (Podowskl et
al., 1979) to 590 mg/kg bw for chlordane of unspecified purity (Ambrose et
al., 1953a,b), when delivered by gavage In lipophilic vehicle (Table V-l).
The values determined for technical grade chlordane (see Chapter II) by
Gaines (1960) of 335 mg/kg bw (confidence limits 299-375 mg/kg bw) for male
Sherman rats, and 430 mg/kg bw (391-473 mg/kg bw) for female rats of the
same strain, are In good agreement with other values of 311+46 mg/kg bw
(Boyd and Taylor, 1969) and 350+22 mg/kg bw (Gak et al., 1976) determined
for technical grade chlordane In rats. A slightly lower value for rats (283
mg/kg bw) was reported by Ben-Oyke et al. (1970). Gak et al. (1976) found
that mice of an unspecified strain were slightly less sensitive (LD^q «
390+35 mg/kg bw) and Golden hamsters were much less sensitive (LD50 =
1720+135 mg/kg bw) to chlordane than rats. Chlordane was not as toxic to
rats when applied dermally (Gaines, 1960; Ben-Dyke et al., 1970). Harbison
(1975) found that neonatal Sprague-Oawley rats were less sensitive than
adult male rats to chlordane (analytical grade) Intoxication when the Insec-
ticide was administered Intraperltoneally. Pretreatment of the neonates
with phenobarbltal, however, reduced the LD^q from 1121 to 539 mg/kg bw,
01800
V-l
03/26/87

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UBl t II I
Acute IO50S for Chlordane
Species/ Se«/
Strain Nuaber	Uetght/Age	Purity
NR/NR
HR/NR
It/70
F/70
Rat/ N/14)
Ulstar
Rat/NR
Rat/
Albino
Rat/
Sheraan
Route
Vehicle
ioso
(ag/kg bw)
nt
Rat/
Charles group
River
NR
NR
SO-60 g/
Sb days
technical
grade
NR
))5 g/90 days technical
grade
?00 g/90 days techntcal
grade
technical
grade
N/6-10/ ' ISO g/b weeks cls-
chlordane
oral
oral
oral
oral
oral
'Innocuous
solvent*
cottonseed
oil
peanut oil
peanut oil
cottonseed
oil
corn oil
IS?
S90
JH
(299-375)
430
(391 473)
311 » 4b
83
Ireaors within IS alnutes, convulsions In ?4
hours persisting until 48 hours. Deaths In
b days.
NC
Survival tlae: 48 hours-9 days for N and f;
treaors, hyperexcttablllty, Irritability and
convulsions. Nuabers In parentheses are
confidence Halts.
Death In 4? 1 18 hours. Clinical syaptoas:
stlaulatlon of CMS Irritability; phonal Ion;
piloerectlon; treaors and convulsions alternating
with depression; hyporefleila; lethargy; diarrhea;
diuresis; anorexia; oligodipsia; hypotherala;
low bw. At necropsy, local gastroenteritis.
nephritis, hepatitis, adrenal and thyaus stress
reactions, vascular congestion, dehydration,
loss of organ weight.
LDi0 - 237 ag/kg bw - estlaated aai.
ID^o • 38b ag/kg bw - estlaated mi,
Oeaths In ?4 hours.
Reference
Lehaan, 19S1
Aabrose et
a).. 19S3a,b
Gaines, I960
Boyd and
Taylor, 1969
99 9.X
Podowskt et
al.. 1979
Rat/NR NR/NR
Nouse/NR NR/NR
Haaster/ NR/NR
Golden
NR
NR
NR
NR
NR
NR
oral
oral
oral
olive oil
olive oil
olive oil
3S0 ~ 22
390 » 35
1720 » 13S
NC
NC
NC
Gak et al..
197b
Gak et al..
197b
Gak et al..
197b

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I ABIE V-l (cont.)
Speclet/
Strain
Se«/
Number
Height/Age
PurIty
Route
Vehicle
lD50
|ag/kg bw)
Coonent
Reference
Rat/NR
NR/NR
MR
i
coMwrclal
foraulatlon
oral
NR
283
NC
Ben-Dyke el
al.. 1970
Rat/NR
NR/NR
NR
ca«Mrclal
formulation
dermal
NR
>600
NC
Ben-Oyke et
al., 1970
Rat/
Sheraan
N/70
175 g/90 days
technical
grade
deraal
xylene
640
(750-941)
Survival tlae:
72 hours - 9 days
Gaines. 1960
1

f/70
200 g/90 days
technical
grade
deraal
xylene
690
(580 821)
96 hours - 25 days


r/70
200 g/90 days
technical
grade
deraal
undiluted
530
(431-652)
120 hours - 15 days
Muabers In parentheses are confidence llalts.

Rat/
Sprague-
Oawley
H/t
10-20/
group
Neonates
2 5 days
analytical
grade
l.p.
corn oil
1121
(984-1289)
Pretreataent with phenobarbltal (40 ag/kg by x 3)
reduced the LDjo to 53' ig/kg bw (428-666).
Niatters In parentheses are confidence llalts.
Harbison.
1975
Rat/
Sprague-
Oawley
N
10-20/
group
120-150 g/
adult
analytical
grade
».p.
corn oil
343
(258-451)
Nuabers In parentheses are confidence llalts.
Harbison.
1975
House/
Swiss-
Webster
H/NR
20 g
cls-
chloi dane
trans -
chlordane
l.p.
l.p.
dlaethyl
sulfoxide
30
130
NC
1
Ivle et al..
1972
NC = No coawnt
HR ¦ Hot reported
t.p. - Intraperitoneal

-------
by enhancing the ability of neonates to metabolize chlordane to Its toxic
Intermediate. Pure cls-chlordane was more toxic than pure trans-chlordane
to male Swiss-Webster mice when administered IntraperItoneally (Ivle et a*).,
1972). The pure c1s-1somer was also more toxic to rats than was technical
grade chlordane. Further details of the LO^q determinations are presented
In Table V-l.
Symptoms of acute Intoxication Include CNS stimulation, as evidenced by
Irritability, tremors and convulsions (Boyd and Taylor, 1969; Stohlman et
al.( 1950; Hyde and Falkenberg, 1976). Boyd and Taylor (1969) described a
wide range of CNS disturbances, Including phonatlon; p1loerectlon; tremors
and convulsions alternating with lethargy; diarrhea; and food and water
refusal. Necropsy of rats revealed vascular congestion, nephritis, hepati-
tis and decreased organ weight.
Other studies of acute exposure, other than LD^q determinations, are
described In Table V-2. Stohlman et al. (1950) have conducted an extensive
study on dose response, vehicle effect and effect of route of administration
of chlordane (described as "specially purified-) on mortality of rats and
rabbits. Chlordane was more toxic when administered orally to rats and
rabbits In Tween-20 than In olive oil, as evidence by greater Incidence of
mortality occurring at shorter periods following treatment. No deaths In
rats occurred at a single oral dose of 100 mg/kg bw 1n Tween-20, although
the same dose resulted 1n three deaths In eight rabbits within 2.5 hours.
Multiple oral doses of 10 mg/kg bw/day 1n olive oil for 16 days resulted In
2 deaths among five rabbits. Increased mortality In rats after Intraperi-
toneal administration and In rabbits after Intravenous administration of
chlordane was also observed (see Table V-2) (Stohlman et al., 1950).
01800	V-4	03/26/07

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TABLE V-2
Acute Toxicity of Chlordane
CD
o
o
Species/ Sex/
Strain Number Uelght/Age	Purity
Route
Vehicle
Dose
Duration
Effects
Reference
Rat/NR
NR/10
130-400
g
octa-klor
oral
Tween-20
0
ag/kg
bw

6


('specially


100
ag/kg
bw

13


purified")


200
ag/kg
bw

21





300
ag/kg
bw

19





1000
ag/kg
bw
Rat/NR
NR/10
134-400
g
octa-klor
oral
olive oil
200
ag/kg
bw

10


('specially


250
ag/kg
bw

10


purified*)


500
ng/kg
bw
Rats/
H/4/
200 g

cts-
oral
corn oil
0.
200

Wtstar
groups


chlordane


ag/kg bw

single dose 0/10 deaths
0/6 deaths
7/13 deaths (53%); treaors and
convulsions.
13/21 deaths (62%); treaors and
convulsions.
18/19 deaths (95%); treaors and
convulsions.
single dose 1/10 deaths (10%) In 30 days
6/10 deaths (60%) In 1-7 days
B/10 deaths (SOX) In 2-5 days
No aentlon of controls was aade
for this series.
single dose Fasting blood glucose Increased
to 147% of control; decreased
liver glycogen (64%), Increased
serua urea (125%); Increased
activities of hepatic pyruvate
carboxylase (183%), fructose-
1,6-dlphosphatase (236%) and
giucose-6-phosphatase (182%).
Increased kidney cortical enzymes
as above: 156%, 178%, 189% and
175%, respectively. Increased
liver and kidney basal and
f1uor1de-stlaulated adenyl
cyclase, and cyclic AMP. All the
changes were significantly
different froa control levels
(p<0.05).
Stohlaan et
al., 1950
Stohlaan et
al., 1950
Kacew and
Slnghal, 1973
Slnghal and
Kacew. 1976
o
CD
O
10

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TABLE V-2 (cont.)
Species/ Sex/
Strain Number Uelght/Age	Purity Route Vehicle	Dose	Duration	Effects	Reference
Rabbit/NR
NR/6
1.6-3.3 kg
•cta-klor
oral
Tween-20
0 ag/kg bw

8
(¦specially

Tween-20
100 ag/kg bw



purified*)




6



Tween-20
200 ag/kg bw

13



olive oil
300 ag/kg bw

9



olive oil
400 ag/kg bw

11



olive oil
600 ag/kg bw

7



olive oil
1000 ag/kg bw
Mouse/
H-F/NR
60-70 days
technical
oral
corn oil
100 ag/kg bw
CD-I


grade


300 ag/kg bw






1000 ag/kg bw
House/
H-F/NR
60-70 days
HCS-3260
oral
corn oil
30 ag/kg bw
CD-I

(98«X)


100 ag/kg bw






300 ag/kg bw






1000 ag/kg bw
Rat/NR
H/12
50r60 g
octa-klor
oral
diet
0.1X. 1000



('specially


ag/kg diet



purified')



Rat/
NR/5
233-335 g
NR
oral
cottonseed
0. 6.25, 12.5
Albino




oil
2S, 50. 100.






200 ag/kg






bw/day
Rat/
H/
80-130 g
technical
oral
diet
0. 5, 10. 20
Mlstar
6/group

grade


or 50 ag/kg






diet
single dose
single dose
single dose
10 days
IS days
14 days
0/6 no effect
3/8 deaths (38X)
dyspnea, treaors,
5/6 (83X) deaths
dyspnea, treaors,
5/13 (38%) deaths
7/9 (78X) deaths
8/11 (73X) deaths
8 days; dyspnea,
convulsions.
7/7 (100X) deaths
4 days; dyspnea,
convulsions.
2-2.5 hours;
convulsions.
2-4 hours,
convulsions.
6-22 days.
4-31 days.
2	hours to
treaors,
3	hours to
treaors.
Stohlaan et
al.. 19S0
No syaptons
Mild hypoactlvlty-no /deaths
100X aortallty
No syaptoas
No syaptoas
Interalttent treaors and ataxia
followed by death.
Interalttent treaors and ataxia
followed by death.
100X aortallty
No observed syaptoas of toxicity
except for slight histologic
changes In the liver of group
exposed to <25 ag/kg/day. In
the next highest group, 2 of the
S anlaals died.
Dose-related emyae induction of
liver aniline hydroxylase,
aalnopyrlne dlaethylase and
hexabarbltal oxidase,
significant at 10-50 ag/kg diet,
10-50 ag/kg diet and 20-50 ag/kg
diet, respectively (p<0.0?5).
No-effect level - 5 ag/kg diet,
lowest-effect level - 10 ag/kg
diet.
Arnold et
al., 1977
Arnold et
al., 1977
Stohlaan et
al.. 1950
Ambrose et
al.. 1953a.b
Den Tonkelaar
and
Van Esch,
1974

-------
TABLE V-2 (cont.)
™ Species/ Sex/
o Strain Nuaber Height/Age	Purity Route Vehicle	Dose	duration	Effects	Reference
Rabbit/NR NR/5
S
4
5
14
12
6
1.6-3.3 kg
Rat/NR
House/
Swiss
Rat/NR
NR/NR
f/
20/
group
octa-klor
('specially
purified')
oral
olive oil
.0-15 days
NR
NR
cls-
chlordane
technical
grade
AAEE
reference
standard
NR
Inhala
Hon
Inhala
tlon
NR
- air
- air
S
10
IS
20
SO
100
200
ag/kg bw/day
200 ag/kg by single dose
current of
air (18 at/
sec) passed
through 10S
at of chlor-
dane In a satu-
ration train
continuous
for 14 days
0/S- no deaths until S3 days
of feeding.
0/S- deaths at 14 days, 2 deaths
at 16 days.
4/4 deaths - 13 days
S/S deaths - 10 days
14/14 deaths - IS days
12/12 deaths - 9 days
6/6 deaths - 8 days
Slight treaors. paralysis of
hind legs, convulsions,
hypothermia
No syaptoas of toxicity for
either grade of chlordane.
Histologically, there was
reticulation and oxyphllla of
liver cytoplasa, congestion
and cell proliferation of
bronchiole lining.
Stohlaan et
al.. 1950
Hrdlna et
al.. 1974
Ingle, 19S3
NR/10
130-400 g octa-klor l.p.
none
160 ag/kg
bw
single dose
3/10 (30X) deaths In 9-10 days Stohlaan et
10
('specially
none
240 ag/kg
bw

8/10 (BOX) deaths In 5-7 days al., 19S0
7
purified')
none
400 ag/kg
bw

6/1 (86%) deaths In 2-6 days

'• -



Pulnonary congestion and






henorrhaglng.
19

Tween-20
0 ag/kg
bw
single dose
2/19 (11X) deaths In 7-9 days;




adhesions and exudates.
10

Tween-20
SO ag/kg
bw

1/10 (10X) deaths In 13 days
20

Tween-20
100 ag/kg
bw

3/20 (15X) deaths In l.S hours-





6 days.
20

Tween-20
200 ag/kg
bw

IS/20 (7SX) deaths In 40 alnutes-
6 days.
Treaors, convulsions.

10
olive
oil
100
ag/kg
bw
single dose
1/10 (10%) deaths 1n B days
o
CD
10
olive
oil
200
ag/kg
bw

4/10 (40X) deaths In 2-3 days
S
O
U>
10
olive
oil
300
ag/kg
bw

7/10 (70X) deaths In 3-6 days

-------
TABLE V-2 (cont.)
Species/ Sex/
Strain Number Uelght/Age	Purity Route Vehicle	Dose	Duration	Effects	Deference
Rat/	N/2	255-275 g	technical l.p. 3* Tween-80 350 ag/kg bw single dose Deaths within 1 hour; alld
Sprague-	grade	Isotonic	treaors and disorientation In
Dawley	saline	minutes; hypersensitivity
progressing to clonic and
recurrent ataxia, Muscular
rigidity, tetanic paralysis,
hyperpnea; changes In EE&.
Hyde and
Falkenburg,
1976
Mouse/
N-F/NR
60-70 days
technical
l.p.
corn oil
100
ag/kg
bw
single dose
No symptoms of toxicity
Arnold et
CD-I


grade


300
ag/kg
bw

Mild hypoactlvlty, 5031 mortality
al.. 1977





1000
ag/kg
bw

100% mortality

Mouse/
M-F/NR
60-70 days
HCS-3260
l.p.
corn oil
30
ag/kg
bw
single dose
No syaptoas of toxicity
Arnold et
CD-I




100
ag/kg
bw

No symptoms of toxicity
al.. 1977






300
ag/kg
bw

Interalttent treaors and ataxia











anong males.







1000
ag/kg
bw

Interalttent treaors and ataxia

Mouse/ F/60/
Balb/ group
c Crl
among sales, no Mortality.
neonate	els-	s.c. sesame oil	0	3 Injections, Significant differences from
chlordane	0.075	1 each on days control: delayed eye opening
100k	ag/mouse	2. 3 and 4	(p<0.001); delayed vaginal
of life	opening (p<0.001), decreased bw
at 4, 6 and 8 weeks.
0.1S ng/»ouse	Decreased bw at 4, 6 and B weeks.
Increased ovary weight (p<0.0S).
trans-	s.c. sesane oil 0.075	3 Injections, Significantly delayed eye
chlordane	ng/aouse	1 each on days opening (p<0.01), decreased bw
99.3%	2, 3 and 4	at 4, 6, 8 and 10 weeks;
of life	decreased pituitary weight
(p<0.05).
0.15 ag/ao use	Delayed vaginal opening (p<0.0S).
decreased bw at 4 weeks,
decreased pituitary weight
(p<0.02). Increased ovary weight
(p<0.02).
Talamantes
and Jang,
1977

-------
TABLE V-2 (coot.)
o					—
g Species/ Se*/
o Strain Number Weight/Age	Purity Route Vehicle	Dose	Duration	Effects	Reference
Gerbll/
h/20/
70-90 g/adult
technical l.m.
petroleum
0.
25. 50.
Indian
group

' grade
ether
75
mg/kg bw
desert






RabbH/NR
NR/1S
1.6-3.3 kg
octa-klor l.v.
Tween-20
0
mg/kg bw

7
(¦specially
Tween-20
10
mg/kg bw

23

purified')
Tween-20
20
mg/kg bw

10


Tween-20
30
mg/kg bw

3


Tween-20
40
mg/kg bw
single dose Dose-related observed hyper-	Simm
glycemla maximal 1 hour after	and Karel,
treatment: blood glucose	1976
levels declined to control
levels 1 week after treatment.
single dose 0/15 deaths	Stohlman,
1/7 (14%) deaths 1n 14 hours	et a).,
17/23 (74X) deaths In 7 minutes 1950
to 4 days.
10/10 (100X) deaths in 7
alnutes to 2 days.
3/3 (100%) deaths In 15-30
minutes. dyspnea, tremors and
convulsions.
NR - Not reported
i
10 l.m. - Intramuscular
l.p. - Intraperitoneal
l.v. - Intravenous
s.c. - subcutaneous

-------
Ambrose et al. (1953a) found no symptoms of toxicity except for tntracyto-
plasmlc bodies 1n the liver cells 1n all albino rats (groups of 5) treated
orally for 15 days with 6.25 or 25 mg chlordane/kg bw/day 1n cottonseed oil.
At doses of 50, 100 or 200 mg/kg bw/day, 2, 5 and 5 animals/group died. A
single oral dose of 200 mg/kg bw c1s-chlordane In corn oil resulted 1n
significantly elevated liver and kidney gluconeogenic enzymes when compared
with controls (Kacew and Slnghal, 1973). Similar results were observed In
gerblls treated Intramuscularly with technical grade chlordane (Saxena and
Karel, 1976).
Single oral doses of technical grade chlordane or of HCS-3260 (see
Chapter III) to CD-I mice resulted 1n no signs of toxicity at 30-100 mg/kg
bw doses (Arnold et al., 1977 ). M1ld hypoactlvlty was observed at 300 mg
technical grade chlordane/kg bw and 100% mortality at a dose of 1000 mg/kg
bw. Similar doses of HCS-3260 resulted 1n tremors, ataxia and death.
Intraperitoneal doses of these preparations produced results similar to
those observed after oral administration. Den Tonkelaar and Van Esch (1974)
determined the dose response of technical grade chlordane administered In
the diet for 14 days to groups of six male Wlstar rats. Significantly ele-
vated (p<0,025) activities of aniline hydroxylase and amlnopyrlne demethyl-
ase occurred at a chlordane concentration of >10 mg/kg diet and of hexo-
barbltal oxidase at >20 mg/kg diet. Enzyme activities at 5 mg/kg diet
levels were not significantly different from control values. Continuous
Inhalations for 14 days of technical grade or AAEE reference standard chlor-
dane (see Table V-2 for exposure) produced no outward signs of toxicity In
Swiss mice; however, mild histological changes were seen In liver and lung
tissues at necropsy (Ingle, 1953).
01800
V-10
03/26/87

-------
Neonatal female rats treated with subcutaneous Injections of ds- or
trans-chlordane (0.075 or 0.15 mg/mouse) on days 2, 3 and 4 of life had
significantly delayed onset of eye opening and vaginal opening, decreased
bw, decreased pituitary weight and Increased ovary weight when compared with
untreated controls (Talamantes and Jang, 1977). A dose-response relation-
ship for these effects was not demonstrated for either Isomer.
Heptachlor and Heptachlor Epoxide — Acute oral L050 values of
heptachlor administered In lipophilic vehicles to rats, mice and hamsters
are 1n good agreement (Table V-3). The range of values for rats was 71 g/kg
bw for male Charles River rats (Podowskl et al., 1979) to 162 mg/kg bw for
female Sherman rats (Gaines, 1960). For male Sherman rats, the LD^q was
100 mg/kg bw. Gak et al. (1976) found that rats and hamsters were equally
susceptible- (LDjq = 105+12 mg/kg bw and 100*11 mg/kg bw, respectively). A
value of 40 mg/kg bw In rats was reported for the less pure commercial for-
mulation (Ben-Dyke et al., 1970). Gak et al. (1976) reported that hepta-
chlor was slightly more toxic to mice (LO^g ¦ 70*7 mg/kg bw). Heptachlor
applied dermally was less toxic than when administered orally to rats
(Gaines, 1960; Ben-Oyke et al., 1970). Harbison (1975) found that upon
Intraperitoneal administration, analytical grade heptachlor was 7.5 times
less toxic to neonatal Sprague-Oawley rats than to adult males. Pretreat-
ment with phenobarbltal, however, reduced the neonatal ID^g from 531 to
133 mg/kg bw by enhancing the neonates' ability to bloactlvate the Insecti-
cide to the more toxic heptachlor epoxide. The acute oral LD5Q of hepta-
chlor epoxide In adult rats was -60 mg/kg bw (NAS, 1977; Sperling and
Ewlnlke, 1969; Podowskl et al., 1979). The Intraperitoneal LD^ of
heptachlor epoxide In mice was 18 mg/kg bw (Iv 1 e et al., 1972). Further
details of the LD^ determinations are presented In Table V-3.
01800	V-ll	04/01/87

-------
TABIC V-3
Acute LDjqs for Heptachlor and Heptachlor Epoxide
GD
Species/
Strain
Sex/
Nuaber
Metght/Age '
Coapound
and/or
Purity
Route
Vehicle
LO50
(ag/kg bw)
Comment
Reference
Rat/NR
NR/NR
NR
¦pure"
heptachlor
oral
"Innocuous
solvent'
90
Treaors and convulsions, deaths occurred up
to 6 days.
Lehaan. 19S1
Rat/
Sheraan
H/60
f/90
175 g/90 days
200 g/90 days
technical
heptachlor
oral
peanut oil
100
(74-13S)
162
(140-188)
Treaors. hyperexcltablHty, Irritability and
convulsions
Survival tlae:
6 hours - 7 days
Survival tlae:
S hours - 11 days
nuobers In parentheses are confidence Halts.
Gainesp 1960
Rat/NR
N/NR
adult
heptachlor
purity NR
oral
NR
112
NC
Sperling and
Ewlnlke. 1969
Rat/
Charles
River
N
6-10/
group
ISO g/6 weeks
heptachlor
99.9*X
oral
corn oil
71
Deaths occurred In 24 hours
Podowskl et
at., 1979
Rat/NR
NR/NR
NR
heptachlor
purity NR
oral
olive oil
105 ~ 12
NC
Gak et al.,
1976
Nouse/NR
NR/NR
NR
heptachlor
purity NR
oral
olive oil
70 ~ 7
NC
Gak et al..
1976
Haaster/
Golden
NR/NR
NR
heptachlor
purity NR
oral
olive oil
100 ~ 11
NC
Gak et al..
1976
Rat/NR
NR/NR
NR
heptachlor
cooaerclal
formulation
oral
NR
40
NC
Ben-Oyke et
al.. 1970
Rat/NR
NR/NR
NR
heptachlor
coanerclal
foraulatlon
dermal
NR
195-250
NC
Ben-Dyke et
al.. 1970
O
00
\
O
CO
\
00

-------
TABLE V-3 (cont.)
o 	.	___			
00
o	Compound
° Species/	Sex/ and/or LOjq
Strain	Nuaber Uelght/Age Purity Route Vehicle (ag/kg bw)	Coanent	Reference
Rat/
Sheraan
Rat/
Sprague-
Oawley
Rat/
Sprague-
Oauley
•
Zl Rat/NR
Rat/
Charles
River
Rat/NR
Mouse/
Swlss-
Uebster
N/100
F/bO
N/f
10-20/
group
N/
10-20/
group
H/NR
N/
6-10/
group
N/NR
F/NR
N/NR
175 g/90 days
200 g/90 days
neonates
2-5 days
120-150 g/
adult
adult
150 g/6 weeks
NR
20 g
technical
heptachlor
dermal xylene
heptachlor
analytical
grade
heptachlor
analytical
grade
heptachlor
epoxide
heptachlor
epoxide
heptachlor
epoxide
reference
heptachlor
epoxide
l.p.
I.p.
oral
oral
oral
l.p.
corn oil
corn oil
NR
corn oil
NR
dlaethyl
sulfoxide
195
(119-320)
250
(200-313)
531
(311-1020)
71
(41-103)
62
60
46.S
61.3
18
Treaors, hyperexcltablllty. Irritability and
convulsions
Survival tlae:
43 hours - 10 days
Treaors, hyperexcltablllty. Irritability and
convulsions
Survival tlae:
96 hours - 13 days
Nuabers In parentheses are confidence Halts.
Pretreataent with phenobarbltal (40 ag/kg bw x 3)
l.p. reduced LOjg to 133 am/kg bw.
Nuabers In parentheses are confidence Halts.
Numbers In parentheses are confidence Halts.
NC
Gaines, 1960
Deaths In 24 hours
NC
NC
Harbison,
1975
Harbison,
1975
Sperling and
Iwlnlke, 1969
Podowskl et
al.. 1979
NAS, 1977
Ivle et al.,
1972
NC- No coanent
NR- Not reported
o
on l.p.. Intraperitoneal
o
to
\
00

-------
Other acute toxicity data For heptachlor and heptachlor epoxide are
presented 1n Table V-4. Symptoms of Intoxication Include tremors, convul-
sions, paralysis and hypothermia (Hrdlna et al., 1974; Yamaguchl et al.,
1980). Oral doses of heptachlor have resulted 1n significantly elevated
levels of serum GPT and serum aldolase coincident with histologically
observed liver damage (Kramplf 1971). These effects were observed In female
Ulstar rats given a single oral dose of 98% pure heptachlor of 60 mg/kg bw
or repeated oral doses of 7 or 12 mg/kg bw/day for up to ?8 days. At 28
days, the serum levels of GPT and aldolase had returned to control levels,
and damage to the liver was less severe. It Is possible that the rats were
developing a tolerance to heptachlor. At a high dose (200 mg/kg bw orally),
which Is above the oral LDgQ, significantly Increased (p<0.05) activities
of gluconeogenic enzymes were observed 1n male Ulstar rats (Kacew and
Slnghal, 1973; Slnghal and Kacew, 1976).
A single day or 5-7 days of dietary administration of heptachlor (96%)
at a level of 10 mg/kg diet to rats resulted In alterations In liver func-
tion, as evidenced by changes In blood glucose, liver glycogen, GPT levels,
acid and alkaline phosphatase activities, etc. (see Table V-4 for other
parameters and directions of changes) (Enan et al., 1982). Single oral or
Intraperitoneal doses (30 or 100 mg/kg bw) of a heptachlor:heptachlor epox-
ide (27:75) preparation to CD-I mice resulted In moderate to severe hypo-
activity, ruffled fur and "partial mortality1 (Arnold et al., 1977). At low
dietary levels of >99* pure heptachlor (2-50 ppm or mg/kg diet) for 14 days,
dose-related Induction of hepatic aniline hydroxylase, amlnopyrlne demethyl-
ase and hexobarbltal oxidase was observed 1n rats (Oen Tonkelaar and Van
Esch, 1974). Details of these studies are given Vn Table V-4.
01800
V-l 4
03/08/87

-------
I ABU V 4
Acute loxlclty of Heptachlor and Heptachlor Epoxide
00
§	Coapound
Species/	Sex/ Height/ and/or
Strain	Huaber Age	Pur It*	Route Vehicle	Dose	Our at Ion	Effects	Reference
Rat/ f/24/ ?00- heptachlor
Ml star group 300 g 98X
Rat/NR HR/MR HR
heptachlor
Rat/ N/4/ 200 g/ heptachlor
Wlstar group adult
HR
oral
Rat/NR F/S 100- heptachlor
150 g 9bX
oral vegetable' 0. 60	single Subgroups were sacrificed at 2, 24 and 72 hours. Kraapl. 1971
oil	ig/k] bw dose	Significant changes (p<0.0S-0.001): elevated
liver GP1 and aldolase at 2 hours, decreased liver
GP1 and aldolase at 72 hours. Elevated serua GP1
at 12 hours and serua aldolase at 24 and 12 hours.
Changes coincided with slight to aoderately severe
histological liver daaage.
NR	200	single Slight treaors, hind leg paralysis, convulsions	Hrdlna et
ag/kg bw dose	and hypotherala.	al.. 1974
corn oil 0, 200 single Increased fasting, blood glucose level (I48X of	Kacew and
ag/kg bw dose	control); decreased concentration of liver	Slnghal.
glycogen In I hour (59X); Increased hepatic	1973
pyruvate carboxylase (199X1, phosphoenotpyruvate
carboxyklnase (200X), fructose-l.b-dlphosphatase Slnghal and
(239%) and glucose-6-pltosphatase (ISM);	Kacew. 19>b
Increased kidney cortical enzyaes as above,
167X. 178%. 18BX and 138X, respectively.
Increased liver and kidney basal and fluoride
stlaulated adenyl cyclase and cyclic AMP. All
changes significantly different froa control
(p<0.0S).
oral diet	0 and 10 1 day of Ho effect on RBC count, significantly Increased	fnan et al.,
ag/kg	feeding MBC count (p<0.0S), significantly elevated serua 1982
diet	bilirubin (p<0.0S) decreased hepatic lipid
content (n.s). Increased blood urea (n.s.) and
Increased serua cholesterol (n.s.), no change In
blood glucose or hepatic protein content,
decreased liver glycogen content (n.s.), decreased
GPl (n.s.). Increased 001 (n.s.). Increased
alkaline phosphatase (p<0.05) and decreased acid
phosphatase (p<0.0S( when caapared with controls.

-------
I ABU V 4 (tonl)
oo Species/ Se*/
§ Strain Nuabcr
Compound
Weight/ and/or
Hqc	Purity
Rat/NR
l/S
100-
ISO 9
Rat/
UVstar
F/24/
gr oup
?00
300 9
i
o«
Route
Vehicle
hejlicklor
oral
itlet
Itepilclilor
90%
oral
vegetable
ol 1
Dose
0 and 10
»9/k99*
heptachlor
fieplachlor
epoxide

-------
TABLE V-4 (cont.)
o
oo
o	Compound
° Species/	Sex/ Height/ and/or
Strain	Number Age	Purity	Route Vehicle	Dose	Duration	Effects	Reference
Rat/NR NR/NR NR
Rat/ N/2/ 160-
Sprague- group 200 g
Oawley
heptachlor
epoxide
hepatchlor
expoxlde
MR
l.p.
NR
NR
100	single - Slight tremors, hind leg paralysis, convulsions,	Hrdlna et
mg/kg bw dose	hypotherala.	al., 1974
0, 200 single Violent convulsions 5 hours after treatment;	Yamaguchl
¦g/kg bw dose	animals were sacrificed - there was Inhibition	et al., 1980
of brain synaptosomal Ca?*-Hg** ATPase
(see Chapter VII).
•The rats were maintained on the test diet for 5 days a week for 4 weeks. It was not clear whether rats were sacrificed after the 7th day of feeding, on
the 9th day of the experiment or after the Sth day of feeding on the 7th day of the experiment.
NR* Not reported
l.p.- Intraperitoneal
« n.s.- not significant
i
-j 6PT- glutamate pyruvate transaminase
60T- glutamate oxaloacetate transaminase
o
CD

-------
Effects of Subchronlc and Chronic Exposures. The majority of the
available studies on the effects of subchronlc and chronic exposure to
chlordane, heptachlor and heptachlor epoxide have been conducted by Incor-
porating the insecticides Into the diets of laboratory rats and mice. Many
of the long-term studies were designed primarily to assess the carcinogeni-
city of these compounds; however, some data on toxicity were available.
Chlordant —
Subchronlc Dietary Exposure. Table V-5 summarizes the studies
discussed In this section.
A preliminary subchronlc study was conducted to determine the dose
levels of chlordane to be administered to Osborne-Mendel rats and B6C3F^
mice for carcinogenicity testing (NCI, 1977a). Hale and female rats, five
of each sex per group, were administered diets containing chlordane (analyt-
ical grade 94.8%) 1n concentrations of 0-1600 ppm (1600 mg/kg diet) for 42
days. The rats were then maintained on chlordane-free diets for 2 weeks.
The only criteria for toxicity were food consumption, body weight gain and
mortality. No effects on body weight gain were observed at <400 mg/kg diet.
01ets containing 800 mg/kg diet resulted 1n deaths of four females. A
dietary level of 1600 mg/kg diet resulted In 100% mortality.
Groups of five male and five female mice received diets containing 0-320
ppm (320 ppm mg/kg diet) chlordane for 42 days, followed by 14 days observa-
tion (NCI, 1977a). No deaths occurred at <80 mg/kg diet. Olets of 160
mg/kg diet resulted 1n two deaths among males, none 1n females. All females
01800
V-18
08/10/84

-------
1ABU V-S
Kfetti In Rals and Hlce of Subchronlc and Chronic Dietary Exposure lo Chlordane
oo
o
o
Species/
Strain
Sex/Nuaber
Height/
Age
Purity
Dose
|ag/kg dltl)'
Duration
tffect
Reference
Rat/
Osborne-
Hendel
House/
BbC3f i
H-t/S/scx/
group
NR
R-f/S/sex/
group
Rat/ N/42/group
Sprague-
Dawley
NR
91 days
iO
Rat/NR
H-f /NR
NR
Rat/NR M-f/NR
NR
analytical
grade
chlordane,
94.B* (71.7*
els-. 23.1*
trans -
chlordane)
analytical
grade 94.8*
technical
grade
technical
grade or
pure trans-
chlordane
technical
grade
0. SO. 100,
?00, 400. 600.
1600
0. 20. 40. 60.
160. 320
0 and 19.5
ag/kg bw/day
based on food
consuaplIon
data
various
dietary levels
0 and 1.2
ag/kg bw/day
based on food
42 days on	No effects or bw gain at <400 ag/kg diet. At BOO ag/kg diet. NCI. 1977a
test diet,	4/5 feaales died, no aales died. but bw gain was reduced
14 days of	during first week only,
observation	At 1600 ag/kg diet, all died.
NCI. 1977a
Shaln el
al.. 1977
42 days on No deaths at <80 ag/kg diet; at 160 ag/kg diet, 2/S aales,
test diet, 0/S females died. At 320 ag/kg diet 2/S aales. S/S feaales
14 days of died,
observation
90	days	No effect on food consuaptlon when coapared with controls.
Decreased aean weekly bw gain coapared with controls.
No significant change In testicular and ventral prostate
weight. Significant Increased nuclear (642X) but not
cyloplasalc androgen receptor site content of ventral
prostate (p<0.0S). decreased ventral prostate protein
content (13X of control). RNA (3SX) and DNA content (25X).
91	days	Dose-related Increased phosphorothloate detoxlcatlon,	Klnoshlla
Increased activities of 0-dlaethylase and N-dlaethylase	and Keapf.
over controls. lh» greatest Increase was observed during	1970
the first week of treataenl. Hale rats were acre sensitive
than feaale rats, the NOEl for enzyae Induction was I ppa
(1 ag/kg diet).
S aonths 2 control rals died. No hlstopathologlcal daaage to lungs, De long and
heart, stoaach, liver, kidney, spleen and testes. One	ludwtg, 19S4
treated rat had adenocarclnoaa unrelated lo treataenl.
o
u
CT
OO

-------
I AM i V-S (cont.)
Species/ Sei/Nuaber Height/	Dose
Strain	Aye Purity	(ag/kg diet)* Duration	Effect	Reference
House/
H/200
S weeks
90*
0
>36 weeks
OX 'benign proliferative lesions* or carclnoMs In liver.
Becker and
CS/B1 6N
controls
,
chlordane;



Sell. 1919

Huober of

in
25

R had"benign proliferative lesions (clearly deaonstratable


test alee NR

heptachlor


foci, areas or nodular lesions which deaonstrated neither







alcrolnvaston nor cytologlcal atypla).





SO

7* had benign proliferative lesions. It was unclear how aany







of the treated alee with benign proliferative lesions also
1






had carclnoMs or elevated a-fetoproteln levels. It was







stated that of the 12 livers with benign proliferative lesions







In the absence of carclnoM, none had elevated a-fetoproteln







levels.

Rat/NR
H/NR
NR
refined
0, 10. ?0, 40,
400 days
Dose-related growth retardation; occasional liver pathology
Aattrose et



technical
BO. 160. 320,

at 40 ag/kg; enlarged livers and liver pathology at >80
al.. 1953a,b



grade
640 and 1280

ag/kg; Increased aortallty at >640 ag/kg


f/NR
NR
refined
0. 10. 20. 40,
400 days
Growth retardation at >120 ag/kg; enlarged livers and liver




technical
80, 160. 320,
pathology at BO ag/kg; Increased aortallty al >640 ag/kg




grade
640 and 1280



House/
H-f/
6 weeks
analytleal
0. 5. 25
18 aonths
No effect on bw gain consuapllon was observed. There was a
Epstein.
CO 1
100/

reference
and SO
Inter la
dose-related Increased aean liver weight In treated Mies and
19)6

sex/

standard

kill at
feMles at 6 aonths and at 18 aonths. These Increases were
(review of

group

(technical

6 aonths
significant for all treated feMle groups, for the SO ag/kg
IRDC, 1973a)



grade)

of 10 alee/
diet Mies at 6 aonths and for the 2S and SO ag/kg diet Mies





sei/group
at 18 aonths. Survival was 14* for Mies and 24* for feMles

at the highest dose-level and SI-73X for all other groups
Including controls, neoplastic lesions of the liver were
observed (see Carcinogenicity Section). Hepatocytoaegaly was
observed In a dose-related Increased Incidence for all treated
Mies and feaales. The Incidence of hyperplastic nodules was
dose-related at the 2 highest levels for Mies and feMles and
significant (p
-------
I ABU V-5 (cont.)
Species/ Sex/Nuatoer
o Strain
CD
o
Height/
Age Purity
Dose
(ag/kg diet)*
Duration
If fee I
Rat/ N/10 aatched. 35 days analytical
Osborne- 60 pooled	grade,
Mendel controls.	94.W
SO treated/
group
f/10 aatched,
60 pooled
controls.
SO treated/
group
35 days
IMA 203.5,
401.0
IMA 120.8,
241.5
109 weeks
80 weeks
on lest
diet, 29
weeks ob-
servation
109 weeks
80 weeks
on test
diet, 29
weeks ob-
servation
Hean bw of high-dose Mies and resales were consistently
decreased fro* controls. Host high-dose feaales had
treaors at 44 weeks. All treated groups had clinical
syaptoas of toilclty throughout the study becoalng
progressively worse with tlae (loss of bw, rough and
discolored hair, palpable Basses and tuaors). Aaong
feaales, there was a significant (p=0.003) dose-related
Increase In aortallty. No significant difference In
aortallty was observed for aales. Histologic signs
of aging were observed with equal frequency In control
and treated rats. Neoplastic lesions were observed
(see Carcinogenicity Section). Hyperplastic lesions
were observed In thyroid glands of rats of all groups.
These were follicular- and C-cell hyperplasia.
Reference
NCI. 1977a
House/ N/20 aatched, 35 days analytical
B6C3fl 100 pooled,	grade
controls.	94.8*
SO treated/
group
F/20 aatched,
80 pooled .
controls.
SO treated/
group
Rat/f-344 80/sex/group
35 days
technical
grade
IMA 29.9. St.2
91 weeks
BO weeks
on test
diet. 10
weeks ob-
servation
91 weeks
B0 weeks
on test
diet. 10
weeks ob-
servation
0, 1, S and 25 130 weeks
ag/kg diet cal-
culated to be
0, 0.045, 0.229
and 1.175 ag/kg/day
IMA 30.1, 63.B
No significant difference In bw gain between treated
aales and feaales and control aales and feaales.
High-dose aales and feaales had treaors at 20 weeks.
A few alee had general or local hair loss and a hunched
appearance at 50 weeks. Abdoalnal distention, aore
prevalent In feaales. was observed In all groups.
Significant differences In aortallty were observed
between low-dose and control aales (p<0.02) and
between high-dose and control aales (p-0.0l»). No
difference In aortallty aaong feaales. Histological
signs of aging were seen In all groups. Neoplastic
lesions were observed (see Carcinogenicity Section).
Hepatocytoaegaly and hyperplastic lesions were observed
Infrequently In all groups.
Significant dose-related hepatocellular necrosis In aales
and hepatocellular swelling In high-dose feaales. Liver
adenoaas In high-dose aales.
NCI, 1977a
Voneaura
et al.. 1983

-------
lABlf
— Specie*/ Sei/Nunber
g Strain
o
Height/
Age Purity
Dose
(•g/kg diet)
OuratIon
Rat/ M-l/20 of
Osborne- each set/
Mendel group
SO-SS g technical
up lo
? years
10
30
i
ro
ro
ISO
300
Dog/Ntf NR/NR
NN
MR
0. 0 3, 3.
IS and 30
2 years
CJ
"v
l\J
•Unless otherwise slated
NR > Not reported
TWA • floe-weighted average
S (cool )
tffect	Reference
No effect on food consumption, growth rate or mortality, no Ingle. 19S?
syaptorn of toalctty, no gross or hlstopathologlcal changes
In 1Itfer, lungs, kidneys, pancreas, stomach, adrenals, thyroid,
thymus, lymph, testes, ovaries, heart and spleen.
fssenttally the same as at S mg/kg diet; however, upon
necropsy, alitor liver damage (occasional hypertrophic cells.
¦Inor bile duct proliferation).
No effect on food consumption, growth rate, mortality or organ
weights. Iretors could be induced after 60 weeks. Slight
liver damage to a lesser degree than at ISO mg/kg diet.
No kidney, lung, heart, adrenal, spleen or Intestinal damage.
Slight reduction In growth rate after 60 weeks, greater in
females, coinciding with periods of anoreila and tremors.
Iremors after ?6 weeks. Increased mortality at 41 weeks.
Kidney and liver hypertrophy at 48 weeks In subgroup of S
rats/sea. Upon histology duodenal ulcers and liver damage
were revealed (centrllobular hypertrophy, nuclear karyor-
rheils, karyolysts, necrosis, fatty degeneration, bile duct
proliferation). Moderate to Barked kidney, lung, myocardium,
adrenal and spleen damage.
Reduced growth rate after 60 weeks, greater In females,
coinciding with periods of anoreila and tremors. Hemorrhaging
around eyes and nose associated with histological changes at
1? weeks. Increased mortality, 0 females and 1 male survived
longer than 84 weeks. More severe liver, kidney, heart,
adrenal, lung, myocardium and spleen damage.
A review panel for WH0/IA0 Indicated that 3 mg/kg diet was a Wazeler, 196/
NOtl. the endpolnts of the sludy were Increased liver weight,
and histopathologic changes In (he liver.

-------
and two males died at the highest exposure level. Histological examinations
were not performed or were not reported. Maximum tolerated doses were
established at 400 and 800 mg/kg diet for female and male rats, respec-
tively, and at 80 and 40 mg/kg diet for female and male mice, respectively.
Shaln et al. (1977) found that feeding of technical grade chlordane to
male Sprague-Dawley rats for 90 days at an average dose of 19.5 mg/kg bw/day
had no effect on food consumption when compared with control-fed rats. Mean
body weight gain was significantly reduced 1n a subgroup of 1? randomly
selected rats. In another subgroup of 24 rats, body weight was not reduced
and no changes were observed In testicular or ventral prostate weights.
Histological examination of the rats was not performed, since the study was
designed to determine the effect of chlordane on prostate homeostasis. Rats
were castrated 24 hours before sacrifice. Significant changes from controls
were as follows: Increased nuclear (642%) but not cytoplasmic androgen
receptor site content, decreased RNA content (36%) and decreased ONA content
(25%). Ventral prostate protein content was also reduced to 13% of control.
Induction of hepatic microsomal enzymes during 13 weeks of feeding vari-
ous levels of chlordane or transchlordane to rats was reported by Klnoshlta
and Kempf (1970) 1n a meeting abstract. Dose-related increases In activi-
ties of enzymes Involved In phosphorothloate detoxlcatlon, 0-demethylase and
N-demethylase activation, maximal during the first week of treatment, were
noted. The no-effect level for enzyme Induction was 1 ppm (1 mg/kg diet).
01800
V-23
03/26/87

-------
DflLong and Ludwlg (1954) found that administration of an average dally
dose of 1.2 ng technical grade chlordane/Vg bw 1n the diet of male and
female rats for 5 months resulted 1n no histopathologic damage to lungs,
heart, stomach, liver, kidney, spleen or testes. The rats were fed dog
pellets that had been treated by fog application of spray concentrations of
chlordane. The average dally dose was calculated by the Investigators from
food consumption data. There was no mention of how many animals were
tested; however, the Investigators stated that two control rats died. One
treated rat had a kidney adenocarcinoma, which was believed to be unrelated
to chlordane treatment.
Chronic Oletarv Exposure. Table V-5 summarizes the studies dis-
cussed 1n this section.
Ambrose et al. (1953a,b) conducted a study on the dietary effects of
technical grade chlordane fed to groups of male and female rats for 400
days. The dietary levels used were 0, 10, 20, 40, 80, 160, 320, 640 and
1280 ppra (mg/kg) diet. Increased mortality was observed 1n the 640 and 1280
ppm groups and retarded growth was observed In all animals at >320 ppm.
Some growth retardation was observed In male rats fed 20, 40 or 160 ppm
chlordane diets, but no growth retardation was observed 1n female rats fed
<160 ppm or In the male rats fed 10 or 80 ppm. Significantly enlarged
livers and liver pathology were found in male and female rats fed chlordane
at >80 ppm and liver pathology was occasionally found 1n male rats fed 40
ppm. Necrotic or degenerative pathology were present In the study animals.
01800
V-24
08/09/84

-------
Ingle (1952) described the dose-response effects in male and female
Osborne-Hendel rats that had been exposed to chlordane 1n the diet for 2
years. High mortality among males and females, reduced growth rates, eye
and nose hemorrhaging and severe histopathologic damage to liver, kidney,
heart, adrenal, lung, myocardium and spleen were observed at 300 ppm (300
mg/kg diet). At a 150 mg/kg diet level, similar but less severe effects
were seen. The effects at the 30 mg/kg diet level Included Inducible
tremors and slight liver damage. Mo effects on food consumption, growth
rate, mortality, organ weights or morphology were observed at this dose
level. A diet containing 10 mg chlordane/kg produced minor liver damage,
such as occasional hepatocytomegaly and mild bile duct hyperplasia. There
was no effect on food consumption, growth rate or mortality at the 5 mg/kg
diet levels. No symptoms of toxicity, gross or histopathologic changes in
liver, kidney, lungs, pancreas, testes, ovaries, heart or spleen were noted.
Several studies that were designed to assess the carcinogenic potential
of chlordane following long-term dietary exposure of rats and mice provide
Information on nontumor pathology. Disagreement among pathologists 1n the
diagnosis and distinction of hepatic carcinoma from pre-cancerous lesions
and hyperplasia In rodents has confounded the assessment of Increased Inci-
dence of hyperplasia as a toxic rather than a carcinogenic effect.
Becker and Sell (1979), 1n an attempt to resolve these differences,
studied elevated alpha-fetoproteln levels as an Indicator of chlordane
carcinogenicity 1n male C57B1/6N mice. The chlordane used was 90% chlordane
and 10% heptachlor and was administered 1n the tilet at concentrations of 0,
25 and 50 ppm (mg/kg diet) for at least 36 weeks to an unspecified number of
01800
V-25
00/09/84

-------
male mice. At 25 and 50 mg/kg diet, 2 and 7%, respectively, of the mice had
benign proliferative lesions defined as 'clearly demonstrable foci, areas or
nodular lesions that demonstrated neither mlcrolnvaslon nor cytologlcal
atypla." No lesions were observed 1n 200 control nice. Elevated levels of
alpha-fetoproteln were observed In treated nice after 38 weeks and was
always associated with primary hepatocellular carcinoma. The Investigators
stated that of 12 mice that had benign lesions without carcinoma, none had
elevated alpha-fetoproteln levels. No growth was detected 3-7 months after
six of the benign lesions were transplanted to compatible hosts, while 5-12
transplanted carcinomas had shown growth after 1.5-3 months. Thus, the
benign lesions did not appear to be premallgnant lesions.
An unpublished report by the International Research and Development
Corporation (IRDC, 1973a) under contract to the Velslcol Chemical Corpora-
tion was reviewed by Epstein (1976). Groups of 100 male and 100 female CD-I
mice were administered technical grade chlordane In the diet at concentra-
tions of 0, 5, 25 or 50 ppm (mg/kg diet) for 18 months. An Interim kill of
10 mice/sex/group was performed at 6 months. No effect on body weight gain
or food consumption was observed. At both 6 and 18 months, mean liver
weights of all treated female groups were significantly Increased In a dose-
related manner. At 6 months, the 50 mg/kg diet level group of males had
significantly Increased mean liver weights when compared with controls.
Survival, although underestimated because of the Interim kill, was 14X for
males and 24% for females 1n the 25 mg/kg diet group and 51-73% for all
other groups. Including controls. Increased Incidence of hepatocytomegaly
in all treated female and male groups occurred 'In a dose-related manner.
01800
V-26
08/09/84

-------
The Incidence of hyperplastic nodules, as reported by IRDC (1973a), was
dose-related at the two highest dose levels (p
-------
TABLE V-6
Design Summary for NCI Bloassay of Chlordane In Osborne-Mendel Rats3
Sex and
Treatment
Group
Initial
No. of
Anlmalsb
Chlordane
1n Diet
(PP«)
Time on
Treated
(weeks)
Study
Untreatedc
(weeks)
Time-Weighted
Average Ooses^
(PPia)
MALE





Matched-Control
10
0
0
109

Low-Dose
50
400
100
50
0
33
14
33
0
29
203.5
Htgh-Oose
SO
BOO
200
100
0
33
14
33
0
29
407.0
FEMALE





Matched-Control
10
0
0
109

Low-Dose
50
200
100
50
0
33
14
33
0
29
120.8

-------
TABLE V-6 (cont.)
Sex and
Treatment
Group
Initial
No. of
Animals^
Chlordane
In Diet
(ppm)
Time
Treated
(weeks)
on Study
Untreated0
(weeks)
Time-Weighted
Average Doses1'
(PP»)
High-Dose
50
400
33

241.5

200
14




100
33




0
0
29

aSource: NCI, 1977a
bA11 animals were 35 days of age when placed on test.
cUhen diets containing chlordane were discontinued, treated rats and their matched controls were fed
plain feed diets (without corn oil) for 12 weeks and then control diets (2% corn oil added) for an addi-
tional 17 weeks.
dT1me-we1ghted average dose = E(dose In ppm x no. of days at that dose)
E(no. of days receiving each dose)

-------
TABLE V-7
Design Summary for NCI Bloassay of Chlordane In B6C3Fj M1cea
Sex and
Treatment
Group
Initial
No. of
Animals^
Chlordane
In Diet
(PPn)
Time on
Treated
(weeks)
Study
Untreatedc
(weeks)
Time-Weighted
Average Doses1'
(ppra)
HALE





Matched-Control
20e
0
0
91

Low-Dose
50
20
30
0
1
79
0
10
29.9
High-Dose
50
40
60
0
15
65
0
10
56.2
FEMALE:


-


Matched-Control
20e
0
0
91

Low-Dose
50
40
30
0
1
79
0
10
30.1

-------
TABLE V-7 (cont.)
Sex and
Treatment
Group
Initial
No. of
An1malsb
Chlordane
In Diet
(ppm)
Time
Treated
(weeks)
on Study
Untreated0
(weeks)
Time-Weighted
Average Doses'1
(ppm)
High-Dose
SO
80
15

63.8

60
65




0
0
10

aSource: NCI, 1977a
bAl1 animals were 35 days of age when placed on test.
cWhen diets containing chlordane were discontinued, mice received the control diet (2% corn oil added)
until termination.
dT1me-we1ghted average dose = i(dose In ppm x no. of days at that dose)
E(no. of days receiving each dose)
e!n1t1aliy 10 animals of each sex were placed on test as matched controls; however, when the study was
restarted, 10 additional animals of each sex were placed on test as matched controls.

-------
was not significantly Increased for male rats. Histopathologic signs of
aging, such as chronic nephritis, biliary hyperplasia and chronic prostati-
tis, were observed with about equal frequency 1n all control and treated
groups. The Incidences of follicular and C-cell hyperplasia 1n thyroid
glands of all treated and control groups were observed In a non-treatment-
related Incidence.
The results In mice Indicated no difference In body weight gains among
groups. Tremors were observed 1n high-dose males and females after 20
weeks. Mortality was significantly Increased among treated males, but not
females, 1n a dose-related manner. Histopathologic signs of aging commonly
seen In mice occurred 1n all groups. Hepatocytomegaly and diffuse hepatic
hyperplasia occurred Infrequently In Individuals of all groups.
In an unpublished study by Yonemura et al. (1983), F-344 rats
(80/sex/group) were fed technical chlordane at dietary levels of 0, 1, 5 or
25 ppm for 130 weeks. Body weight, food consumption and water Intake were
monitored at regular Intervals. Clinical laboratory studies were performed
and organ weights were measured on eight an1mals/sex/group at weeks 26 and
52, and on all survivors at week 130. Gross and microscopic pathology were
performed on all tissues. Oally dose levels of 0.045, 0.229 and 1.175 mg/kg
for the 1, 5 and 25 ppm treatment groups, respectively, were calculated from
food consumption and body weight data. No effects were observed for hema-
tology, clinical chemistry and urinalysis endpolnts, and no treatment
related effects were reported for body weight and mortality. Hepatocellular
necrosis was observed In 3, 13, 11 and 27 males (64/group) In the 0, 1, 5
and 25 ppm groups, respectively. The Increased Incidence was statistically
01800
V-32
03/08/87

-------
significant for all treatment groups. Liver adenomas were found In the
high-dose males. The only significant effect In females was hepatocellular
swelling 1n the 25 ppm group.
Increased 1Iver-to-body weight ratios were reported for male and female
mice fed chlordane for 2 years at 0.76 ppm (0.09 mg/kg/day), the lowest dose
administered (Inul et al., 1983). Liver necrosis was observed at 0.43 and
1.1 mg/kg/day for males only.
Four-week pilot studies 1n rats and mice were performed to establish the
dose levels 1n these two studies. Both dietary studies resulted in liver
lesions In the low-dose animals (50 ppm for rats and 10 ppm for mice).
An additional unpublished chronic study performed In dogs by Wazeter
(1967) was reported by Vettorazz! (1975). The dogs were maintained for 2
years on diets containing chlordane at levels of 0, 0.3, 3, 15 or 30 ppm.
The effects observed were Increased liver weight and histologic changes 1n
the liver. As reported by Vettorazz1 (1975), a scientific review panel for
WH0/FA0 examined this study and concluded that no effects were observed at
exposures of 3 mg/kg diet or less. Other specific data on the experimental
design or results of this study were unavailable.
Other Routes of Exposure. No data were available on Inhalation or
dermal exposure; however, several Investigators (Karel and Saxena, 1976;
Slnghal and Kacew, 1976; Hyde and Falkenberg, 1976) have described effects
following Intraperitoneal and Intramuscular Injection of chlordane. Details
of the studies are summarized in Table V-8. Karel and Saxena (1976) found
01800
V-33
03/26/87

-------
00
o
o
1ABLI v-a
Effects of Subchronlc Exposure to Chlordane by Other Routes
Species/	Sex/ Weight/
Strain	Nuaber	Age	Purity	Route	Vehicle	Dose	Duration	Effects	References
Gerblt/
Indian desert
H/
64 group
70-80 g/
adult
technical
grade
60-75X
pure
Intra-
auscular
petroleua
ether
0 and 2.5
ag/kg bw
every 3 days
45 days
Increased blood glucose. serua
protein, serial alkaline phos-
phatase and serua actd phos-
phatase levels of treated
gerblls coapared with controls.
Karel and
Saxena.
Rat/MR
i
1
NR/
4/group
NR
cts-
chlordane
Intra-
peritoneal
corn oil
0 and 25
•g/kg bw/day
45 days
Significantly Increased blood
glucose, serua urea, decreased
liver glycogen. Increased
kidney and liver pyruvate
carboxylase, phosphoenolpyruvate
ca'rboiyklnase. fructose-) ,6-
dlphosphatase and glucose-6-
phosphatase |p<0.0S).
Slnghal and
Kacew. 197b
Rat/
Sprague-
Oawley
N/
4/group
255-275 g
technical
grade
Intra-
peritoneal
lween-80-
Isotonlc
saline
0. 0.15. 1.75.
and 25.0 ag/kg
bw/day
48 days
Dose-related changes In EEGs
Increasing as exposure Increased
without syaptoas of toxicity
Hyde and
falkenberg.
1976
NR - Not reported

-------
Impaired liver function 1n gerblls, as evidenced by Increased blood glucose,
serum protein, add and alkaline phosphatase, after Intramuscular Injection
with 15 doses of chlordane (60-75% pure) 2.5 mg/kg bw over 45 days. Similar
results were observed for Increased activities of gluconeogenic enzymes In
rats following Intraperitoneal Injections of cls-chlordane at 25 mg/kg
bw/day for 45 days (Slnghal and Kacew, 19761. Dose-related changes 1n EEG
without symptoms of toxicity were observed 1n rats treated Intraperitoneal^
with <25.0 mg/kg bw/day for 48 days {Hyde and Falkenberg, 1976).
Heptachlor and Heptachlor Epoxide —
Dietary Exposure. Table V-9 sumnarlzes the studies discussed In
this section.
A preliminary subchronlc study was conducted to determine the dose
levels of heptachlor to be administered to Osborne-Mendel rats and B6C3F^
mice for carcinogenicity testing (NCI, 1977b). Hale and female rats, five
of each sex per group, were administered diets containing technical grade
heptachlor (-73% heptachlor, 22% transchlordane, 5% nonachlor) In concentra-
tions of 0-320 ppm (320 mg/kg diet) for 42 days. The rats were then main-
tained on heptachlor-free diets for 2 weeks. The only criteria for toxicity
were food consumption, body weight gain and mortality. No effects on body
weight gain or food consumption were observed at <40 mg/kg diet. At 80
mg/kg diet, female rats had reduced body weight during the first week.
Diets containing 160 mg/kg diet resulted 1n deaths of four females. A
dietary level of 320 mg/kg diet resulted In the deaths of two male and five
female rats.
01800	V-35	03/26/87

-------
TABLE V.)
Effects In Kits ind Nice of Subchroolc And Chronic Dietary Exposure to Heptachlor and Heptachlor Epoilde
oo
o
o
Species/Strain
Sex/
Nrtftir
Height/
Ago
Compound
and/or
Purity
Oose
(mg/kg diet)'
Duration
Effects
Rat/Osborne- N and F/	HI	TicMI(l) hepta-
Nindil	S/sei/	chlor (73X hepta-
group	chlor, 22X trans-
chlordane, 1%
nonachlor
0
20
40
00
HO
320
42 days on tost
diet. 14 days
observation
No effect or bw gain or food con-
sumption compared with controls.
Mo offoct on bw gain or food con-
sumption compared with controls,
fMales had docroastd bw gain
during first week.
4/S faults died, no offoct on bw
gain or food consumption of males.
2/S Mies died, S/S footles died.
ttCI. 1977b
Nouse/ftbCSFj
i
u>
PI and f/	MR	Technical hepta-
S/sox/	chlor (73X hepta-
group	chlor, 22% trans-
chlordane, 5%
nonachlor
0
20
40
00
42 4ays on test
diet, 14 days
observation
Mo effect on bw gain or food con-
sumption.
Mo effect on bw gain or food con-
sumption.
S/S males. 2/S females died.
MCI, 1977b
Dawlay
H/
42/gfo'ep
91 days 99.OX heptachlor
v.1
o
CO
n and f/
Hft
MR
Heptachlor 90.IX
0 and 1.29
mg/kg bw
(based on food
consumption
data)
90 days
t mg/kg bw* S? weeks
Moderate but significant (pO.Ol)	Stain et
reduction In food consumption coo-	al., 1977
pared with control rats. Decreased
mean bw gain (p<0.01). Ho signifi-
cant change in testicular or ventral
prostate weights. Significantly In-
creased cytoplasmic, but not nuclear,
androgen receptor site content of
ventral prostate |p<0.0S). Oocrease
In available cytoplasmic androgen
receptor site content (0.0S
-------
UllC V-9 (cot.)
oo
o
o
Sai/
Spaclas/StraU Muabar
Ualght/
Coapaund
and/ar
Purity
Dost
fag/kg dial)*
IvrittM
trricti
KtfirMci
Moutt/CO-1
n and f/
100/saa/
graap
? Miki
Haptach1or:hapt;
apoilda <2S:J5|
0, 1.0, 5.0
and 10.0
IS aoaths, Utirta
hill at I »atbs
of 10 alca/sai/group
i
CO
imiIii at 10 19/19 dlat lad
dacraasad, kit not airM, bv
galp coaparad with controls, na
dlffaraacas In food consuaptlon.
lhara was a dasa-ralatad Incraasad
¦tan llvar Might In traatad
faaalas aad aspoclally ailit at t
and II aonths. Thasa Incraasai
wara significant for S and 10 ag/kg
dlat ailos and faaalas at 16 Mnlkt

-------
TABLE ¥-9 (cont.)
Sa«/	Height/
Spacles/Stratn Nuaftar	Aga
Coapound
and/or
Purity
Dos*
(ag/kg dial)*
Ouratlon
(Meets
rna-
Nandal
N/10
Batched,
60 poo I ad
contralt
35 dayt
Technical haptachlor
(-73* haptachlor. 2»
trans-chlordane,
5* nonachlor
IHA 38.9.
77. 9
F/10
aatchad,
M poolad
contrail
35 days
IMA 25.7,
SI.3
Nouto/BiC3f |
N/20
¦atchad,
100 pooiad
controli
35 day*
Tachnlcal haptachlor
(tee above)
TWA 6.1.
13.8
,10/aatched
80 pooiad
controls
35 days
TUA 9.0,
18.0
III Maakt
80 Maakt on tait
diet, 21 Maakt
obiervatlon
111 waakt
80 waaki on tatt
dtat, 21 waakt
obiervatlon
10-91 waakt
80 Maakt on tatt
dtat, 10 Maakt
90-91 Maakt
80 Maakt on tatt
dlat, 10 week!
observation
Contlttantly dacraatad bn In bath	NCI, 1917b
htgh-dota aalet and feaalat coapared
Mtth contrail; a graatar effect In
aw let. Tha bw of loM-doia groupt
Mara tlallar to controlt. Both
traatad and control group! developed
•dverte clinical lyaptoat. In toaa
fe^alei of both traatad groupt,
vaginal blaadlng davalopad after
80 Maakt on tatt dtat. Soaa low-
dota feaalat had ainry tuaort.
A doia-ralatad. however not tlg-
nlflcant (p-0.17) Incraata In
aortallty Mat obtarvad In aalat.
A dota-ralatad tlgnlflcant Incraat^
In aortallty (p-0.04) Mat obterved
batMaan traatad feaale groupt.
Hlttologlc signs of aging
occurrad with equal frequency In
control and traatad rati. Neo-
plattlc latlont Mara obterved and
ara dltcuttad In tha Carclnogonlclty
Sac Hon. Hyparplattlc latlont Mara
obtarvad In thyroid glandt of rati
of all groupt. Thata wara follicu-
lar- and C-call hyparplatlat.
Ho appraclabia dlffarancat In bw	NCI, 1977b
batMaan traatad and control alco Mara
obtarvad. Nlca of all groupt had
torat and hair lot! during flrtt
yaar. High dota faaalat davalopad
abdoalnal dtitantlon. Nlca froa all
groupt had rough coati, hair lott,
torat and palpabta aattat. Thara
Mat no apparant dlffaranca In aila
aortallty aaong groupt. Thara wat a
tlgnlflcant dota-ralatad Incraata In
aortallty (p»0.02) batMaan traatad
faaila groupt. Hepatocytoawgaly.
diffuse hyperplasia and aodular
hyperplasia wara seen In livers of
control and treated alca. Non-
neoplastic lesions occurrad In other
organs randoaly aaong all groups.

-------
1ABIE V 9 (cont.)
OD
o
Specles/Straln
Sex/
Number
Weight/
Age
Cuapound
and/or
Purity
Dose
(ag/kg dlel)a
Duration
Effects
Reference
House/C3H
«/
)] control
81 treated
3 i*cek» Heptachlor purity NB
0 and 10
7 years
Bouse/C3H
N and f/
100/group
3 weeks Heptachlor purity MR 0 and 10
2 years
i
CJ
No hepatic vein or cardiac atrial	Rcuber.
thruahosls In controls. I3X of	197/a, 1918
treated alee (10X of Mies and 15X	(histologic
of females) had venous occlusion	slides were
with recent liver Infarcts. Car-	froai vtudy
clnoaas and cirrhosis were also	of Davis,
observed In soae alee with throabl.	1965)
Of the alee with thrortl. 11* did
not have carclnoaas. A few alee with
hepatic vein throobosls had cardiac
atrial throabosls. Hyperplasia and
hyperplastic nodules were observed
tn controls and treated alee.
Survival was 341 for controls. 30X	Epstein,
for treated alee. "Hepatic hyper-	197b
plasla and benign tiaurs* were seen	(review of
tn ?- to 3-fold greater frequency	Davis,
In treated alee. Histologic re-	1965)
evaluation of slides by other
pathologists resulted In decreased
Incidences of hyperplasias and
nodules In favor of Increased In-
cidence of earclnoaa (see the
Carcinogenicity Section).
Rat/Cf
H/?(T/group 10 weeks Heptachlor purity NR
o
CO
M
o>
0
1.5
3.0
5.0
7.0
10.0
110 weeks
Mortality
~ 4 OX
70*
b'.X
MIX
4 OX
/•>x
low of bw tn treated
aales, aost predominant
In III ag/kg diet group.
Increased liver weights
In treated aales
especially tn highest
dose group. Reduced food
consuaptlon In 10 rag/kg
diet group. Liver lesions
described as 'Chlorinated
hydrocarbon" type seen tn
38X of 7.0 ag/kg diet
group. 17X tn 10 ag/kg
diet group and OX In other
groups.
tpstrtn,
1976
(review of
Hitherup
et al..
1955)

-------
TAIL! V-9 (cont.)
oo			
o
0	Coapound
Sei/	Weight/	and/or	Dot*
Species/Strain Muaber	Ago	Purity	(ag/kg diet)* Duration	Hficti
Rat/Cf (cont.) f/20/group 10 Miki Hap
0
l.S
3.0
$.0
7.0
10.0
110 weeks
Rat/CO
f/
54 control
IS treated/
group
MB
99.9X heptachlor:
Ml, hoptacklor
epoxide (75:251
0
S
7.5
10.0
12.S
2 years
Houio/CM
«/
79 troatad
f/
•1 troatad
3 weeks
Heptachlor oipoilde
purity Ml
0 and 10 ppa
2 yaari
o
a
s
o
00
N.
CO
WorU»m»
40X lost of bw, incroatod liver
40X weight, not taan In foaales.
S5X 'Chlorinated hydrocarbon'
35X typo liver lotions start
40* In I7X of 7.0 ag/kg diet
SSX group. SOX of 10 ag/kg
diet group and OX In other
groups. The Incidence of
aallgnant tuaors Is dis-
cussed In the Carcinogen-
icity Section.
2IX Age related lesions seen	fpstoln.
39X In all groups. Spontane-	1974
25X ous lesions and tuaors uin (review of
43X in all groups (Multiple	Jolley
SOX cell typo hypertrophy and ot al.,
telangiectasia In anterior I9U)
pituitary and adrenal
hyper trophy|. Hopatoeylo-
se gal y exceeded control
levels at 7.S, 10.0 and
12.S ag/kg diet levels.
¦o hepatic vein or cardiac atrlua	Reuber,
throabl. no cirrhosis of liver In	1977a, 1970
controls. I0X of treated alee (7X	(histologic
of aales. 1IX of featles) developed	slides
hepatic vein throabosls; 9X (S aales. obtained
9 foaales) treated alee had venous	froa study
occlusion. Carclnoaas and cirrhosis	of Davis,
Mere observed In soae alee with	19(5)
throabl. Of the alee with throabl,
1IX did not have carcinosis. A few
had cardiac atrlua throabosls as
well. 12/70 treated aales. IS/01
treated feaulos had cirrhosis.
Reproduced from
best available copy.

-------
lAHIt V 9 (cont .)
CO
o
o
Specles/Straln
Sex/
Nuaber
Weight/
Age
Coapound
anil/or
PurIty
Dose
(ag/kij diet)4
Dm at lun
Huuse/C3H
N and f/
100/ijroup
NR
Heptarhlor epoxide
purlly MR
Rat/CfN
M/?S/group J weeks
Heplachlor epoxide
pur My NR
F/?Vgroup I weeks
0 and 10
0
OS
?.S
S.O
l.'j
10.0
0
0.5
2.5
i.O
/. i
10.0
2 years
110 weeks
110 weeks
Effects
Survival was 34X for controls, 9.5X
for treated alee. Premature death
was high. "Hepatic hyperplasia and
benign tuaors* were seen In 2 fold
greater frequency In treated alee.
Histologic re-evaluatIon of the
slides by other pathologists re-
sulted In decreased Incidences of
hyperplasias and nodules In favor of
Increased Incidences of carcinomas
(see Carcinogenicity Section).
Mortality
37X No difference In food
48X consumption or growth
'y/% rale. Hepatic cell vacuo-
48X lliallon In all treated
4(1% groups. Hepatocyto-
44X aegaly, degeneration and
regeneration In unspeci-
fied groups, luaor data
are discussed In the
Carcinogenicity Section.
?4X No food consumption
44X differences, no differ-
36X ences In growth rate.
XHX Hose-related Increase In
S?X liver weights. Hepatic
48X cell vacuolliatlon In
all treated groups.
Hepatocytoaegaly, degen-
eration and regeneration In
unspecified groups. Tuaor
data are discussed In the
Carcinogenicity Section.
Reference
19 lb
(review of
Oavls,
196S)
ipvteln,
1916
(review of
Ultlierup
et al.,
I9S9)
o
u>
o>
dag/kg diet unless otherwise stated
bll Is unclear fro* the study whether or not this Is a dally dose and adalnlstered by feeding. Ihe author stated that heplachlor was 'administered with
food* and 'the applied dose was 6 ag/kg bw*.
NR - Not reported; TMA = Tlae-weighted average

-------
Groups of five male and five female mice received diets containing 0-80
ppm (0-80 mg/kg diet) heptachlor for 42 days followed by 14 days observation
(NCI, 1977b). No deaths and no effects on body weight gain or food consump-
tion occurred at <40 mg/kg diet. At 80 mg/kg diet, all male and two female
mice died. Histological examinations of the rats or the mice were not per-
formed or were not reported. Maximum tolerated doses were established at
160 and 80 mg/kg diet for male and female rats, respectively, and 40 mg/kg
diet for male and female mice.
Shaln et al. (1977) found that feeding of heptachlor (99.8X) to male
Sprague-Dawley rats for 90 days at an average dose of 1.29 mg/kg bw/day
significantly reduced the food consumption and mean body weight gain In a
subgroup of 12 randomly selected rats. In another subgroup of 22 rats, body
weight was not reduced and no changes were observed 1n testicular or ventral
prostate. Histological examination of the rats were not performed, since
the study was designed to determine the effect of heptachlor on prostate
homeostasis. Rats were castrated 24 hours before sacrifice. Cytoplasmic,
but not nuclear, androgen receptor site content of the ventral prostate was
significantly Increased. Ventral prostate protein content was reduced to
13% of control levels and cell number was reduced to 18% of control.
Induction of hepatic microsomal enzymes during 13 weeks of feeding vari-
ous dietary levels of heptachlor or heptachlor epoxide to rats was reported
by KlnoshHa and Kempf (1970) In a meeting abstract. There were dose-
related Increases in the activities of enzymes Involved In phosphorothloate
detoxlcatlon and activities of 0-den»ethylase and N-demethylase, maximal
during the first week of treatment. The no-effect level for enzyme
Induction was reported to be 1 ppm (1 mg/kg diet).
01800	V-42	03/08/87

-------
Mestltzova (1967) found that heptachlor (98.1%) administered with food
at a dose of 6 mg/kg bw for 52 weeks resulted In markedly decreased litter
sizes, decreased lifespan of pups and the development of cataracts In parent
rats and pups. It Is unclear whether the 6 mg/kg bw dose was administered
dally or whether 1t represented a total dose consumed over 1 year. In the
former case, the dose would be quite high, roughly corresponding to a con-
centration of 120 mg/kg diet, assuming that a rat consumes 5% dally of Its
body weight as food. In the latter case, the dose would be quite low,
resulting In a dally average of -0.016 mg/kg bw/day.
As with chlordane, several long-term feeding studies with heptachlor or
heptachlor epoxide were designed as carcinogenicity studies but provide
toxicologic data as well, and there has been disagreement among pathologists
on the distinction between malignant and benign lesions of the liver Induced
by heptachlor or Its epoxide.
In the unpublished report by IROC (1973b) reviewed by Epstein (1976), a
25:75 mixture of heptachlor:heptach1or epoxide was administered to groups of
100 male and 100 female C0-1 mice 1n the diet at 0, 1.0, 5.0 and 10.0 ppm
(mg/kg diet) for 18 months. An Interim kill of 10 mlce/sex/group was per-
formed at 6 months. Female mice at the 10 mg/kg diet level had decreased
body weight gain; otherwise, no other effect on body weight or food consump-
tion was observed. Hean liver weight increased significantly In a dose-
related manner In treated females and males at 6 and 18 months. This
Increase was more marked in males. Survival, although underestimated due to
the Interim kill, was 29% for males and 30% for females at the highest dose
level and 51-66% for all other groups. Including controls. Increased
01800
V-43
03/08/87

-------
Incidence of hepatocytomegaly 1n all treated female and male groups occurred
In a dose-related manner. The Incidence of hyperplastic nodules, as
reported by IRDC ( 1973a), was dose-related at the two highest levels for
males and females and was highly significant (pdxlO**). Re-evaluation of
histologic slides by six other pathologists resulted In several different
Interpretations; however, the overall result was a decreased Incidence of
hyperplasia 1n favor of a greater Incidence of carcinoma. Further discus-
sion of this study Is presented In the Carcinogenicity Section.
Epstein (1976) reviewed the results of several other unpublished
reports: 1) Davis (1965), an FDA report on heptachlor and heptachlor
epoxide; 2) Wltherup et a1. (1955), a study by the Kettering Laboratories on
heptachlor; 3) Jo 1 ley et al. (1966), a study by Kettering on a heptachlor
and heptachlor epoxide mixture; and 4) Wltherup et al. (1959), a Kettering
study on heptachlor epoxide. Discussion of these studies with respect to
toxic effects follows. Further discussion of the carcinogenic effects 1s
presented 1n the Carcinogenicity Section.
Davis (1965) administered 0 and 10 ppm (10 mg/kg diet) heptachlor or
heptachlor epoxide to groups of 100 male and 100 female C3H mice for 2
years. For heptachlor, low survival was observed for treated (30%) as well
as control (34%) mice. For heptachlor epoxide, survival was 9.5%. A 2-fold
Increase over controls 1n the Incidence of hepatic hyperplasia and benign
tumors way observed for both compounds, although re-evaluat1on of the slides
by four other pathologists changed the Interpretation In favor of a greater
number of hepatomas.
01800
V-44
03/06/87

-------
Wltherup et al. (1955) studied the effects of heptachlor on groups of 20
male and 20 female CF rats. The Insecticide was administered In dietary
concentrations of 0-10 ppm (10 mg/kg diet). Mortality among test groups was
not dose-related (see Table V-9). Loss of body weight, decreased food con-
sumption and Increased liver weight were seen among treated males, but not
females, and were most marked at 10 mg/kg diet. Liver lesions described as
'chlorinated hydrocarbon" type and considered to be non-neoplastlc were
observed In 17% of males and 50% of females at 10 mg/kg diet, and 38% of
males and 17% of females at 7 rag/kg diet. At dose levels at or below 5.0
mg/kg diet, none of these lesions was observed. This study was not re-
evaluated by other pathologists.
Jolley et al. (1966) administered a 75:25 mixture of heptachlor: hepta-
chlor epoxide to groups of 25 female CO rats in the diet at concentrations
5-12.5 ppm (12.5 mg/kg diet) for 2 years. A group of 54 rats received diets
free of Insecticide. Mortality was Increased 1n a somewhat dose-related way
(see Table V-9). Comprehensive histological evaluations were performed.
Spontaneous lesions and tumors were present in all groups and Included
multiple cell type hypertrophy, telangiectasia In the anterior pituitary and
adrenal hypertrophy. The Incidence of hepatocytomegaly was Increased over
control levels at 7.5, 10.0 and 12.5 mg/kg diet. This study was not re-
evaluated by other pathologists.
Wltherup et al. (1959) studied the effect of heptachlor epoxide In
groups of 25 male and 25 female CFN rats for 110 weeks. The dietary concen-
trations ranged from 0.5-10,0 ppm (mg/kg diet). Control rats received
heptachlor epoxlde-free diets. No differences were observed with respect to
01800
V-45
03/08/87

-------
food consumption or growth rate. There was a dose-related Increase In liver
weight In females. Hepatic cell vacuolization occurred 1n treated males.
Hepatocytomegaly, degeneration and regeneration In unspecified groups were
reported. Mortality was higher than control level for all treated groups,
but a dose-response relationship was not observed. This study was not re-
evaluated by other pathologists.
Reuber (1977a, 1970), using slides from the study of Davis (1965), found
hepatic vein thrombosis and cirrhosis among the heptachlor and heptachlor
epoxide (10 mg/kg diet) treated mice. These conditions were not observed In
any of the 127 control slides available for review. For heptachlor-treated
mice, 13% (10% of males, 15% of females) had hepatic vein thrombosis and 6%
had venous occlusion with recent liver Infarcts. Thrombosis of the cardiac
atrium was also present 1n some mice with hepatic vein thrombosis. The
Incidence of cirrhosis was 2/86 treated males and 5/77 treated females. For
heptachlor epoxide, 10% of treated mice (7% of males, 11% of females) had
hepatic vein thrombosis and 9% had venous occlusion. Cardiac atrium throm-
bosis was present 1n some mice. The Incidence of cirrhosis was 12/78
treated males and 15/81 treated females. In addition, liver carcinomas were
also observed In the treated mice.
In the NCI (1977b) bloassay of the carcinogenicity of heptachlor, groups
of 50 male and 50 female Osborne-Mendel rats and groups of 50 of each sex
B6C3F1 mice were maintained on diets containing technical grade heptachlor
for 80 weeks plus 21 and 10 weeks of observation for rats and mice, respec-
tively. Controls for the rats consisted of 10 matched and 60 pooled
untreated rats (controls from concurrent and recent bloassays of other
01800
V-46
03/08/87

-------
related compounds) per sex. For male mice, 20 matched and 100 pooled con-
trols were used, and for female nice, 10 matched and 80 pooled controls were
used. The doses were determined from the previously described preliminary
subchronlc study. Changes In dose levels were necessitated by developing
symptoms of toxicity. A description of these adjustments, the dosing sched-
ule and the resultant TWA doses, hereafter referred to as high doses and low
doses, are presented In Tables V-10 and V-ll for rats and mice, respectively.
For rats, mean body weight of high-dose males and females were con-
sistently depressed especially In males. The low-dose groups had growth
rates similar to controls. Adverse clinical signs such as loss of body
weight, rough and discolored hair and palpable masses, developed In treated
and untreated groups. In some females from both dose groups, vaginal bleed-
ing developed after 80 weeks. A dose-related, but not significant, Increase
In mortality was observed for treated males. The Increase was significant
between treated female groups. Histopathologic signs of aging, such as
chronic nephritis, biliary hyperplasia and chronic prostatitis, were
observed with about equal frequency In all control and treated groups. The
Incidence of follicular- and C-cell hyperplasia In thyroid glands of treated
and control groups were observed to be nontreatment-related.
The results In mice Indicated no differences In body weight gains among
groups. Sores and hair loss occurred In treated and control mice during the
first year- Abdominal distention and hair loss was prevalent In high-dose
females. Adverse clinical signs developed 1n mice of all groups. Hale
mortality was unaffected; however, there was a significant dose-related
Increase in mortality between treated female groups. Hepatocytomegaly and
diffuse hyperplasia occurred Infrequently In Individuals of all groups.
01800	V-47	03/08/87

-------
2	TABLE V-10
CD
O
°	Design Summary for NCI Bloassay of Heptachlor In Osborne-Hendel Rats3
Sex and
Treatment
Group
Initial
No. of
An1ma1sb
Heptachlor
In Diet
(ppm)
Time on Study
Treated
(weeks)
Untreated0
(weeks)
Time-Weighted
Average 0osesd
(PP«)
o
CJ
N
o
GO
HALE
Matched-Control
Low-Dose
Hlgh-Oose
. i
FEMALE
Matched-Control
Low-Dose
10
50
SO
10
50
80
20
10
0
160
40
20
0
40
60
20
10
0
31
14
35
0
31
14
35
0
22
9
14
35
0
111
30
30
111
38.9#
77.9
25.7
30
00

-------
TABLE V-10 (cont.)
Sex and
Treatment
Group
Initial
No. of
Animals^
Heptachlor
In Diet
(ppm)
Time on
Treated
(weeks)
Study
Untreated0
(weeks)
Time-Weighted
Average Doses'1
(PP«)
High-dose

80
22

51.3


120
9




40
14




20
35




0
0
30

aSource: NCI, 1977b
bAll animals were 35 days of age when placed on test.
cWhen diets containing heptachlor were discontinued, treated male rats and their matched controls were fed
plain feed diets (without corn oil) for 11 weeks, then control diets (2X corn oil added) for an additional
18 weeks; treated female rats and their matched controls were fed plain feed diets for 9.5 weeks, then
control 
-------
TABLE V-ll
Design Summary for NCI Bloassay of Heptachlor In B6C3F] H1cea
Sex and Initial Heptachlor 	Time on	Study		Tine-Weighted
Treatment	No. of	In Diet	Average Doses'!
Group An1ma1sb (ppn) Treated	Untreatedc	(PP*)
(weeks)	(weeks)
MALE
Hatched Control
Low-Dose
High-Dose
FEMALE
Matched-Control
Low-Dose
20e
50
SO
10
50
10
5
0
20
10
0
20
10
5
0
18
62
0
17
50
0
17
13
50
0
90-91
10
10
90-91
6.1
13.8
9.0
10

-------
TABLE V-ll (cont.)
Sex and
Treatment
Group
Initial
No. of
Animals'*
Heptachlor
1n Diet
(ppm)
Time
Treated
(weeks)
on Study
Untreatedc
(weeks)
Time-Weighted
Average Oosesd
(PP")
High-Dose
50
40
17

18.0


20
13




10
SO




0
0
10

aSource: NCI, 1977b
bAl1 animals were 3S days of age when placed on test.
cWhen diets containing heptachlor were discontinued, mice received control diet (2% corn oil added) until
termination.
^Tlme-weighted average dose - Kdose In ppm x no. of days at that dose)
l(no. of days receiving each dose)
eIn1t1ally 10 males were placed on test as matched controls.

-------
Other Routes of Exposure. No data were available on Inhalation or
dermal exposure; however, several Investigators (Kacew et al.. 1973; Slnghal
and Kacew, 1976; Hrdlna et al., 1974) have described effects following
Intraperitoneal and Intramuscular Injections of heptachlor and heptachlor
epoxide. Details of the studies are summarized in Table V-12. Kacew et al.
(1973) administered heptachlor or heptachlor epoxide to Wlstar rats Intra-
muscularly for 45 days. The doses were 0, 3 or 15 mg/kg bw/day for hepta-
chlor and 0, 1 or 5 mg/kg bw/day for heptachlor epoxide. For both com-
pounds, liver weights were decreased In all treated rats when compared with
controls. Dose-related Increases In enzymes of gluconeogenic metabolism In
livers and kidneys were significant. Similar results were obtained follow-
ing Intraperitoneal injections of heptachlor (Slnghal and Kacew, 1976).
Hrdlna et al. (1974) administered 0, 3 or 15 mg/kg bw/day heptachlor or
heptachlor epoxide by an unspecified route to rats for 45 days and observed
significantly decreased cerebro-cortlcal acetylcholine levels.
Target Organ Toxicity
Chlordane.
Effects on the Liver — Histopathologic changes In the liver were
observed In rats at dietary levels of chlordane >18 mg/kg diet for males and
females (Ambrose et al., 1953a,b). At exposures as low as 10 mg/kg diet,
female rats had slight Increases In liver weight. Increased Incidences of
hepatocytomegaly, hepatic hyperplasia and nodules were reported In a car-
cinogenicity study (IRDC, 1973a); however, re-examination of slides indi-
cated that the nodules were hepatomas (Epstein, 1976). Chlordane feeding
Induced benign proliferative lesions in the liver of a strain of mice that
historically and concurrently did not develop lesions when untreated (Becker
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TABIE V-l?





tffecls
of Subchronlc Exposure to Heptachlor and Heptachlor fpoxlde by Other Routes

Species/
Strain
Sei/
Nuaber
Height/
Age
Coapound
and/or
Purity
1
Route Vehicle
Oose
(ag/kg bw day) Duration
iffects
Reference
Rat/
Millar
N/4/
group
100 g
heptachlor
purity MR
I.*. corn oil
0, 3 or IS 4S days
Ho significant effect on bw, kidney,
adrenal, lltyaus or heart weights at either
dose level. Significantly decreased liver
weights at both dose levels (p<0.0S) and
significantly decreased testes weight at
IS ag/kg bw/day (p<0.0S) when coapared with
controls. Dose-related levels significantly
Increased over control levels of kidney and
liver pyruvate carboxylase, phosphoenol-
pyruvate carboxyklnase, fructose-1,6-
d(phosphatase and glucose-6-ptwsphatase
(p<0.0S). Dose-related, significantly
elevated levels of blood glucose and serua
urea and decreased liver glycogen (p<0.05).
Kacew el
al.. 1973
1
Rat/MR
MR/
(/group
MR
heptachlor
purity MR
l.p. corn oil
0 and IS 4S days
Significantly Increased blood glucose,
serua urea, liver and kidney levels of
pyruvate carboxylase, pkosphoenolpyruvate
carboxyklnase, fructose-l,6-dtphosphatase
and glucose-b-phosphatase; significantly
decreased liver glycogen compared with
controls (p<0.0S).
Slnghal
and Kacew,
197b
Ral/NR
NR/NR
MR
heptachlor
purity NR
MR MR
0, 3 and IS 4S days
Significantly decreased cerebro-cortlcal
acetylcholine; no change In braln-stea
norepinephrine; significantly decreased
brain stea S-hydroxytryptophan.
Hrdlna et
al., 1974

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I All I V-12 (cont.)
Coapound
Species/ Se«/	Height/ and/or	Dose
Strain Nuaber Age	Purity	Saute Vehicle (ag/kg bw day) Duration	Effects	Reference
Rat/
Ulstar
N/4/
group
100 g
MflKklpr l.i
epoxide
purity MR
corn oil
Rat/NR
MR/MR
NR
heptachlor
epoxide
purity NR
NR
NR
0. 1 or i	4S days No significant effect on bw, kidney,	Kacew et
adrenal, thyaus testes or heart weights	a)., 1973
at either dose level. Significantly
decreased liver weights at both dose
levels. Oose-related levels significantly
Increased over control levels of liver
and kidney pyruvate carboxylase,
phosphenopyruvate carboxyklnase, fructose-
l.t-dlphosphatase and glucose-6-phosphatase
|p<0.05). Dose-related, significantly
elevated levels of blood glucose and
serua urea; decreased liver glycogen
(p<0.0S).
0, 1 or S	(S days Significantly decreased cerebro-cortlcal	Mrdlna et
acetylcholine level, no change In brain	al., 19)4
stea norepinephrine levels.
NO* Not reported
I.a.- Intraauscular
I.p.- Intraperitoneal

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and Sell, 1979). The Incidence of hepatocytomegaly and hyperplasia was not
Increased over control Incidence In B6C3F1 mice (NCI, 1977a). Dose-related,
Increasingly severe liver damage was observed by Ingle (1952) in rats
treated chronically with dietary levels of chlordane. The damage Included
centrllobular hypertrophy, nuclear karyorrhexls, karyolysls, necrosis, fatty
degeneration and bile duct proliferation. Reticulation and oxyphllla of
liver cytoplasm was observed after acute Inhalation exposure (Ingle, 1953).
Several Investigators noted changes 1n liver function, as evidenced by
enzyme activity changes. K1nosh1ta and Kempf (1970) found that the activi-
ties of the microsomal enzymes, such as phosphorothloate detoxlcatlng
enzyme, O-demethylase and N-demethylase, were significantly Increased In
rats following technical grade chlordane or transchlordane feeding. Induc-
tion of Hver aniline hydroxylase, amlnopyMne demethylase and hexobarbltal
oxidase 1n rats was observed by Den Tonkelaar and Van Esch (1974). Enhance-
ment of gluconeogenesls was described 1n rats, as evidenced by Increased
blood glucose. Increased serum urea, decreased liver glycogen. Increased
activities of hepatic pyruvate carboxylase, phosphoenolpyruvate carboxy-
lase, fructose-1,6-d1phosphatase and glucose-6-phosphatase (Slnghal and
Kacew, 1976; Kacew and Slnghal, 1973).
Effects on Endrocrlne System and Reproductive Organs — Hyperplastic
lesions, such as fol11cular-cel1 and C-cell hyperplasia of the thyroid, were
observed In rats treated with 120-407 mg/kg bw of chlordane (NCI, 1977a).
Shaln et al. (1977) found changes in ventral prostate homeostasis of rats at
dose levels of -20 mg/kg bw, as evidenced by Increased nuclear androgen
receptor site content and decreased protein, RNA and DNA content. Delayed
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vaginal opening. Increased ovary weight and decreased pituitary weights were
seen In neonatal mice treated with 0.075 mg/mouse (Talamantes and Jang,
1977).
Effects on CNS — Clinical and behavioral manifestations of chlordane
poisoning have been noted In rats and mice. Tremors, hyperexc1tab1l1ty,
Irritability, phonatlon, plloerectlon and convulsions were commonly observed
after acute exposures (Lehman, 1951; Gaines, 1960; Boyd and Taylor, 1969;
Stohlman et al., 1950; Arnold et al., 1977; Hrdlna et al.t 1974; Hyde and
Falkenberg, 1976) and chronic exposures {Ingle, 1952; NCI, 1977a). Hyde and
Falkenberg found dose-related (between 0.15 and 25 mg/kg bw/day) changes In
EEGs of rats given dally Intraperitoneal Injections of chlordane.
Effects on Other Organs — Moderate to marked histopathologic damage
to kidneys, lungs, myocardla, adrenals and spleen In rats fed chlordane at
150 mg/kg diet became more severe at 300 mg/kg diet (Ingle, 1952). Duodenal
ulcers and hemorrhaging around eyes and nose were also observed. Congestion
and proliferation of bronchiole lining cells were seen In lungs of mice
after acute Inhalation exposure to chlordane (Ingle, 1953). Increased
activities of kidney cortical pyruvate carboxylase, phosphoenolpyruvate
carboxyklnase, fructose-1,6-d1phosphatase and glucose-6-phosphatase were
observed In rats after acute oral exposure to chlordane (Kacew and Slnghal,
1973).
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Heptachlor and Heptachlor Epoxide.
Effects on the Liver — Moderate to severe histopathologic changes In
the liver were accompanied by elevated serum GPT and serum aldolase levels
1n rats after acute or subchronlc oral exposures to heptachlor up to 14
days. At 28 days, however, the serum enzyme levels returned to control
levels, and liver damage was less severe (Krampl, 1971). Similar findings
by Enan et al. (1962) also Included elevated serum bilirubin and serum
cholesterol, decreased hepatic lipid content, decreased liver glycogen.
Increased serum glutamate-oxalacetate transaminase, Increased alkaline phos-
phatase and decreased acid phosphatase levels after oral exposure of rats to
heptachlor. Increased Incidences of hepatocytomegaly, diffuse hepatic
hyperplasia and hyperplastic nodules were common findings In mouse livers
after chronic feeding of heptachlor and heptachlor epoxide (IRDC, 1973b;
NCI, 1977b;' Davis, 1965; Wltherup et al., 1955, 1959; Jolley et al., 1966);
however, re-evaluatIon of slides from the IRDC (1973b) study and of the
Davis (1965) study, indicated that the hyperplastic conditions had been
overdlagnosed by the original Investigators and that the conditions of hepa-
tocellular carcinoma had been underdiagnosed (Epstein, 1976). Cirrhosis and
hepatic vein thrombosis were observed by Reuber (1977a) In mice treated with
heptachlor or heptachlor epoxide.
Several Investigators noted changes In liver function, evidenced by
changes In enzyme activities. The microsomal enzymes of phosphorothloate
detoxlcatlon, O-deraethylase and N-demethylase activities Increased In rat
livers after dietary exposure to heptachlor or heptachlor epoxide (K1nosh1ta
and Kempf, 1970). Induction of liver aniline hydroxylase, amlnopyrlne
demethylase and hexobarbltal oxidase occurred In fats after oral exposure to
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heptachlor (Den Tonkelaar and Van Esch, 1974). Decreased liver weights were
accompanied by enhanced gluconeogenesls In rats following Intramuscular or
Intraperitoneal Injections of heptachlor or heptachlor epoxide (Kacew et
al., 1973; Slnghal and Kacew, 1976). Increased liver pyruvate carboxylase,
phosphoenolpyruvate carboxyklnase, fructose-1,6-d1phosphatase and glucose-
6-phosphatase activities; Increased blood glucose and serum urea; and
decreased liver glycogen were evidence of enhanced gluconeogenesls.
Effects on Endocrine System and Reproductive Organs — Increased but
not significantly dose-related Incidence of hyperplastic lesions of the
thyroid. Including foilicular-cel1 and C-cell hyperplasia, were observed In
rats following chronic dietary heptachlor exposure (NCI, 1977b). Increased
cytoplasmic nuclear androgen receptor site content of the ventral prostate,
decreased prostate protein content and cell loss were Indicative of changes
In ventral prostate homeostasis In rats exposed to heptachlor (Shaln et al.,
1977). Kacew et al. (1973) observed decreased testes weights In rats fol-
lowing Intramuscular Injections of heptachlor. Testes weight was not
Increased following heptachlor epoxide Injection. Vaginal bleeding devel-
oped In female rats after chronic dietary exposure to heptachlor (NCI,
1977b); however, the Incidence was low and not significantly different from
controls.
Effects on CNS — Clinical and behavioral manifestations of heptachlor
and heptachlor epoxide poisoning were observed In rats and mice. Tremors,
hyperexcltabmty, irritability, convulsions, hind leg paralysis, hypo-
activity and hypothermia were common findings after acute exposures to hep-
tachlor and heptachlor epoxide (Lehman, 1951; Gd'lnes, 1960; Hrdlna et al.,
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1974; Arnold et al., 1977; Yamaguchl et al., 1980). Hrdlna et al. ( 1974)
found decreased levels of cerebrocortlcal acetylcholine and brain stem
5-hydroxy-tryptophan associated with tremors and convulsions.
Effects on Other Organs -- In addition to the hepatic vein thrombosis
observed 1n mice following heptachlor or heptachlor epoxide feeding, a few
cases of thrombosis of cardiac atrium were observed (Reuber, 1977a). In the
kidney cortex, enzymes associated with gluconeogenesls have Increased
activities In rats after exposures to heptachlor or heptachlor epoxide
{Kacew et al., 1973). Mestltzova (1967) reported that rats fed heptachlor
developed cataracts of the eye.
Other Effects
Studies' on the carcinogenicity, mutagenicity, teratogenicity and repro-
ductive effects of chlordane, heptachlor and heptachlor epoxide exposure In
laboratory animals are discussed In this section.
Carcinogenicity.
Animal Studies-Chlordane — Chlordane has been studied 1n four mouse
and four rat long-term carcinogenesis bloassays. Tables V-13 and V-14
present a summary of the experimental design and tumor results. Each study
1s described 1n more detail In the following sections.
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TABU V-13
Summary of Mouse Dietary Carcinogenicity lests for Chlordane
Strain
No./Sex
Dose
(PP»)
Duration of .
Exposure/
Observation
Tumor Results
Reference
C57B1/6N
NS
25 or SO
18 months
Hepatocellular carcinomas
In 27X (16) of the
survivors. No other
Information on tumor
Incidences and time of
death was presented.
Becker and
Sell. 1979
CD-I
100/H&F
5, 25 or 50
18 months
tlver nodules at 25 and
50 ppm (1RDC); hepato-
cellular carcinomas at
25 and 50 ppm (Reuber)
IROC, 1973a;
Epstein, 1976
B6C3F1
50/N&F
29.9 and 56.2. H;
30.1 and 63.8, F
80 weeks
plus 10 weeks
observation
Hepatocellular carcinomas
In both males and females
NCI, 1977a
ICR
80/M&F
1. 5 or 12.5
24 months
Hepatocellular adenomas
and hemangiomas In 12.5
ppm males; nontumor liver
lesions In females at 5
and 12.5 ppm and males
at 5 ppm
RIASBT, 1983a
NS = Not specified

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TABLE V-14
Summary of Rat Dietary Carcinogenicity Tests for Chlordane
Duration of -
Strain	No./Sex	Dose	Exposure/	Tumor Results	Reference
(ppm)	Observation
Albino
Osborne-
Hendel
3-5/MM
20/H&F
10, 20. 40.
80. 160. 320
640. 1280
5. 10. 30. 150
or 300
400 days
2 years
No treatment-related
tumors; liver lesions
at 80 ppm and above
High toxicity at ISO and
300 ppm; no treatment-
related tumors
Ambrose et
al., 1953a,b
Ingle, 1952
Osborne-
Hendel
50/N&F
203.5 and
407 ppm, H;
120.8 and
241.5 ppm. F
80 weeks
plus 29 weeks
observation
Toxicity In all groups;
high mortality In
females. Neoplastic
nodules of liver In low-
dose females; possible
thyroid tumors In both
males and females
NCI. l
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Studies with Mice.
Becker and Sell 11979) — A 90:10 mixture of chlordane/heptachlor
was fed to an unspecified number of male C57B1/6N mice at concentrations of
25 or 50 ppm (-3.57 or 7.14 mg/kg bw) for 18 months. Specific Information
as to treatment and observation periods and time of death was not provided.
The C57B1/6N mouse rarely develops spontaneous liver lesions and In a group
of 200 control mice no liver tumors or nodular lesions were found over 18
months of observation. In mice receiving the chlordane/heptachlor diet,
many liver lesions were seen. Including both benign proliferative lesions
and hepatocellular carcinomas. Of those surviving to the end of the experi-
ment, 27% (16 mice) had primary hepatocellular carcinomas, with the first
appearing at 36 weeks. No other Information was presented as to early
deaths and associated tumor Incidences. An even greater number of mice had
benign proliferative lesions. Data relating tumor Incidence to dose were
not available. Elevated levels of alpha-fetoproteln were associated with
animals with primary hepatocellular carcinomas. Cells from the carcinomas
grew when transplanted, producing tumors that were histologically similar to
the primary hepatocellular carcinoma. Cells from the benign lesions did not
grow when transplanted. The authors concluded that the benign lesions did
not appear to be premallgnant lesions.
In a companion study, acetylamlnofluorene was administered to the same
strain of nice (C57B1/6N) at 0.045 or 0.03% diet. After 61 weeks, seven
(18%) of .the survivors had liver carcinomas, and three showed growth at
2.4-4 months when transplanted. On a comparison basis, chlordane was con-
sidered to be a more potent carcinogen than acetylamlnofluorene. Another
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key finding In this study was that chlordane Induced hepatocellular carci-
nomas In a strain of mice that does not spontaneously develop hepatocellular
carcinomas.
The International Research and Development Corporation (IRDC.
1973a) — IRDC, under contract to the Velslcol Chemical Corporation,
administered analytical grade chlordane In the diet at concentrations of 0,
5, 25 or 50 ppm (-0, 0.71, 3.57 or 7.14 mg/kg bw) for 18 months to groups of
100 male and 100 female CD-I mice. The mice were 6 weeks of age when
exposure began. A 6-month Interim sacrifice of 10 mice/sex/group did not
reveal compound-related lesions. No effect of chlordane on body weight gain
or food consumption was observed. However, the survival was greatly reduced
at the high-dose levels. A large number of animals were also lost due to
autolysis. Only about one-half of the mice were histologically examined. A
dose-related Increased incidence of hepatocytomegaly was seen In all treated
female and male groups. The Incidence of hyperplastic liver nodules, as
reported by IR0C (1973a), was dose-related at the two highest dose levels
(p<0.01). For males, the Incidences were: 1/47 (2%), 34/52 (65%) and 38/50
(76%) at 0, 25 and 50 ppm, respectively; whereas for females, the Incidences
were 0/57 (0%), 32/51 (63%) and 36/51 (71%), respectively. The number of
hepatomas was higher, although not statistically at p«0.05, In the 25 ppm
group (12) vs. that In the control (5) or 5 ppm groups (6). Only four
hepatomas were diagnosed at 50 ppm; however, early deaths and a large amount
of autolysis markedly reduced the number of mice at risk. Dr. Reuber re-
examined the IRDC slides and found highly significant |p
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50 mg/kg diet levels. Three other Independent pathologists (Drs. R. Squire,
H. Stewart and H. Popper) examined subsets of the same slides examined by
Reuber and were 1n close agreement with the diagnosis. Table V-15 presents
a breakdown of liver lesions as diagnosed by Reuber. The Incidences of
hepatic carcinoma, as determined by Reuber, are presented In Table V-16,
along with those found In the NCI study to be discussed next.
National Cancer Institute (1977a) « In the NCI Moassay, groups of
50 male and 50 female B6C3F1 mice were fed chlordane consisting of 71.7%
cls-chlordane, 23.1% trans-chlordane, 0.3% heptachlor, 0.6% nonachlor, 1.1%
hexachlorocyclopentadlene, and 0.25% chlordene Isomers In the diet for 80
weeks, at two doses -- a predicted maximum tolerated dose (MTD) and 1/2 MT0.
This was followed by a 10-week observation period. As upward or downward
adjustments were made In dose levels, the doses are expressed as time-
weighted average (TWA) concentrations. The TWA concentrations for male mice
at the high and low doses were 29.9 and 56.2 ppm (-4.27 and 8.03 mg/kg bw),
respectively, and 30.1 and 63.8 ppm (-4.3 and 9.11 mg/kg bw), respectively,
for female mice. Controls consisted of 20 matched control mice of each sex
and 100 and 80 pooled male and female control mice, respectively. The
results revealed no differences in body weight gain among groups. Tremors
were observed 1n high-dose males and females after 20 weeks. A dose-related
Increase In mortality was seen In treated males, but not In females. A
statistically higher (p<0.001) Incidence of hepatocellular carcinoma was
found In both males and females (see Table V-16).
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TABLE V-15
Incidence of Liver Lesions and Tumors In CO-1 Nice Following Dietary
Administration of Chlordane*
No. of Mice Exhibiting Liver Lesions and Tumors^
Dose
(ppm)
Sex
No. of H1ce
Examined

H

N

SC
LC
TC
0
H
33
20
(61)
1
(3)
0

3
19)
3 (9)

F
45
26
(58)
0

0

0

0
5
M
55
34
(62)
6
(11)
3
(5)
2
(4)
5 (9)

Fc
61
32
(52)
1
(2)
0

0

0
25
H
52
7
(13)
3
(6)
9
(17)
32
(62)
41 (79)

F
50
13
(26)
4
(8)
7
(H)
25
(50)
32 (64)
50
n
39
7
(18)
0

4
(10)
28
(72)
32 (82)

F
37
11
(30)
0

2
(5)
24
(65)
26 (70)
aSource: IROC, 1973a, breakdown of liver lesions diagnosed by Reuber
(Epstein, 1976)
bH = hyperplasia; N * nodules; SC ¦ small carcinomas (including hyper-
plastic nodules with focal carcinomas); LC = large carcinomas >5 mm; TC =
total carcinomas
cTwo liver sarcomas diagnosed In the 5 ppm female group
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TABLE V-16
Incidence of Hepatocellular Carcinoma In Nice Following
Chronic Dietary Administration of Chlordane
Dose
(ppm)
No. of Tumor-Bearing M1ce/No. of
(% positive)
Mice Examined
Hale
Female

Strain CD-la


0 .
3/33 (9%)
0/45
(0%)
5
5/55 (9*)
0/61
(0%)
25
~1/52 (79%)
32/50
(64%)
50
32/39 (82%)
26/37
(70%)

Strain B6C3F1&


0 (pooled)
17/92 (18%)
3/78
(4%)
0 (matched)
2/18 (11%)
0/19
(0%)
29.9
16/48 (33%)
NA

30.1
NA
3/47
(6%)
56.2
43/49 (88%)
NA

63.8
NA
34/49
(69%)
aIR0C, 1973a; tumor Incidence as determined by Reuber. Tumor Incidences
significantly different from controls for 25 and 50 ppm In the diet

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Research Institute for Animal Science 1n Biochemistry and Toxicology.
Japan (R1ASBT. 1983a) — In this study, conducted for the Velslcol Chemical
Corporation, technical grade chlordane (distribution of Isomers not
specified) was fed to groups of 80 male and 80 female ICR mice at levels of
0, 1# 5 or 12.5 ppm {-0, 0.14, 0.71 or 1.79 mg/kg bw) for a period of 24
months.
Each group (sex and dose level) consisted of 80 mice of which 8 were
sacrificed at 52 weeks. There was np apparent effect of dosing on survival
or body weight gain. The mean liver weight was significantly Increased for
the eight males receiving 12.5 ppm sacrificed at 52 weeks, and the liver-
to-body weight ratios were significantly Increased for all dosed groups of
males when compared with controls. At terminal sacrifice (104 weeks), the
mean weight and organ-to-body weight ratio of the liver were statistically
significantly Increased In both males and females receiving 12.5 ppm. In
addition, the 11ver-to-body weight ratios of females receiving 1 and 5 ppm
chlordane were statistically significantly greater than In controls.
A significant Increase (p<0.001) In the Incidence of hepatocellular
adenoma and hemangioma of the liver was found In the 12.5 ppm male group In
animals dying between 19 and 24 months or at terminal sacrifice, as
described In Table V-17. There was no Increase In hepatic tumors In female
mice. Other than for the liver tumors In male mice, there were no signifi-
cant differences In tumors at other sites related to chlordane exposure.
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TABLE V-17
Neoplastic and Toxic Lesions of the Liver In ICR Mice fed Chlordanea
Males/Dose (ppm)	Females/Dose (ppm)

0
1
5
12.5
0
1
5
12.5
No. of tissues examined
71
71
12
12
12
72
71
72
Hepatocellular adenoma
13
13
15
28b
1
1
3
1
Hepatocel lu'lar adenoma -
carcinoma
3
3
7
9
0
0
0
1
Hemangioma
4
1
8
14b
0
2
1
0
Hepatocellular swelling
and degeneration
5
8
58b
59b
3
2
24b
59b
Fatty degeneration
2
0
3
9C
1
2
9
9
Necrosis
6
7
23c
l9t>
14
8
15
20
aSource: RIASBT, 1983a
^Statistically significantly different from control value (p<0.001)
c$1gn1ficantly different from control value (p<0.01)
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In addition to liver tumors, there were remarkable Increases 1n liver
lesions. There were also significant increases (p<0.01) In the Incidence of
hepatocellular swelling, degeneration and necrosis in males at 5 and 12.5
ppm and a significant Increase (p<0.01) in fatty degeneration at 12.5 ppm.
A significant Increase (p<0.001) 1n hepatocellular swelling and degeneration
was found In females at doses of 5 and 12.5 ppm. In summary, these liver
lesions were observed after 12 months; dose-related Increased incidences of
hepatocellular swelling and degeneration; fatty degeneration, and necrosis
1n the livers of males, with a less distinct trend 1n females.
Studies with Rats.
Ambrose ct al. (1953a.b) « In this study, technical grade chlor-
dane was fed to groups of 3-5 male and female albino rats for 400 days at
dietary levels of 0, 10, 20, 40, 80, 160, 320, 640 or 1280 ppm (-0, 0,5.
1.0, 2.0, 4.0, 8.0, 32.0, 64.0 or 128.0 mg/kg bw). Increased mortality was
observed In the 640 or 1280 ppm groups, and retarded growth was observed In
all animals at >320 ppm. No growth retardation was observed In female rats
fed <160 ppm or In male rats fed 10 or 80 ppm. Significantly enlarged
livers and other liver changes were found In male and female rats fed chlor-
dane at >80 ppm, and pathologic changes In the liver were occasionally found
In male rats fed 40 ppm. No treatment-related Increase 1n tumors was
found. The study duration (400 days) 1s considered too short and the number
of animals too small for this to be a valid carcinogenicity study.
Ingle (1952) « Six groups of 20 male and 20 female Osborne-Hendel
rats were fed chlordane for up to 2 years at dietary dose levels of 5, 10,
30, 150 or 300 ppm (-0.25, 0.5, 1.5, 7.5 or 15.0 mg/kg bw). Marked toxicity
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was encountered at 300 ppm In both males and females. This Included high
mortality, reduced growth rates, eye and nose hemorrhaging, and severe
histopathologic damage to the liver, kidneys, heart, adrenals, lungs, myo-
cardium and spleen. At 150 ppm, similar but less severe effects were seen.
The effects at 30 ppm Included Inducible tremors and slight liver damage.
At 10 ppm, only minor liver damage such as occasional hepatocytomegaly and
mild bile duct hyperplasia was seen. No symptoms of toxicity, gross or
histopathologic changes In the liver, kidneys, lungs, pancreas, testes,
ovaries, heart or spleen were noted at 5 ppm. No treatment-related tumor
Incidence was found.
National Cancer Institute (1977a) — In the NCI carcinogenicity
study, groups of 50 male and 50 female Osborne-Mendel rats were fed chlor-
dane (94.8% pure) in the diet for 80 weeks, at two dose levels -- a pre-
dicted HT0 and 1/2 NTD. This was followed by a 29-week observation period.
As upward or downward adjustments were made 1n dose levels, the doses are
expressed as TWA concentrations. The TWA concentrations for male rats at
the low and high doses were 203.5 and 407 ppm (-10.2 and 20.4 mg/kg bw),
respectively, and for females, 120.8 and 241.5 ppm (-6.04 and 12.08 mg/kg
bw), respectively. Ten rats of each sex served as matched controls, and 60
rats of each sex served as pooled controls. Complete necropsies and histo-
logic examinations were performed, except 1n the cases of a few spontaneous
deaths.
The mean body weight of high-dose males and females was consistently
lower than controls. All treated groups had symptoms of toxicity, Including
loss of body weight, rough and discolored hair, palpable masses, and tumors.
01800
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that became progressively worse as the study continued. Among female rats,
there was a highly significant (p-0.003) dose-related Increase In mortal-
ity. Mortality was not significantly Increased for male rats. Only two
hepatocellular carcinomas were observed, one In a low-dose male and one
among the pooled controls. A significant (p<0.05) Increase In neoplastic
nodules of the liver was seen 1n the low-dose females but not In the high-
dose females or 1n either the high- or low-dose males. A dose-related trend
(p<0.05) was found for neoplastic lesions (adenomas and carcinomas) of the
thyroid glands (fol11cular-cel1 and C-cells) for females when compared with
the matched controls. However, the results were ambiguous and Internally
Inconsistent. NCI discounted the Importance of these findings because the
Incidences were comparatively low and were known to be variable In popula-
tions of control rats.
In a more recent review of tumors In Osborne-Mendel rats In the NCI
studies (>900 of each sex), Goodman et al. (1980) presented data Indicating
7.IX follicular cell tumors In control males and 3.4% 1n control females.
These data provide additional support for NCI's decision to discount the
Importance of an apparent Increase In thyroid tumors. A highly significant
dose-related Increase In the Incidence of fibrous histiocytoma (p=0.0007)
was observed for male rats. This was based on an Increase only ^n the
high-dose male group (7/44) as compared with 1/44, 0/8 and 2/58 for the
low-dose, matched control and pooled control groups, respectively. The
Investigators discounted this finding because they did not believe these
lesions to be treatment-related, as they had occurred spontaneously through-
out the bloassay program. All other tumors were common for this strain of
rat, and were not treatment-related.
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The Research Institute for Animal Science In Biochemistry and
Toxicology. Japan (RIASBT. 1983b) — In this study, conducted for the Vel-
slcol Chemical Corporation, chlordane (distribution of Isomers not speci-
fied) was fed to groups of 80 male and 80 female Fischer 344 rats at levels
of 0, 1, 5 or 25 ppm {-0, 0.5, 0.25 or 1.25 mg/kg bw) for a period of 130
weeks. Each group (sex and dose level) consisted of 80 rats, of which sub-
sets of eight rats were sacrificed and studied at 26 and 52 weeks. The dose
levels were set on the basis of a pilot study In which groups of 5 male and
5 female Fischer 344 rats were fed diets containing 0, 50, 100, 200, 400 or
800 ppm technical grade chlordane for 4 weeks. Hepatocellular swelling and
fatty degeneration 1n the liver were found in both male and female rats at
50 ppm, the lowest dose tested. One ppm was set as the no-effect level,
based on an 18-month study 1n mice (provided by Velslcol) In which changes
in the liver were evidenced at 5 ppm chlordane.
In the 130-week study, there were no dose-related effects on mortality,
food consumption, water consumption, hematology, clinical chemistry, or uri-
nalysis. Virtually all of the toxic effects were restricted to the Hver.
The weight of the liver In females receiving 25 ppm was significantly
Increased at weeks 26 and 52 but not at week 130, whereas In males receiving
5 and 25 ppm, the liver weight was Increased at week 130 but not at the 26-
or 52-week sacrifice.
At necropsy, enlargement of the liver was noted in 19 control males and
19, 26, and 32 males dosed at 1, 5 and 25 ppm, respectively. Table V-18
presents the tumor and nontumor lesions of the liver. There was a signifi-
cant increase In adenomas of the liver In males receiving 25 ppm as compared
01800	V—7 2	03/08/87

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TABLE V-18
Liver Tumors and Nonneoplastic Lesions In Fischer 344 Rats
Fed Chlordanea»k



Hales/dose (pom)
Females/dose i
[ppmj


0
1
5
25
0
1
5
25
No. of tissues
examined
64
64
64
64
64
64
64
64
Hepatocellular
adenoma
1
1
3
9C
0
2
0
0
Mesenchymoma

0
3
1
2
0
0
0
0
Hepatocellular
swelling
5
15d
14
42c
7
2
8
38c
Hepatocellular
necrosis
3
13d
11
27c
0
0
1
1
Hepatocellular
degeneration
fatty
26
15d
19
22
20
16
19
2
Focal hepatocellular
hyperplasia
5
7
3
11
5
10
1
1
^Source: RIASBT, 1983b
°The animals sacrificed at weeks 26 and 52 are not Included
cS1gn1fIcantly different from control value (p<0.001J
dS1gn1fIcantly different from control value (p<0.05)
01800
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with controls, but no corresponding effect occurred In females. All of
these tumors were found after 104 weeks (mean time to tumor death was 121.8
weeks). There was also a significant increase in fibroadenomas of the
mamnary gland In females receiving 1 ppm as compared with controls but no
significant Increase at 5 or 25 ppm.
Nonneoplastic lesions occurred frequently. There was a dose-related
Increase 1n the Incidence of hepatocellular swelling and necrosis In male
rats. When compared with controls, the Incidence of hepatocellular swelling
was significantly Increased In all dosed males, and the Incidence of hepato-
cellular necrosis was significantly Increased In males receiving 1 and 25
ppm. The Incidence of hepatocellular swelling was significantly higher In
females receiving 25 ppm than 1n controls. There was also an Increase In
focal hepatocellular hyperplasia 1n males receiving 25 ppm, but the Increase
was not significantly different compared with controls. Most of these
lesions of the liver occurred after 78 weeks of the study. A slight
Increase In nonneoplastic liver lesions was seen 1n the 26- and 52-week
sacrifice groups (Table V-19).
Chlordane was	considered positive for oncogenicity by the authors, since
the Incidence of	hepatic adenomas was significantly Increased (p<0.001) In
males In the 25	ppm group (9/64 vs. 1/64 In controls). The historical
Incidence of this	tumor 1n F344/CRJ rats for the testing laboratory was 2.5%
In males and 2.3%	1n females. The control Incidence 1n this study was 1.6%.
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TABLE V-19
Liver Lesions 1n Fischer 344 Rats Fed Chlordane for 26 or 52 Weeks3'**
Lesion
Males/Dose (ppm)	Females/Pose (ppm)
0 1 5 25	0 1 5 25
26 weeks
Hepatocellular fatty	0 0 0 1	0 0 10
degeneration
Focal necrosis	0 0 0 0	0 10 0
Bile duct proliferation	0 0 0 1	10 0 1
52 weeks
Hepatocellular fatty	0	2	0 1	0 0 0 0
degeneration
Hepatocellular swelling	0	0	0 2	0 10 3
Focal necrosis	0	0	11	0 10 1
B11e duct proliferation	0	0	0 0	0 0 10
Small granuloma	0	0	0 0	0 0 1 2
^Source: RIASBT, 1983b
*>LWers from eight rats/group were examined.
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An Independent review of the liver hlstopathology, conducted by Dr. Gary
H. Williams, differed somewhat In that three of the neoplasms of the liver.
Identified as adenomas by the report authors, were diagnosed as carcinomas
by Dr. Williams. In addition, three neoplasms (adenomas) were found that
were not diagnosed by the testing laboratory (Table V-20). It was noted
that only two slices of liver, one from the median lobe and one from the
left lobe, were taken from rats without grossly observed tumors, and Dr.
Williams felt that the number of neoplasms might have Increased had more
sections been evaluated. In his opinion, the small Increase In benign liver
neoplasms was considered as weak evidence for the oncogenicity of chlordane
In rats.
The Increased Incidence of mammary fibroadenomas In females receiving 1
ppm chlordane was not considered to be compound/dose related because mammary
fibroadenomas were absent In females dosed at higher levels.
Nonneoplastic changes 1n the liver of both males and females were
increased 1n dosed animals compared with controls. The principal changes
consisted of hepatocellular swelling and necrosis. The liver changes were
accompanied by an Increase In liver weights In. males receiving 5 and 25 ppm
at 130 weeks and 1n females receiving 25 ppm at weeks 26 and 52.
It was concluded that, under the conditions of the study, technical
grade chlordane caused a significant Increase in the Incidence of benign
hepatocellular tumors when fed at a level of 25 ppm 1n the diet to male F344
rats for 130 weeks. There was also a significant Increase in nonneoplastic
lesions of the liver 1n both male and female rats, namely, an increased
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TABLE V-20
Liver Neoplasms In Male Fischer 344 Rats Fed Ch1ordaned»b
	Dose (ppw)	
Neoplasm
0 1	5 25
Adenoma lc 1	4 8C
Carcinoma lc 0	0 2C
Total neoplasms 2 1	4 10*1
aSource: RIAS0T. 1983b
^Pathology by Gary H. Williams, M.D., dated March 9,	1984
c0ne animal had both an adenoma and carcinoma.
^Significantly different from control value (p<0.05)
01800
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Incidence of hepatocellular swelling 1n males receiving 1, 5 or 25 ppm and
In females receiving 25 ppm. In addition, there was a significant Increase
In hepatocellular necrosis In males receiving 1 or 25 ppm, but no corre-
sponding effect occurred In females. The histologic changes In the liver
were accompanied by Increased liver weights In males at 130 weeks (5 and 25
ppm groups) and females receiving 25 ppm at weeks 26 and 52. A NOEL and
LOEL for chronic toxicity In females based on nonneoplastic changes in the
liver are 5 and 25 ppm, respectively; the LOEL 1n males Is 1 ppm.
Other Species.
Mazeter (1967) — In this unpublished study, Wazeter, as cited in
Vettorazz! (1975), performed a chronic study with dogs that were fed a diet
containing chlordane at levels of 0, 0.3, 3, 15 or 30 ppm (-0, 0.008, 0.075,
0.375 or 0.75 mg/kg bw) for 2 years. Increased liver weight and histologic
changes In the liver were reported along with a NOEL of 3 ppm. No tumors
were reported. The study duration (2 years) 1s considered Inadequate for a
carcinogenicity assay In dogs.
Animal Studles-Heptachlor — Heptachlor has been studied In three
mouse and five rat long-term carcinogenesis bloassays; Tables V-21 and V-22
present a summary of the experimental design and tumor results for these
studies. One long-term chronic study using dogs has also been conducted.
These studies are described 1n more detail in the following sections.
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TABLE V-21
Summary of Mouse Dietary Carcinogenicity Tests for Heptachlor and Heptachlor Epoxide
Strain
No./Sex
Dose
(PP»)
Duration of
Exposure/
Observation
Tumor Results
Reference
C3H
100/N&F
10
2 years
Benign liver tumors (FDA);
hepatocellular carcinomas
In both males and females
(Reuber)
FDA (Davis. 1966);
Reuber. 1977b
B6C3F1
50/H&F
6.1 and
13.8. N;
9 and 18. f
80 weeks plus
10 weeks
observation
Hepatocellular carcinomas
In both males and females
NCI. 1977b
CD-I
lOO/N&f
J 'j
1. 5 and 10
18 nonths
observation
Nodular hyperplasia at 5
and 10 ppa In both males
and females (1RDC)
Hepatocellular carcinomas
at 5 and 10 ppm In both
males and females (Reuber)
IRDC. 1973b
Reuber. as cited
In Epstein. 1976

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TABLE V-22
Summary of Rat Dietary Carcinogenicity Tests for Heptachlor and Heptachlor Epoxide
Duration of
Strain	No./Sex	Dose	Exposure/	Timor ResuTts	Reference
Observation
Wis tar 95/H&F no.
of each sex
In dose group
not specified
TO mg/kg
(5 doses)
S dally
doses then
observation
for 106-110
weeks
No treatment-related
Increase In tumors
Cabral et al.
1972
Osborne-
Nendel
CD
CF
CFN
50/N&F
25/H&F
20/M&F
25/H&F
38.9 and
77.9 ppm, H;
25.7 and
SI.3 ppm, F
5, 7.5, 10
and 12.5 ppm
1.5. 3, 5, 7
and 10 ppm
0.5, 2.5,
5.0, 7.5
and 10 ppm
80 weeks plus
30 weeks
observation
2 years
110 weeks
108 weeks
No liver tumors; neoplastic
nodules In treated and
control groups; thyroid
tumors In females
No Increase In tumors;
some liver lesions at
7.5 ppm and above
No dose-related Increase
liver lesions at 7 and
10 ppm
No dose-related Increase
In tumors (Kettering);
slight Increase In liver
nodules and carcinomas
(Reuber)
NCI, 1977b
Kettering
(Jolley et al.,
1966)
Kettering
(Ultherup
et al., 1955)
Kettering
(Ultherup
et al., 1959);
Epstein, 1976

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Studies with Mice.
The Food and Drug Administration (Davis. 1965) « The carcinogenic-
ity of heptachlor and heptachlor epoxide (purity not specified) was studied
using groups of 100 male and 100 female C3H mice fed a diet mixture contain-
ing 0 or 10 ppm (-1.43 mg/kg bw) for 2 years. Survival was generally low:
50% of the controls, 30% of the heptachlor-treated, and 9.5* of the
heptachlor epoxlde-treated mice survived the 2-year period; early deaths
were due to tumors. F0A pathologists found a 2-fold Increase In benign
liver lesions (hepatic hyperplasia and benign tumors) In the treated animals
over the controls, although the Incidence of malignant liver tumors was less
(Table V-23).
A reevaluatlon was performed by Reuber (1977b), resulting In a change In
diagnosis for many benign tumors to liver carcinomas. Hepatic carcinomas 1n
the treated groups were generally large, especially In the epoxide groups,
and were frequently multiple. In contrast to carcinomas in control groups,
which were smaller and solitary. The Incidences of liver carcinoma are
presented In Table V-24. Reuber's findings yielded a highly significant
(p=5xl0_i to <1x10~») Increase In Incidences of liver carcinoma In both
sexes for heptachlor and heptachlor epoxide. Four other Independent pathol-
ogists (Drs. R. Squire, R. Sternberg, H. Stewart and G. Williams) reviewed
a sample of 19 slides and generally concurred In the Reuber diagnoses
(Epstein, 1976).
In addition to liver tumors, Reuber also diagnosed nontumor liver
lesions, primarily hyperplasia, nodules, hepatic vein thrombosis, and
cirrhosis, 1n the heptachlor and heptachlor epoxlde-treated mice.
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TABLE V-23
Incidences of Liver Lesions 1n C3H Mice
Treated with Heptachlor or Heptachlor Epoxide3
Dose Group
Hepatic
Hyperplasia
Benign Liver
Tumors Only
Malignant Liver
Tumors Only
Controls
38/200
30/200
21/200
Heptachlor (10 ppm)
108/200
51/200"
10/200
Heptachlor
epoxide (10 ppra)
65/200
8S/200C
13/200
aSource: Davis, 1965
^Statistically significant (p
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TABLE V-24
Incidences of Hepatocellular Carcinoma 1n C3H Mice
Treated with Heptachlor or Heptachlor Epoxide*
Dose group	Males	Females
Controls	22/78 (28%)	2/54 (4*)
10 ppm heptachlor	64/87 (73%)	57/78 (73%)
10 ppm heptachlor epoxide	73/79 (92%)	77/81 (95%)
'Source: Davis, 1965 (as diagnosed	by Reuber, 1977b)
01800	V-83	03/08/87

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National Cancer Institute (1977b) -- In the NCI carcinogenicity
bloassay conducted at Gulf South Research Institute, technical grade hepta-
chlor (-73% heptachlor, 22% trans-chlordane and 5% nonochlor) was fed to
groups of 60 86C3F1 mice of each sex for 80 weeks, followed by an observa-
tion period of 10 weeks. The TWA high-dose and low-dose concentrations were
6.1 and 13.8 ppm (-0.87 and 1.97 mg/kg bw) for males and 9.0 and 18.0 pprn
(-1.29 and 2.57 mg/kg bw) for females, respectively. Controls consisted of
20 male and 10 female matched control mice, and 100 male and 80 female
pooled control mice.
Hepatocellular carcinoma was the most frequently observed neoplasm. The
Incidence In high-dose males was significantly higher (p«0.001) when com-
pared with matched controls, while the Incidence 1n low-dose males was com-
parable to that of the control groups. The difference In hepatocellular
carcinoma Incidence between high-dose females and matched controls was also
significant (p<0.005), and there was a highly significant (p<0.0001) dose-
related Increase In the Incidence of hepatocellular carcinomas for female
mice due to the difference between the high- and low-dose groups. Other
tumor types were observed with low frequency among all groups. The inci-
dence of hepatocellular carcinoma 1n mice 1s presented in Table V-25. There
were also many nodules 1n the mice that did not have liver carcinomas.
International Research and Oevelopnent Corporation (1973b) — IROC,
under contract with Velslcol Chemical Corporation, fed a 25:75 mixture of
heptach1or:heptach1or epoxide In the diet, at concentrations of 1, 5 or 10
ppm (-0.14, 0.71 or 1.43 mg/kg bw) for 18 months to groups of 100 male and
100 female CD-I mice, starting at 7 weeks of age. Similar control groups
01800
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TABLE V-25
Incidence of Hepatocellular Carcinoma In B6C3F1 Mice Following
Chronic Dietary Exposure to Heptachlor/Chlordane H1xturea*b
Groups (ppm diet)	Incidence*5

MALES


0 (pooled)

17/92
(18%)
0 (matched)

5/19
(26%)
6.1

11/46
(24%)
13.8

34/47
(72%)c

FEMALES


0 (pooled

3/78
(~X)
0 (matched)

2/10
(20X)
9.0

3/47
(6%)
18.0

30/42
|71%)<1
aSource: NCI, 1977b
^Incidence expressed as No. of tumor-bearing mice
No. of tissues examined
dose-
dose-
related increase in female mice (p<0.000l).
Statistically different from matched controls (p«0.0001); also
related Increase In male mice (p<0.0001).
^Statistically different from matched controls (p<0.005), also
01800
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were fed an Insectlc-lde-free diet. The Incidences of hepatomas were lower
among the higher dose groups than 1n the 1 mg/kg diet group and controls.
However, the Incidences of nodular hyperplasia (Table V-26) were highly
significant at 5 and 10 ppm in both males and females when compared with
controls. Upon reexamination of the slides, Reuber diagnosed more hepatic
carcinomas and less hyperplasia and hyperplastic nodules (Epstein, 1976).
Five other pathologists (Drs. J. Rust, P. Newberne, R. Squire, H. Stewart
and G. Williams) examined a portion of the slides and agreed with Reuber
that the Incidence of hepatic carcinoma was considerably underdiagnosed In
the original analysis (Epstein, 1976). The Incidences of liver carcinoma as
diagnosed by Reuber are presented In Table V-27.
Studies with Rats.
Cabral et al. (1972) -- Heptachlor (96.BX pure) In corn oil was
administered by gavage to Wlstar rats 1n five doses of 10 mg/kg bw each on
alternate days starting at 10 days of age. The heptachlor-treated group
contained 95 Wlstar rats, 7 of which died before weaning. The controls con-
sisted of 19 males and 27 females treated with corn oil alone. Many rats
were lost because of high mortality of both treated and control rats and to
an Interim sacrifice at 60 weeks. Twenty-nine females and 30 males remained
after 60 weeks and comprised the carcinogenicity test groups. All surviving
rats were sacrificed at 106-110 weeks. There was no Indication of a treat-
ment-related Increase In tumors.
Kettering Laboratory (Jollev et al.. 1966) -- In this study, a
75:25 mixture of heptachlorrheptachlor epoxide was administered to groups of
25 female CO rats In the diet at concentrations of 5, 7.5, 10 and 12.5 ppm
01600
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TABLE V-26
Incidence of Nodular Hyperplasia In CD-I Mice
Exposed to Heptachlor/Heptachlor Epoxide (25:75) Mixture*
Dose
(ppm)
Males

Females
0
0/50
(OX)
1/67 (1%)
1
2/53
(4%)
0/63 (0%)
5
24/57
(~»)
9/56 (16%)
10
53/69
(77%)
28/46 (61%)
•Source: IRDC, 1973b
01800
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TABLE V-27
Incidence of Hepatic Carcinoma In CD-I Mice Following
Chronic Dietary Exposure to Heptachlor:Heptachlor Epoxide <25:75)a
Groups	Incidence^
(ppm diet)

HALES


0

0/62
(OX)
1.0

2/68
(3%)
5.0

18/61
(26%)
10.0

52/80
(65%)c

FEMALES


0

6/76
(8%)
1.0

1/70
(10%)
5.0

6/65
(9%)
10.0

30/57
(53%)c
a8ased on Reuber's reevaluatlon of IRDC (1973b) slides (Epstein, 1976)
^Incidence expressed as No. of tumor-bearing mice
No. of tissues examined
cS1gn1ficantly different from control value (p<0.001).
01800
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(-0.25, 0.375, 0.5 and 0.625 mg/kg bw) for 2 years. A dose-related Increase
In mortality was observed. A comprehensive histological evaluation revealed
spontaneous tumors, such as mammary adenomas or fibroadenomas, with random
frequency among treatment and control groups. No malignant lesions of the
liver were observed, although hepatocytomegaly was Increased at 7.5, 10 and
12.5 ppm.
National Cancer Institute (1977b) -- In the NCI bloassay conducted
at Gulf South Research Institute, technical grade heptachlor was fed for 80
weeks to groups of 50 male and 50 female Osborne-Hendel rats at a TWA
dietary level of 38.9 or 77.9 ppm (-1.95 or 3.90 mg/kg bw) for males and
25.7 or 51.3 ppm (-1.29 or 2.57 mg/kg bw) for females. Animals were main-
tained for an additional 30 weeks on a heptachlor-free diet. Ten rats of
each sex served as the matched controls, and 60 rats of each sex served as
pooled controls.
Overall, the Incidence of neoplastic liver lesions was somewhat greater
In the heptachlor-treated groups than that observed after chlordane treat-
ment. However, no hepatocellular carcinomas were observed In any of the
rats, with one cholanglocarclnoma diagnosed 1n one low-dose male. Neo-
plastic nodules were observed In all treated and control groups, with no
statistically significant (p<0.05) dose-related trend. The Incidences are
presented In Table V-28. A statistically significant exact test for a
dose-related trend (p<0.002) was found for fol1Icular-cel1 carcinomas of the
thyroid of females, but not In males, when they were combined with adenomas.
01800
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TABLE V-28
Incidences of Neoplastic Nodules In Osborne-Mendel Rats
Following Chronic Oletary Exposure to Heptachlora»b
Groups (ppm diet)	Incidence0

MALES


0 (pooled)

2/58
(3%)
0 (matched)

1/10
(10%)
38.9

3/44
(7%)
77.9

6/49
(12%)

FEMALES


0 (pooled)

5/59
(8%)
0 (matched)

1/10
(10%)
25.7

9/48
(19*)
51.3

5/46
(11*)
^Source: NCI, 1977a
bNot significant (p<0.05) by either exact test or life-table adjustment
cInc1dence expressed as No. of nodule-bearing rats
No. of tissues examined
01800
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However, this Finding was discounted by NCI because the Incidences of
carcinomas were low and because of the variability of thyroid tumors In
control rat populations. The more recent review by Goodman et al. (1980) of
historical control tumor Incidences In the Osborne-Mendel rat used In the
NCI studies lends further support to the NCI decision.
Kettering Laboratory (Mltherup et al., 1955) — In this first
Kettering study, heptachlor (purity not specified) was administered to
groups of 20 male and 20 female CF rats at dietary levels of 1.5, 3, 5, 7
and 10 ppm (-0.075, 0.15, 0.25, 0.35 and 0.5 mg/kg bw) for 110 weeks.
Similar groups of 20 rats served as controls. Benign and malignant tumors
were randomly distributed among test and control groups, with greater
Incidences observed for females, especially at 5 and 7 ppm. Liver lesions,
described as the 'chlorinated hydrocarbon" type, were observed with high
Incidence at 7 and 10 ppm In the diet, but no liver lesions were found at
lower dose levels. The authors did not believe that the liver lesions were
neoplastic. The analysis Indicated that the Incidence of tumors In treated
rats was not significantly different from control Incidence.
Kettering Laboratory (Mltherup et al., 1959) — In the second
Kettering study, heptachlor epoxide (purity not specified) was administered
in the diet to groups of 25 male and 25 female CFN rats at concentrations of
0.5, 2.5, 5.0, 7.5 and 10 ppm (-0.025, 0.125, 0.25, 0.375 and 0.5 mg/kg bw)
for 108 weeks. Similar groups of controls were maintained on heptachlor
epoxlde-free diets. In the Kettering analysis, malignant and benign tumors
occurred randomly among the test groups and were not related to heptachlor
epoxide treatment. A reexamination of the histologic slides was conducted
01800
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by two pathologists," Ors. H. Reuber and G. Williams. Dr. Reuber concluded
that the Incidence of hepatic carcinomas was significantly Increased above
control Incidence at 5 and 10 ppm In female rats. Dr. Williams found more
hepatic nodules at the 10 ppm level 1n males. Table V-29 presents a summary
of Reuber's and Williams' diagnoses of liver carcinomas and nodules. Three
other pathologists reviewed the Kettering studies and also diagnosed more
carcinomas than reported by Wltherup (Epstein, 1976}.
Studies with Dogs.
Kettering Laboratory (U.S. EPA. 1977) — In this study, groups of
two males and three females were exposed to dietary doses of 0, 0.5, 2.5, 5
and 7.5 ppm heptachlor epoxide (purity not Indicated) for 60 weeks, at which
time they yere sacrificed and autopsled. No tumors were reported. While
the liver weights of both males and females tended to Increase logarithmic-
ally 1n proportion to the amounts of heptachlor epoxide 1n the diet, only
one male at the highest dose had observable hepatic damage. The damage was
characterized by cloudy swelling of the cells with slight clumping of the
cytoplasm. The study duration (60 weeks) Is considered too short and the
number of animals too small for this to be a valid carcinogenicity study.
Mutagenicity.
Chlordane — Chlordane (unspecified purity, technical grade and com-
mercial formulations) has been tested for mutagenicity and related effects
In a number of systems (Table V-30). Negative results have been obtained In
reverse mutation assays using nine strains of Salmonella tvohlmurlum and two
strains of Escherichia col 1 with and without metabolic activation systems
(Probst et al., 1901; Gentile et a 1.. 1982). Chlordane was negative when
01800
V-92
03/26/87

-------
TABLE V-29
Incidence of Hepatic Carcinoma and Neoplastic Nodules 1n CFN Rats
Following Chronic Dietary Exposure to Heptachlor Epox1dea*b
	Rcuber			Will lams	
Dose
(ppm) Carcinomas	Nodules	Carcinomas	Nodules




HALES




0
1/24
(4%)
6/24
(25%)
0/24
(0%)
0/24
(0%)
0.5
1/22
(5%)
6/22
(27%)
1/23
(4%)
0/23
(0%)
2.5
0/19
(0%)
7/19
(37%)
0/21
(0%)
0/21
(0%)
5.0
1/23
(4%)
9/23
(39%)
1/22
(5%)
0/22
(0%)
7.5
1/25
(4%)
2/25
(8%)
0/25
(0%)
1/25
(4%)
10.0
4/22
(18%)
7/22
(32%)
1/22
(5%)b
4/22
(18%)c




FEMALES




0
0/17
(0%)
7/17
(41%)
0/17
(0%)
4/17
(24%)
0.5
3/22
(14%)
8/22
(36%)
0/20
(0%)
5/20
(25%)
2.5
3/18
(17%),
4/18
(22%)
0/18
(0%)
2/18
(11%)
5.0
7/22
(32%)d
10/22
(45%)d
1/22
(5%)
2/22
(9%)
7.5
3/21
(14%)
14/21
(67%)
0/23
(0%)
5/23
(22%)
10.0
5/19
(26%)d
8/19
(42%)d
1/19
(5%)
1/19
(5%)
aSource: WUherup et al., 1959. Data from the Reuber/wm 1a«s diagnoses
presented In Epstein, 1976.
^Incidence 1s expressed as No. of tumor-bearing rats
No. of rats examined
Statistically significant (p-0.019) for combined Incidence of carcinomas
and nodules (Epstein, 1976).
^Statistically significant (p=0.05) for combined Incidence of carcinomas
and nodules (Epstein, 1976).
01800
V-93
03/26/87

-------
I ABU V-30
Mutagenicity Testing of Chlordane
CO
o
o
Assay
Indicator/
OrganIsa
Coapound and/or Purity Application
Concentration
or Dose
Activating
Systea
Response
ent
Reference
Reverse
autatlon
Reverse
autatlon
Salaonella
typhlaurlua
646. 1A1535,
TAI000, C3076.
TAIS37, D30S?.
TA1S38, 1A98
S. tmhlaurlia
TAIS3S, TA1537.
IA1538, 1A98,
IA100
unspecified purity
erdil foraulatlon
plate
Incorporation
plate
Incorporation
four gradient	»S9
plates to give
a 10-fold concen-
tration range/
plate up to
10,000-fold range
NR	»S9
- In all
tester strains
at all concen-
trations
- In all
strains
Probst
•t al..
1981
Gentile
et al.,
198?
Reverse
autatlon
S. twphlaurlua
TA1S35, TA1S37.
1AIS38, TA98.
1A100
technical grade
plate
Incorporation
MR
»S9
- In all
strains
Gentile
et al..
198?
i
Reverse
autatlon
Escherichia
coll WP2.
UP? urvr
unspecified purity
Mitotic gene Saccharoavces
conversion cerevti»ae
04
erclal foraulatlon
plate
Incorporation
Incorporated Into
log-phase cell
suspension
four gradient	»S9
plates to give
10-fold concen-
tration range/plate
up to 10,000-fold
range
NR	»S9
- In both
strains al all
concentrations
• with aeta-
bollc activa-
tion In a dose-
related aanner
Probst
et al.,
1981
Gentile
et al..
198?
Mitotic gene S. cerevlslae
conversion
technical grade
Incorporated Into
log-phase cell
cell suspension
MR
~ S9
~ with S9
activation
In a dose-
related aanner
Gentile
et al..
198?
Reverse
autatIon
lei aavs
M?? hoaoiygous
for wk-C allele
erclal foraulatlon
applied p
seedling
ergence
?.?4 kg/ha
HA
none
Gentile
e! al.,
198?
o
CO
o
OD
CO
Reproduced trom
best available copy-

-------
1ABU V-30 (conl.)
g Assay	Indicator/	Conpound and/or Purity Application	Concentration	Activating Response Coment	Reference
O	Organ Isa	or Dose	Systea

UDS
adult rat
pwrtty unspecified
cell culture
8 doses 0.*-1000.0
NA
chlordane was
Probst


prlaary hepato-
Incorporation
naoles/at

toxic to cells
et al..


cytes




at >100 naoles/
ai
1901

UDS
adult iale rat
purity unspecified
applied to cover -
10"»-10"' H
NA
chlordane was
Haslansky


prlaary hepato-

slip cell cultures


toxic to cells
and


cytes




at >10-4 N
Mil Haas,








1901

UOS
adult Bale
purity unspecified
applied to cover -
10"«-10"» N
NA
chlordane was
Haslansky


¦ouse prlaary

slip cell cultures


toxic to cells
and


hepatocytes




at >10* H
Milllaas.








1981

UOS
adult Mle
purity unspecified
applied to cover-
10"»-I0~* n
NA
chlordane was
Haslansky


haaster prlaary

slip cell cultures


toxic to eel Is
and


hepatocytes




at >10"* H
Milllaas.
<
1







1981
10








LT>
Doaliunl
¦ouse CD-I
technical grade
gavage or 1 .p.
0. SO. 100 ag/kg
NA
- at all
Arnold

lethal


Injection
bw

coneentratIons
et al..








1977

Dominant
¦ouse CD-I
coaaerclal foraulatlon
gavage or l.p.
0, SO. 100 ag/kg
NA
- at all
Arnold

lethal


Inject Ion
bw

concentration
et al..








1977

UOS
SV 40 trans-
unspecified purity
cell culture
1. 10. 100 and
~S9
~ at each
Ahaed


foraed huaan

Incorporation
1000 hN

> dose (P<0.05)
et al.,


fibroblasts




only without
1977


(M-«)




aetabollc








activation;








>3-fold over








control at 1 |A


Inhibition
Hela cells
Ortho Klor 74
cell culture
0.0075-0.POO Ng/M
NA
» 99 100% Inhi-
Blevlns

of cell

(44.4X chlordane)
IncorporatIon

bition with
and

growth





all concentra-
Sholes,
O






tions tested
1978
CJ








^ MR - Not reported; NA = Not applicable; UOS * Unscheduled DMA synthesis; I.p. = Intraperitoneal
CD

-------
assayed for unscheduled DNA synthesis In rat, mouse and hamster primary
hepatocyte cultures. Concentrations of >10~4 M chlordane were toxic to
the cells (Probst et al.f 1981; Maslansky and Williams, 1981). Commercial
and technical grades of chlordane were negative In the dominant lethal assay
In CD-I mice following either Intragastric or Intraperitoneal administration
(Arnold et al., 1977).
Positive results were obtained for mitotic gene conversion assays In
Saccharomyces cerevlslae with but not without metabolic activation (Gentile
et al., 1982). Ahmed et al. (1977) reported a >3-fold Increase (p<0.05)
over controls 1n unscheduled DNA synthesis by SV-40 transformed human fibro-
blast cultures (VA-4) treated with 1 pM chlordane, but only In the absence
of the S-9 metabolic activating fraction. Ortho-Klor 74 (44.4% chlordane
commercial formulation) Inhibited by 99-100% the growth of HeLa cells at
concentrations of 0.0075-0.200 yl/l (Blevlns and Sholes, 1978). Final-
ly, chlordane was positive In a reverse mutation assay using a strain of Zea
mays homozygous for wx-allele (Gentile et al., 1982). This assay In a plant
was Included for the sake of completeness since It was positive. Further
details of these assays are given In Table V-30.
Heptachlor and Heptachlor Epoxide — Heptachlor has been tested for
mutagenicity In bacteria, yeast, maize, fruit flies, mice and rats, and for
unscheduled DNA synthesis In mammalian cell cultures (Table V-31). Negative
results have been obtained In the reverse mutation assay In 10 strains of
Salmonella typhlmurlum and three strains of Escherichia col 1 for heptachlor
tested with and without metabolic acltvatlon (Morlya et al., 1983; Probst et
al., 1981; Marshall et al., 1976) and In the rec assay In two strains of
Sac 111 us sub111 Is In which no activating system was used (Shlrasu et al.,
01800	V-96	03/08/87

-------
TABU W-31
Mutagenic I ly Testing of Heptachlor and Heptachlor (poxlde
ao
o
o
Assay
Indicator/
Organ Is*
Coapound and/or Purity
Application
Concentration
or Dose
Activating
Systea
Response
nt
Reference
Reverse
autatIon
Salaonella
typhlaurlua
IAT00, TA98,
TA1S3S, TAIS37,
TA1538
1
plate
Incorporation
O-SOOO pg/plate
»S9
- In all
strains at
all concen-
trations
Horlya
et al.,
1983
Reverse
autatlon
S. UphlMirtin
G4b, TA1S35,
TA1000. C3076,
TAIS37. 0305?.
TA1S38, TA9B
heptachlor/NR
plate
Incorporation
four gradient
plates to give
10-fold concentra-
tion range/plate
up to 10,000-fold
range
~S9
- In all
strains at all
concentrations
Probst'
. et al.,
1981
Reverse
¦utatlon
S. tvohlaurlua
TA1S35, TA1S36,
TA1S37, TA1538
heptachlor/reference
standard pure
plat*
Incorporation
SO-IOOO tig/plate
»S9
- In all
strains at all
concentrations;
toxic level
>?S00 |ig/plate
(-S9); >1000
iig/plate |»S9)
Marshall
et al..
1976
Reverse
¦utatlon
S. tvDhtaurlua
TA1S35, TA1S37,
IAI538, IA98,
1A100
heptachlor/technlcal
grade
plate
Incorporation
range NR
~S9
t aaxlaua ~ In
strains TA98
and TAI00 with
S9 at 10 »g/
plate
Gentile
et al..
198?
Reverse
autatton
S. tvohlaurlua
TA1S3S, TA1537.
TAIS38, IA98,
IAIOO
heptachlor/coaaercla 1
foraulatIon
plat*
IncorporatIon
MR
fS9
- In all
strains
Gentile
et al.,
198?
Reverse
¦utatlon
Escherichia
coll
UP? her
heptachlor/MR
plate
Incorporation
O-SOOO pg/plate
»S9
Hone
Morlya
et al..
1983
Reverse
¦utatlon
i. coll
MP?. UP? uvrA"
heptachlor/NR
plate
Incorporation
four gradient
plates to give
10-fold concen-
tration range/plate
up to 10,000-fold
range
»S9
- In both
strains at
concentratIons
Probst
et al..
1981
o
cj
CD
N.
CD

-------
TABLE V 31 (conl.)
10810
Assay
Indicator/
Organ 1sa
Coapound and/or Purity
AppllcalIon
ConcentratIon
or Dose
ActIvatIng
Syslea
Response
Coaaent
Reference
o
Dec assay
Bacillus
subtllls
M17 Rec\
IMS Ret
heptachlor/NR
spot test
NR

none
-
none
Shlrasu
el al..
1976

Nilotic gene
reversion
Saccharoavces
cerevlslae
0«
heptachlor/technlcal
grade
Incorporation
Into log-phase
cell suspension
NR

iS9
-
none
Gent lie
et al..
1)62,

Hllotlc gene
reversion
S. cerevlslae
D4
heptachlor/coaaerclal
foraulatlon
Incorporation Into
log-phase cell
suspension
NR

»S9
-
none
Gentile
et al.,
198?

Reverse
Mitallon
Tea Bam
U?? howiygous
for wx-ca)lelle
heptachlor/coaaerclal
foraulatlon
applied prior to
seedling eaer-
gence
1.1? kg/ha

NA
»
none
Gentile
et al..
198?
V-98
Recessive
lethal
Orosophlla
aelanoaaster

Injection Into
abdoaen
S x 10~*X In
water

NA

concentration
was the
highest sub-
lethal concen-
tration
¦enes
and
Sraa.
1969

UOS
adult rat
prlaary hepato-
cytes
heptachlor/NR
cell culture
O.S-IOOO naole/at
NA

heptachlor
was toitc to
cells at >10
naoles/at
Probst
et al.,
1981

UDS
adult ult
rat prlaary
hepatocytes
heptachlor/M
applied to
coversltp cell
cultures
10"» to 10"'
N
NA

heptachlor.
Mas toxic to
cells at
>10"« N
Haslansky
and
Milllaas.
1981

UDS
adult aale
aouse prlaary
hepatocytes
heptachlor/NR
applied to
covers lip cell
cultures
10"» to 10~»
H
NA

heptachlor was
toxic to
cells at
>10 • H
Haslansky
and
Mil llaas.
1981
o
CJ
UDS
adult aale
haaster prlaary
hepatocytes
heptachlor/NR
applied to
coversllp cell
cultures
10"* to 10~«
H
NA

heptachlor Mas
toxic to cells
at >10~« N
Haslansky
and
HI 1llaas,
1981
o
GO
\
CD

-------
lABlf W-31 (conl.)
Assay
Indicator/
Organlsa
Coapound and/or Purity
Application
Concentration
or Dose
Activating
Systea
Response
Coaaent
Reference
Doalnant
lethal
ral/3 genera-
tions
i
diet for 3
generations
1 and 5 ppa (ag/kg
diet)
HA
~
Increased
nuaber of
resorbed
fetuses
Cerey
et al..
19)3
Chroaosoae
iberrilIon
rat bone
¦arrow
diet for 3 genera-
tions
1 and 5 ppa
(ag/kg diet)
NA
NA
~
Increased
nuabers of
abnoraal alto-
ses, cltroaa-
tlds, pulver-
izations and
translocations
Cerey
et al.,
19)3
Doalnanl
lethal
aouse CO-l
heptachlor/technical
grade
gavage or l.p.
Injection
0. 7.5 and IS
ag/kg bw
HA
-
- at either
dose by either
route
Arnold
et al..
19))
UDS
SV-40 trans-
foraed huaan
fibroblasts
heptachlor/MR
cell cultures
Incorporation
100, 1000 tJI
~S9
»
» with S9 at
both concen-
trations
(p<0.05)
Ahaed
el al.,
19))
Reverse
aulatIon
S. tvohtaurlua
TAIOO. 1A98,
1A1535, TA153).
1A1538
heptachlor epoilde/NR
plate
Incorporation
0 5000 Hg/plate
.S9

- for all
strains at all
concentrations
Horlya
et al..
1983
Reverse
autallon
S. typlllaurlua
1A1535.'TAU36.
IA153), IA1538
heptachlor epoxide/
Reference Standard
pure
plate
IncorporatIon
50-1000 tig/plate
»S9

toxic level
>2500 |ig/plate
(-S9); >1000
iig/plate (»S9)
Marshal 1
et al.,
19)6
Reverse
autatIon
I. coll UP2 her
heptachlor epoxlde/NR
plate
Incorporation
0-5000 tig/plate
iS 9
-
none
Norlya
et al..
1983
Recessive
lethal
0. aelanoqaster
heptachlor epoxide
Injection Into
abdoaen
2.5 i 10 ~«X In
water
NA

concentration
was the high-
est sublethal
concentratlun
Benes
and
Sraa.
1969

-------
Assay	Indicator/	Compound and/or Purlly
Organ tin
Oualnanl	aouse CP-1	hepUchlor eposlde:
lelhal	ktftKklor |7S:2S|
UOS	SV-40 trans-	beptachlor epoxide/HA
forced fibro-
blasts
MR < Hot reported
MA - Hot applicable
UOS * Uatcheduled DMA synthesis
TABU V 31 (cont.)
Application	Concentration	Activating Response Coaaent	Reference
or Oose	Syslct
gavage or l.p.
Injectloa
0, 7.5 and IS
ag/kg bw
MA

- at either
dose by either
route
Arnold
et al.
197/
cell culture
Incorporation
10. 100. 1000 »*
~s*
»
» with S9 at
each concen-
tration
(p<0.0S>
A teed
et al.
197;
I

-------
1976). Gentile et a 1. (1982), however, reported a maximum positive
response for reverse mutation 1n strains TA98 and TA100 of S. typhlmurlum at
10 wQ technical grade heptachlor/plate with S-9 activation. Negative
results were obtained In these strains when a commercial formulation of
heptachlor was tested. Heptachlor, whether technical or commercial grade,
was also negative for mitotic gene reversion In Saccharomyces cerevlslae In
the presence and In the absence of S-9 (Gentile et al., 1982). Negative
results were obtained 1n the recessive lethal assay In DrosophUa melano-
gaster (Benes and Sram, 1969) and for unscheduled DNA synthesis by rat,
mouse and hamster primary hepatocyte cultures (Probst et al., 1981;
Maslansky and Williams, 1981). The dominant lethal assay in CD-I mice
receiving heptachlor either by gavage or Intraperitoneal Injection also gave
negative results (Arnold et al., 1977); however, the dominant lethal assay
in rats fed heptachlor at 1 and 5 mg/kg diet for three generations resulted
In Increased numbers of resorbed fetuses (Cerey et al., 1973). Increased
Incidences of chromosome aberrations were also noted In the bone marrow of
these rats. Ahmed et al. (1977) reported positive results (p<0.05) for
unscheduled DNA synthesis 1n SV-40 transformed human fibroblasts (VA-4)
with, but not without, the S-9 fraction at 100 and 1000 yM heptachlor.
Heptachlor epoxide was negative in reverse mutation assays in several
strains of S. typhlmurlum and E_. coll with and without metabolic activation
(Morlya et al., 1983; Marshall et al., 1976) In the recessive lethal assay
In D. meTanoqaster (Benes and Sram, 1969) and in the dominant lethal assay
In CD-I mice (Arnold et al., 1977). Ahmed et al. ( 1977 ) reported that
heptachlor epoxide was positive 1n Inducing unscheduled DNA synthesis 1n the
SV-40 transformed human fibroblasts at concentrations of 10, 100 or 1000
mM in the presence, but not the absence, of S-9.
01800	V-101	03/08/87

-------
Teratogenicity and Other Reproductive Effects.
Chlordane -- Ingle (1952) found no fetotoxlc or teratogenic effects In
neonatal Osborne-Nendel rats following Jm utero exposure to technical chlor-
dane. Female rats, two each from groups receiving chlordane at continuous
dietary levels of 5, 10, 30, 150 or 300 ppm (mg/kg diet), were mated, one
during the 24th week and the other during the 48th week of treatment. No
effects were noted on fetal mortality. Utter size or health of the pups.
Three pups from each Utter were allowed to suckle from foster dams that had
not been exposed to chlordane; another three from each Utter remained with
their treated mothers. No differences 1n growth rates of pups resulted from
suckling with exposed or non-exposed dams at 5, 10 or 30 mg/kg diet levels.
No differences In growth rates were noted when these pups were compared with
control pups that had not been exposed either Ui utero or by suckling.
Hyperexc1tab1l1ty and mild tremors were present In pups born to and nursed
by dams at the 150 mg/kg diet level that were mated at 48 weeks. One male
pup died. No symptoms of toxicity developed In equivalent pups nursed by
nontreated foster mothers. Of pups bom to 300 mg/kg diet level dams, those
transferred to non-treated foster dams did not develop toxic symptoms; those
remaining with their treated natural mothers (24-week mating) developed
tremors after nursing for 4 days. One male and two female sucklings died.
One weanling male died and was underweight. Mortality was 100% within 5
days of nursing by dams treated at the 300 mg/kg diet level (48-week
mating). Pups born to untreated dams but nursed by treated dams were
similar to" the pups born to and nursed by dams treated at each respective
dietary level.
01800
V-102
03/08/87

-------
Welch et al. (1971) studied the effect of chlordane on mouse fertility.
Female Swiss-Webster mice were treated IntraperUoneally with 25 mg/kg bw
technical chlordane In corn oil once a week for 3 weeks. Controls received
only corn oil. The mice were mated on the last day of injection and killed
before the gestational period was complete. No effect was observed on
Utter size for pregnancy; however, the number of mice that became pregnant
was appreciably reduced. Of 104 control mice, -40 (38%) became pregnant.
Of 105 treated mice, -17 (16%) became pregnant. Microsomes prepared from
ovaMectomlzed mice treated intraperitoneally with 25 mg/kg bw chlordane
once a week for 3 weeks Increased the metabolism of estradiol by 3-5 times
the control level. Chlordane pretreatment also Inhibited the J_n vivo utero-
tropic action of estrogens 88-98%. Welch et al. (1971) also studied the
effects of chlordane pretreatment on estrogenic effects 1n Sprague-Oawley
rats. Female rats (19-20 days of age) were treated IntraperUoneally with
technical chlordane or transchlordane (>99%) in corn o 11 dally for 7 days.
At 25 mg/kg bw/day of either compound, the J_n vitro metabolism of estrone-
4-i4C by hepatic microsomes prepared from the treated rats was signifi-
cantly Increased. Pretreatment with technical grade chlordane at 10 or 50
mg/kg bw/day for 7 days significantly enhanced the Wi v1 vo metabolism of
IntraperUoneally administered estrogens. The uterotropic response of
estrone was Inhibited 93 and 98% by technical and transchlordane, respec-
tively. The Inhibition was significant (p<0.05) at >5 mg/kg bw/day of tech-
nical grade chlordane.
Heptachlor and Heptachlor Epoxide -- Mo data on the teratogenicity of
heptachlor or heptachlor epoxide were available. Hestltzova (1967) studied
the effect of heptachlor on the fertility of rats. Heptachlor (98.1% pure)
01800
V-103
03/30/87

-------
was "administered with food" at an "applied dose" of 6 mg/kg bw to an
unspecified strain of rats. It Is unclear whether this was a dally dose or
a total dose administered over the duration of the treatment. There was a
marked reduction In Utter size In generations and 1n one genera-
tion. Suckling pups had a high rate of mortality. During the first week
after birth a mean percent mortality of the exposed pups was 46% as compared
with 12% 1n controls. Cataracts developed 1n treated adults as well as 1n
pups.
Welch et al. (1971) studied the effect of heptachlor on the metabolism
of estrogen by Sprague-Dawley rats. Immature female rats (19-20 days of
age) were treated 1ntraper1toneally with heptachlor (>99% pure) at 10 mg/kg
bw/day for 7 days. The Wi vitro metabolism of estrone-4-**C by microsomes
prepared from the treated rats was significantly enhanced 2.5-fold over
control levels. Pretreatment of rats by the same dose schedule Inhibited
the in vivo uterotropic response of estrone by 67%.
Summary
Chlordane. Values for acute oral LD^q* of chlordane range from 83
mg/kg bw for cls-chlordane (Podowskl et al., 1979) to 560 mg/kg bw for tech-
nical grade chlordane (Ambrose et al., 1953a,b), when administered In lipo-
philic vehicle. Gaines (1960) determined an oral LO^q of 335 mg/kg bw for
male and 430 mg/kg bw for female Sherman rats. Mice had about equal sensi-
tivity (LOgQ = 390 mg/kg bw), while hamsters were much less sensitive
(LD^q 1720 mg/kg bw) (Gak et al., 1976). Neonatal rats were less sensi-
tive than adults with an Intraperitoneal L05Q of 1121 mg/kg bw (Harbison,
1975). Cls-chlordane was more toxic than trans-chlordane to mice (Iv1e et
01800
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03/26/87

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al., 1972). Symptoms of acute Intoxication Include CNS stimulation, as
evidenced by Irritability, tremors and convulsions (Boyd and Taylor, 1969;
Stohlman et al., 1950; Hyde and Falkenberg, 1976). At sublethal doses,
hepatic microsomal enzyme Induction (Den Tonkelaar and Van Esch, 1974) and
enhanced activities of hepatic and kidney cortical gluconeogenic enzymes
(Kacew and Slnghal, 1973) were observed. Delayed onset of eye and vaginal
opening occurred in neonatal mice treated subcutaneously with 0.075 mg/mouse
(Talamantes and Jang, 1977).
Maximum tolerated subchronlc dietary concentrations were 800 and 400
mg/kg diet for male and female Osborne-Mendel rats, respectively, and 40 and
80 mg/kg diet for male and female B6C3F1 mice, respectively (NCI, 1977a).
Subchronlc dietary exposure to chlordane has resulted 1n changes In prostate
homeostasis In rats (Shaln et al., 1977). The no-effect level for microso-
mal enzyme Induction was <5 mg/kg diet (Den Tonkelaar and Van Esch, 1974).
Dally oral Intake of 1.2 mg/kg bw chlordane by rats resulted In no histo-
pathologic damage to lungs, heart, stomach, liver, kidneys, spleen or testes
(DeLong and Ludwlg, 1954). Ingle (1952) defined a no-effect level In
Osborne-Mendel rats at 5 mg/kg diet chlordane for hlstopathologlcal changes
1n liver, kidneys, lungs, pancreas, stomach, adrenals, thyroid, thymus,
lymph, testes, ovaries, heart and spleen, as well as for food consumption,
growth rate and mortality. A dietary level of 10 mg chlordane/kg diet
produced hepatocytomegaly and mild bile duct proliferation. At higher con-
centrations, progressively more severe hlstopathologlcal damage to liver and
other organs occurred. Increased Incidences of hepatic hyperplasia and
hepatocytomegaly In mice were reported In a carcinogenicity study (IRDC,
1973a), but histological re-evaluation of slides by Independent pathologists
Indicated that the hyperplastic nodules were carcinomas (Epstein, 1976).
01800	V-105	03/26/87

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The Incidence of liver hyperplasia was not Increased 1n mice or rats 1n the
NCI (1977a) bloassay, but benign proliferative lesions In chlordane-treated
mice were found by Becker and Sell (1979).
Four chlordane carcinogenesis bloassays 1n mice have been reported. The
strains tested Include C57B1/6N, CD-I, B6C3F1 and ICR. In C57B1/6N mice fed
25 or SO ppn for 10 months, hepatocellular carcinomas were observed In 27%
(16) of the survivors. This mouse strain rarely develops spontaneous liver
lesions. For CD-I mice fed 5, 25 or 50 ppm for 18 months, liver nodules/
hepatocellular carcinomas were observed In the 25 and 50 ppm groups. In
B6C3F1 mice fed -30 and 60 ppm for 80 weeks and then held for 10 weeks,
hepatocellular carcinomas were observed In both males and females. For ICR
mice fed 1, 5 or 12.5 ppm for 24 months, hepatocellular adenomas and
hemangiomas were significantly Increased (p<0.001) In males receiving 12.5
ppm and nonneoplastic liver lesions were present In males fed 5 ppm and In
females fed 5 or 12.5 ppm.
Four chlordane carcinogenesis bloassays In rats have been reported. The
strains tested Include albino, Osborne-Mendel and Fischer 344. Three of
these studies were considered adequate and one was Inadequate. In albino
rats fed 10, 20, 40, 80, 160, 320, 640 or 1280 ppm for 400 days, there were
no treatment-related tumors. In Osborne-Mendel rats fed 5, 10, 30, 150 or
300 ppm for 2 years, hepatic toxicity was noted at 150 and 300 ppm, but no
liver turvors were noted. In Osborne-Mendel rats fed 203.5 or 407 ppm
(males) or 120.8 or 241.5 ppm (females), respectively, for 80 weeks and held
for an additional 29 weeks, no liver tumors were noted, but thyroid tumors
were significantly Increased. In light of historical data for Osborne-
Mendel rats, the thyroid tumors were not considered to be treatment-related.
01800	V-106	03/26/87

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In Fischer 344 rats fed 1, 5 or 25 ppm for 130 weeks, there was a statis-
tically significant Increase 1n hepatocellular adenomas, which was con-
sidered by the authors as weak evidence for carcinogenicity in males fed 25
ppm. Hepatocellular swelling was significant In females fed 25 ppm. The
hepatocellular adenomas occurred only 1n males surviving longer than 104
weeks.
Negative results for mutagenicity of chlordane were obtained In nine
strains of Salmonella typhlmurlum and two strains of Bacillus subtIlls for
reverse mutation with or without metabolic activation {Probst et al., 1981;
Gentile et al., 1902); In rat, mouse and hamster primary hepatocyte cultures
for unscheduled DNA synthesis (Probst et al., 1981; Haslansky and Williams,
1981); and In mice for the dominant lethal assay (Arnold et al., 1977 ).
Positive results were obtained In Saccharomvces cerevlslae for mitotic gene
conversion with, but not without, metabolic activation (Blevlns and Sholes,
1978) and 1n maize for reverse mutation (Gentile et al., 1982).
Ingle (1952) found no fetotoxlc or teratogenic effects In neonatal
Osborne-Mendel rats born to dams that were continuously fed chlordane at
5-300 mg/kg diet. Pups bom to and nursed by treated dams developed dose-
related symptoms of toxicity. Pups nursed by non-treated dams did not
develop toxic signs. Pups bom to non-treated dams and nursed by treated
dams were comparable with pups born to and nursed by treated dams. Welch et
al. (1971)- found that approximately half as many mated female mice, which
had been treated Intraperitoneal^ with three weekly doses of 25 mg/kg bw/
week of chlordane, became pregnant, as did untreated mice. Chlordane pre-
treatment In mice and rats resulted In enhanced Yn vitro and y1v° metabo-
lism of estrogen and Inhibited the uterotropic response to estrogen.
01800	V-107	03/08/87

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Hcotachlor and -Heptachlor Epoxide. Heptachlor Is more toxic than
chlordane to laboratory animals. Acute oral LD^ values In rats for
heptachlor range from 40 mg/kg bw for a commercial formulation (Ben Dyke et
al.» 1970) to 162 mg/kg bw for female Sherman rats for technical grade
heptachlor (Gaines, 1960). Oral LO^s were 70 mg/kg bw for mice and 105
mg/kg bw for hamsters (Gak et al., 1976). The Intraperitoneal LD^Q for
neonatal rats was 531 mg/kg bwt but was reduced to 133 mg/kg bw by pretreat-
ment with phenobarbltal. It was suggested that the enhanced metabolic
capacity of the neonate after phenobarbltal dosing resulted In the metabo-
lism of heptachlor to the more toxic heptachlor epoxide (Harbison, 1975).
The acute oral LD^ for heptachlor epoxide In adult rats was -60 mg/kg bw
(NAS, 1977; Sperling and Ewlnlke, 1969; Podowskl et al., 1979). Symptoms of
acute Intoxication Include tremors, convulsions, paralysis and hypothermia
(Hrdlna et al., 1974; Yamaguchl et al., 1980).
Moderate liver damage, accompanied by Increased levels of serum GPT and
serum aldolase, was observed In rats following single (60 mg/kg bw) or
repeated oral doses (7 or 12 mg/kg bw/day) for 3-14 days (Krampl, 1971);
however, tolerance appeared to develop at 20 days. Enhanced activities of
gluconeogenic enzymes, such as pyruvate carboxylase and glucose-6-phos-
phatase, In rats treated with a 200 mg/kg bw dose (above the LD^Q) was
observed by Kacew and Slnghal (1973). Dietary levels of 2-50 mg/kg diet for
14 days resulted In Induction of hepatic microsomal enzymes (Den Tonkelaar
and Van Esch, 1974).
Maximum tolerated subchronlc dietary concentrations were 160 and 80
mg/kg diet for male and female Osborne-Mendel rats, respectively, and 40
mg/kg diet for male and female B6C3F-J mice (NCI, 1977b). Subchronlc
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dietary exposure to heptachlor has resulted In changes In prostate homeo-
stasis 1n rats (Shaln et al., 1977) and Induction of hepatic microsomal
enzymes (Mnoshlta and Kempf, 1970).
A dose-related Increase In liver weight In male and female mice treated
with 1-10 rag/kg diet levels of a heptachlor:heptachlor epoxide (25:75) mix-
ture was reported In an unpublished carcinogenicity study (IROC, 1973b,
reviewed by Epstein, 1976). Increased incidences of hepatocytomegaly and
hyperplastic nodules were also reported to be significantly Increased. The
histologic slides from this study were re-evaluated by six Independent
pathologists. The overall Interpretation was that many oF the hyperplastic
nodules diagnosed by IROC (1973b) were actually carcinomas (Epstein, 1976).
Epstein (1976) examined other unpublished reports of heptachlor and hepta-
chlor epoxide carcinogenicity (Oavls, 1965; Wltherup et al., 1959) that
reported Increased Incidences of hepatic hyperplasia or nodules. Reevalua-
tlons by Independent pathologists again resulted In an Interpretation 1n
favor of Increased incidences of carcinomas rather than hyperplastic
nodules. Other unpublished reports (UHherup et al., 1955; Jolley et al.,
1966) were reviewed by Epstein (1976) and describe Increased incidences of
hepatocytomegaly and hepatic hyperplasia In rats and mice after chronic
feeding with heptachlor and heptachlor epoxide. The Incidence of 11ver
hyperplasia was not Increased In mice or rats In the NCI (1977b) bloassay.
Decreased body weight gain was observed In male and female rats treated at
77.9 and SI.3 mg/kg diet levels, respectively. Reuber (1977a), In reevalu-
ating the slides from the Davis (1965) study, found appreciable Incidences
of hepatic vein thrombosis and cirrhosis of the liver In male and female
i
mice treated at 10 mg/kg diet levels of heptachlor or heptachlor epoxide for
2 years. These conditions were not observed in any of the 127 controls.
01800	V-109	03/08/87

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Epstein (1976) reviewed several unpublished reports of chronic dietary
carcinogenicity studies of heptachlor and heptachlor epoxide In rats and
mice (Davis, 1965; Wltherup et al., 1955, 1959; Jolley et al., 1966; IRDC,
1973b) In an article based on a Statement of Suspension Testimony at EPA
Hearing on Heptachlor/Chlordane. Epstein (1976) also presented results of
Independent statistical analyses and re-evaluations of histologic slides.
Increased Incidences of nodular hyperplasia were reported for mice after
heptachlor or heptachlor epoxide exposure (Davis, 1965; IRDC, 1973b). Inde-
pendent re-evaluat1on of slides resulted In statistically significant
Increased Incidences of hepatocellular carcinoma in mice and rats {Epstein,
1976). The dietary levels of heptachlor or heptachlor epoxide in these
studies were 0.5-12.5 rag/kg diet.
Three heptachlor/heptachlor epoxide carcinogenesis bloassays 1n mice
have been reported. The strains studied Include C3H, B6C3F1 and CD-I mice.
In C3H mice fed 10 ppm of both heptachlor and heptachlor epoxide for 2
years, benign liver tumors/hepatocellular carcinomas were reported In both
male and female mice. Hepatocellular carcinomas In treated groups were
generally large and frequently multiple tumors, especially In the epoxide
group In respect to the controls. For 86C3F1 mice fed technical grade (con-
taining 22% chlordane) at concentrations of 6.1 or 13.8 ppm (males) or 9 or
18 ppm (females), respectively, for 80 weeks and held for an additional 10
weeks, hepatocellular carcinomas were significantly (p<0.001) increased In
both male and female mice. In CD-I mice fed a mixture of heptachlor epox-
ide/heptachlor (75:25) at concentrations of 1, 5 or 10 ppm for 18 months,
nodular hyperplasia/hepatocellular carcinomas were noted at 5 and 10 ppm In
both male and female mice.
01800
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Five heptachlor/heptachlor epoxide carcinogenesis bloassays In rats have
been conducted. The strains of rats studied Include Wlstar, Osborne-Mendel,
CO and CFN. In Wlstar rats given 5 doses of 10 mg/kg bw of heptachlor and
held for 106 to 110 weeks, no treatment-related tumors were observed. For
Osborne-Hendel rats fed technical grade heptachlor at concentrations of 38.9
or 77.9 (males) or 25.7 or 51.3 (females) ppm, respectively, for 80 weeks
and held for 30 weeks, no liver tumors were noted, although neoplastic
nodules were found 1n both treated and control rats. In CD rats fed a mix-
ture of heptachlor/heptachlor epoxide (75:25) at concentrations of 5, 7.5,
10 or 12.5 ppm for 2 years, no liver tumors were noted, although nonneo-
plastic lesions were noted In the livers of rats fed 7.5, 10 or 12.5 ppm.
In one study using CFN rats fed 1.5, 3, 5, 7 or 10 ppm of heptachlor for 110
weeks, the Incidence of liver tumors was not statistically different 1n
treated and control animals. In a second study using CFN rats fed 0.5, 2.5,
5, 7.5 or 10 ppm of heptachlor epoxide for 108 weeks, treatment-related
liver carcinomas were noted by several pathologists.
Heptachlor was negative for reverse mutation In 10 strains of Salmonella
typhimurium and 3 strains of Escherichia coll with and without metabolic
activation (Norlya et al., 1983; Probst et al., 1981); 1n 2 strains of
Bad! 1 us subtil Is In the rec assay (Shlrasu et al., 1976); In Saccharomvces
cerevlslae for mitotic gene conversion In the presence of S-9 (Gentile et
al., 1982); In Orosophlla melanoqaster for recessive lethality (Benes and
Sram, 196?); for unscheduled DNA synthesis In rat, mouse and hamster primary
hepatocyte cultures (Haslansky and Williams, 1981); and for the dominant
lethal assay In mice (Arnold et al., 1977). Positive results were obtained
01800
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In the dominant lethal assay In rats (Cerey et al., 1973) and for unsched-
uled DNA synthesis In SV-40 transformed human fibroblasts with metabolic
activation (Ahmed et al., 1977).
Heptachlor epoxide was negative for reverse mutations In S. typh1mur1um
and E,. col 1 with and without metabolic activation (Morlya et al., 1983;
Marshall et al., 1976); recessive lethal assay In D. melanogaster (Benes and
Sram, 1969); and the dominant lethal assay In mice (Arnold et al., 1977).
Heptachlor epoxide was positive for unscheduled ONA synthesis In SV-40
transformed human fibroblast cultures (Ahmed et al., 1977).
Heptachlor was reported to cause
rats when administered In the diet
1967). The dose 1n this study was not
a marked reduction In Utter size of
for several generations (Hestltzova,
clearly defined.
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VI. HEALTH EFFECTS IN HUMANS
Clinical Case Studies
Many clinical case studies describing the acute and chronic toxic
effects of technical grade chlordane, containing heptachlor, are available
In the literature. The three effects seen most frequently are CNS effects,
blood dyscraslas and neuroblastoma. In an attempt to substantiate the
toxicity associated with chlordane exposure 1n clinical case studies, Owlght
DeLong subjected himself twice, during two separate time Intervals, to 7%
chlordane vapors for IS minutes every 3 days for 12 weeks (DeLong and
Ludwlg, 1954). No treatment-related effects were detected during repeated
medical examinations, described as thorough but not further specified by the
authors.
CNS Effects Associated with Exposure to Chlordane and Heptachlor. Ten
clinical case studies describing CNS effects, along with miscellaneous other
effects, following oral, dermal and Inhalation exposure to technical grade
chlordane containing heptachlor are summarized in Table VI-1. These effects
frequently Included, but were not limited to. Irritability, salivation,
labored respiration, muscle tremors, convulsions and death with or without
an Immediately preceding period of deep depression.
Five of these case studies described toxic effects occurring after oral
exposure. Several CNS effects were observed In a 32-year-old woman (Case
No. 1) who had Ingested 104 mg chlordane/kg bw (Oerbes et al., 1955), and In
an 16-year-old woman (Case No. 2) who had Ingested 32 mg chlordane/kg bw
(estimated to be 10 mg/kg after vomiting) (Dadey and Kammer, 1953). Similar
effects on the CNS were observed following Ingestion of estimated chlordane
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TABLE Vl-1
Sumury of Citai with CMS effects Associated with Eiposure to Chlordane or Heptachlor,
Either Alone or In Combination with Other Agents
Case
No.
Case
Description
Route of
Eiposure
Oose
forMutation
Duration of
Eiposure
Possible
Confounding
factors
Syaptoas
Deference
32-year-old.
chronically
depressed,
white, widowed
woaan, weighing
-58 kg
oral
(Ingestion)
6 g chlordane
(104 ag/kg bw)
toxlchlor
dust con-
taining 5X
chlordane
and related
chealcalt
and MX talc
single eiposure
none
Clinical tyaptoas inclu-
ded chealcal burns
of the aouth. severe
heaorrhaglc gastritis,
shock, anuria, convul-
sions, death; at autopsy,
pathological findings
included cerebral con-
gestion and edeeu, lower
nephron nephrosis, con-
fluent bronchopneumonia
and ureala.
Oerbes et
al.. USS
IB-year-old.
white, feaale
student weigh-
ing -50 kg
oral
(Ingestion)
1.6 or 32 ag/kg
(estlaated that
SOQ ag or 10 ag/kg
bw was retained
after voatttng)
40V oil solu-
tion contain-
ing 241 chlor-
dane
single exposure
none
Starting at l.S hours
after Ingestion: nausea,
voaltlng. diplopia,
blurred vision, twitching
of the eitrealtles,
generallied convulsion,
no abnoraal blood or
urinalysis values, costo-
vertebral angle tender-
ness.
Oadey and
Kaaaer, 19S3
15-aonth-old,
feaale child,
weight 9 kg,
and In jgood
health before
Ingestion of
chlordane
oral
(Ingestion)
Not quantified
(estlaated to
be <100 ag or
<11.1 ag/kg bw)
wettable
powder (SOX
chlordane);
1 tablespoon
chlordane
diluted to
1 quart with
water
single eiposure
none
Kenerallied treaor.
Incoordination, ataxia,
convulsions (alternating
tonic and clonic phases);
Irregular respirations;
Increased pulse rate;
dilated pupils; Irrita-
bility; rtsus sardonlcus;
opisthotonos, bilateral
ankle clonus, and gener-
allied hyperactive reflex-
es; alld hypochroaattc
aneata (not clearly asso-
ciated with eiposure In
the literature).
lensky and
Evans, 19S2

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TABIC Vl-l (cont.)
Case Case	Route of	Ouralion of	Possible
No. Description	Exposure	Ooso	formulation	Exposure	Confounding	Symptoms
factors
4-year-old,
Spanish Ameri-
can, female
child weighing
11 kg (24 pounds
oral
(Ingestion)
Hoi quantified
(based on (I
absorption
estimated to bo
>g.*2 ag or >0.15
m'U
4SX chlor-
dane eaulsl
flable con-
centrate
single exposure
Clonic convulsions;
coordination loss; hypo-
flexla; Increased exci-
tability; sinus tachy-
cardia and right axis
deviation, as evidenced
by €C6; Increased body
temperature (100.3#f) on
the day after exposure;
absence of spontaneous
vomiting.
Aldrlch and
Holmes, 19*9
20-month-old,
white, male
child, weigh-
ing 12.7 kg
(2B pounds)
oral
(Ingestion)
Hot quantified
oil solution
containing
74% techni-
cal grade
chlordane
single exposure
Subject had a hypo-
chromic microcytic
anemia, which Is
usually associated
with a history of
poor Iron nutri-
tion.
Vomiting; frightened
appearance; short Inter-
rupted seliures (both
general and focal) accom-
panied by brisk deep
tendon reflexes In all
extremities; transient
Increased body tempera-
ture; hemic heart murmurs;
transient Increased
white blood cell count;
abnormal serum alkaline
phosphatase and thymol
turbidity levels at 3
months after exposure.
Cur ley and
Gerrettson,
1969
47-year-old,
married, male
nurseryman;
considered to
be a "fairly
heavy pipe
(tobacco)
smoker*
dermal (skin
contact with
soil contain
Ing chlor-
dane)*
Hot quantified
2 years
concurrent exposure
to DOT, hormone
sprays and arsenic
pesticides
Jacksonlan and grand
mal convulsions; 1(6
revealed a generalized
dysrhythmia with no
localising features;
liver function tests
were normal.
Barnes, 1947

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1ABIE VI-1 (conl.)
Case
Case
Route of


Duration of
Possible


Ho.
DescrIptIon
i xposure
Dose
foraulatlon
Ixposure
Confounding
Syaptoas
Reference






factors


7
?3-year-old.
deraal
Mot quantified
suspension
single exposure
occupational expo-
Confusion; convulsions;
Derbes et

woaan, who had


of ?S pounds

sure to chlorinated
at autopsy, nonspecific
al.. 19SS

worked with


of DDI. 39

hydrocarbons for 2
pathological changes


chlorinated


pounds of

years before acute
were seen In the ktdneys.


hydrocarbons


velslcol, AR

exposure, but with
brain and lungs.


(Including


SO and 10

no syaptoas of pre-


chlordane) for


pounds of

vious drug Intoxi-



2 years pre-


trlton x-100

cation; acute expo-

1

ceding acute




sure also Included



exposure




DDI. velslcol. AR








SO and trlton 1-100


Bb
S6-year-old,
deraal and
Mot quantified
wet table pow-
-3-S hours over
'occasional* use of
Starting at -S aonths
Furle and

•ale dentist
Inhalation

der; 74*
2 aonths for
S ag dlaiepaa
after Initiation of expo-
Irubowlti,

In excellent


technical
deraal; -300
(vallua) and 200 ag
sure: tachycardia; night-
1976

health until


grade
hours over S
ethchlorvynol
sweats; fatigue; short-


-S aonths


chlordane
aonths for
(plac Idyl) before
ness of breath; pale


after Initia-


diluted 1:90
Inhalation;
and during exposure
conjunctivae; presence


tion of expo-


with water
exposures began

of Sj gallop; occult


sure



simultaneously

blood In stool; persis-








tent elevated body temp-








erature (38.8-39.«*C)








with prostration, chills.








and sweat; pallor of








skin and aucous aeabranes;








cholestatic hepatitis.

9
Mo case
deraal
Mot quantified
73.3X tech-
NR
MR
dlulness. nausea and
(leaner et

descrIptIon


nical grade


voaltlng
al.. 1977

In the litera-


chlordane In





ture


petroleua








distillate




10
3 aonth-old
deraal
Not quantified
NR
NR
concurrent exposure
lethargy
Klemer et

Infant
and/or



to aldrIn

al.. 1977


Inhalation






'this subject handled ptpe tobacco after handling soil contaminated with chlordane, probably resulting In Inhalation exposure In addition to the known
deraal exposure.
blhls case Is also discussed In Table VI-? (Case Ho. 17).
NR ' Not reported

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doses of <11.1 mg/kg by a 15-month-old Infant (Case No. 3) (Lensky and
Evans, 1952) and >0.15 mg/kg by a 4-year-old child (Case No. 4) (AldMch and
Holmes, 1969). Curley and Garretson (1969) observed CNS effects 1n a
20-month-old Infant (Case No. 5), but the dose level was not quantified.
Dose levels for exposures by the dermal and/or Inhalation routes were not
quantifiable (Barnes, 1967; Derbes et al., 1955; FuMe and Trubowltz, 1976;
Klemmer et al., 1977).
Blood Dyspasias Associated with Exposure to Chlordane and Heptachlor.
Exposure to chlordane and heptachlor by skin contact and/or Inhalation has
been associated with several blood dyscrastas, Including four cases of
aplastic anemia (Infante et al., 1978; Klemmer et al., 1977 ), one case of
refractory megaloblastic anemia (Furle and Trubowltz, 1976), one case of
acute stem cell leukemia (Infante et al., 1978), one case of acute lympho-
blastic leukemia (Infante et al., 1978) and one case of acute myelomonocytlc
leukemia (Infante et al., 1978). Exposures were a result of Indoor or
outdoor applications or a combination of the two. These clinical case
studies are summarized In Table VI-2.
Three cases (Nos. 1, 2 and 5) were mixed exposure with other pesticides,
which may also be associated with blood dyscraslas In humans.
In addition to the clinical case studies described In Table VI-2, Mutr-
head et a-1. (1959) reported one case of hemolytic anemia associated with
exposure (route of exposure not specified) to chlordane, heptachlor, dlel-
drln and toxaphene, and five cases of aplastic anemia, of which two were
associated with exposure (routes of exposure not specified) to chlordane and
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IABIE VI?
Suaaary of Cases of Blood Dyscraslas Associated ullh Exposure lo Chlordane or Heplachlor, Itlher Alone or with Other Agents
o
CD
o Ciie
No.
Cdse Description
Description of Exposure
Exposure to
Other Agents
lype of
Blood Pyscrasla
Reference
11
lie. IS years old
Deraal by contact with outdoor	Isotox (contain-
spray for S Months and Inhalation	Ing carbaryl)
by Indoor application of chlor-
dane.
aplastic aneala
Infante et al.
1978
1?
13
15
16
¦ale, 28 year old, self-
eaployed realtor
¦ale, 68 years old
14	feaale, 9 years old
¦ale, 23 years old, eaployed
by a lawn care fir*
lie, 37 yeajrs old
i(tensive use of a 7SX chlordane
foraulatlon for 6 Months, before
onset of syiptoas (exposure
not otherwise specified).
Use of chlordane outdoors and
Indoors (74% chlordane) aore than
once during the 3 years preceding
the onset of syaptoas.
Eiposure to chlordane applied to
the Interior of her hose Initially
at the age of -6 Months and
annually thereafter.
Spraying lawns with chlordane
and other pesticides for 3 years
before onset of syaptoas.
Use of a 44% chlordane foraula-
latlon for 10 years before
onset of syaptoas, "frequent*
use Indoors of full strength
(application with a paint brush)
and several tlMes/year outdoors
of diluted chlordane.
dlailnon, various
paints, thinners
undeterMlned
Banvel D, dlailnon,
Bursban, 2,4-0,
paints, strippers,
thinners
aplastic aneala
aplastic aneala
acute slea cell
leukeMla
acute ly^ihoblastlc
leukeala
acute MyeloMonocytlc
leukeala
Infante et al.
1978
Infante et al.
1978
Infante et al.
197B
Infante et al.,
1978
Infante el al.,
1978
o
CJ
N
i\J
~J
S
as
-j

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TABLE VI-2 (cant.|
ClH
Ho.
Cis* Description
Description of iiposure
Exposure to
Other Agents
Type of
Blood Oyscrasla

17*
nil. 56 years old, dentltt
Exposure to 74* technical grade
chlordane diluted 1:90 with
water for -3-S hours skin
contact over 2 Months and -300
hours by Inhalation over S
¦oaths; exposures began simul-
taneously.
'Occasional' use
of 5 ag dlaiepa*
(vallua) and 200
¦g ethchlorvynol
(placldyl) before
and during expo-
sure
refractory aegalo-
blastlc aneala
furle and Trubowlti,
1976
IB
engineer; no othor dotal Is
provided
Treated an area under a house
with 250 gallons of 4.5* chlor-
dane eaulslon.
not specified
*
aplastic aneala
K lower et al.,
1977
•This cm 1i «1so discussed In Table ¥1-1 (Cat* Ho. 6).

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heptachlor only and three to chlordane, heptachlor and unspecified other
drugs. Also, Loge (1965) reported 12 cases of aplastic anemia with pancyto-
penia, of which three were associated with chlordane exposure only and nine
1n conjunction with other drugs, and one case of thrombocytopenia and two
cases of leukopenia agranulocytosis associated with exposure to chlordane 1n
conjunction with other drugs.
The American Medical Association, Council on Orugs, as reported 1n the
Registry on Blood Dyscraslas, concluded that there 1s evidence that a
"specific cause-effect relationship exists' between exposure to chlordane
and resulting blood dyscraslas, particularly pancytopenia, thrombocytopenia,
leukopenia and anemia (Anonymous, 1962).
Neuroblastoma Associated with Pre- and/or Postnatal Exposure to Chlor-
dane and Heptachlor. Infante et al. (1978) described five cases of neuro-
blastoma In children with a pre- and/or postnatal history of exposure to
technical grade chlordane containing -36.5% chlordane and 3-7% heptachlor.
These cases are surrmarUed 1n Table VI-3. Nine additional cases of neuro-
blastoma were diagnosed during the same time period (December 1974 to
February 1976) at the same pediatric hospital, but pre- and/or postnatal
exposure to chlordane and heptachlor were not ascertained. That effects may
be Induced by prenatal exposure Is consistent with the documented trans-
placental .transfer of chlordane In humans (Curley et al., 1969; Wasserman et
al., 1972, 1974; Zavon et al., 1969).
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TABLE VI-3
Cases of Neuroblastoma Associated with Exposure to Chlordane and Heptachlor*
Case
No.
Case Description
Description of Exposure and
Confounding Factors
Pathological
Condition
19
female, age 2 years and 8 months
Technical grade chlordane was
applied to both the Interior and
exterior of the 28-year-old mother's
home during the first trimester of
gestation. Application to the base-
ment, where the mother spent 25-30
hours/week typing. Included sealing
chlordane In holes drilled In cement
blocks. The house was not well venti-
lated, because of cold weather. The
mother took two vallum tablets and
received general anesthesia (thio-
pental sodium, halothane and nitrous
oxide) early In the pregnancy. The
exterior of the house was sprayed
with chlordane a second time one year
later.
Neuroblastoma
(Stage III)
20
male, age 4 years and 5 months
Technical grade chlordane was applied
to both the Interior and exterior of
the home every 6 months from one year
before conception. The mother took
aspirin during pregnancy.
Neuroblastoma
(Stage IV)

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TABLE VI-3 (cont.)
Case
No.
Case Description

Description of Exposure and
Confounding Factors
Pathological
Condition
21
female, age 4 years and
4 months
Technical grade chlordane was applied
to the Interior of the home when the
girl was 23 months old.
Neuroblastoma
(Stage IV)
22
male, age 3 years and 9
months
Technical grade chlordane was applied
to both the Interior and exterior of
the home 2 years before the child was
born and when he was 2 years old.
Between the two applications of chlor-
dane, application of a commercial
pesticide was Intermittent.
Neuroblastoma
(Stage IV)
23
female, age 6 years and
5 months
Chlordane dust was applied to the
exterior foundation of the house
when the child was 3 years and 8
months old (autumn of the year) and
again In the spring of the next year.
Neuroblastoma
(Stage IV)
~Source: Infante et al., 1978

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Epidemiological Studies
Wang and MacHahon (1979a) conducted a retrospective cohort mortality
study of 16,126 professional pesticide applicators that was undertaken to
study the mortality patterns of these workers. The cohort was selected from
three pest control companies with offices 1n over 40 states. Personnel
records for all persons In Job categories potentially Involving exposure to
various pesticides and employed between January 1t 1967 and June 30, 1976,
were reviewed for two of the companies. For the third company, employee
records were not available for the period prior to January 1, 1968; there-
fore, this date was used as the earliest date of eligibility for this
company, with a closing date of December 31, 1976. From 44,083 records,
16,126 subjects were Identified who met the following criteria: 1) male;
2) employed for 3 months or more; and 3) social security number, date of
birth, and employment dates were also available. Of those not eligible, a
sample of 4000 were examined; 11% had been excluded because they were
female, 71% because they were employed for <3 months, and 18% because of
missing Identifying Information.
Individual follow-up was not attempted. Identifying Information for the
16,126 eligible subjects was submitted to the Social Security Administration
(SSA). The SSA Identified 311 deaths, of which 269 death certificates were
obtained from the states or registration areas. The authors stated that
because there was no reason to believe that the 42 deaths for which death
certificates could not be found were distributed differently than those for
which certificates were found, observed numbers were "corrected" by dls-
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trlbutlng the 42 cases according to the distribution of obtained death
certificates and adding the results to the observed values for those with
obtained death certificates.
In order to develop some measurement for Intensity of exposure to pesti-
cides, Job titles were classified as either normal, moderate or heavy expo-
sure. Particular Interest was focused on those applicators exposed to
chlordane and heptachlor by conducting separate analyses for persons ever
holding jobs as termite control operators, a Job category likely to have
exposure to these two pesticides. If a worker held more than one Job he was
classified under the category of exposure corresponding to the Job held for
the longest period of time.
Expected numbers of deaths by cause were calculated by means of a com-
puter program by Munson (1974), using national mortality rates of white
males specific for 5-year categories of age and calendar year. SMRs were
calculated along with 95% confidence Intervals by an extension of the
Cornfield method. The authors presented SMRs based on the corrected number
of observed deaths. For this review, SHRs based on the true numbers of
observed deaths were calculated whenever possible. Based on the corrected
observed numbers, the SHR for all causes was 84 (observed-311, 95% CI
75-94). The SHR for all malignant neoplasms was 83, which was statistically
nonsignificant. Increased SMRs were observed for sk 1 n cancer (SMR=173t
statistically nonsignificant) and for bladder cancer (SMR=277, corrected
observed«3.5, 95% CI 101-761). Statistically significant SHR deficits were
observed for malignant neoplasms of the digestive organs and peritoneum
(SMR=46, corrected observed=6.9, 95% CI 22-95), respiratory system diseases
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(SMR.29, corrected observed*4.6, 95X CI 12-70), diseases of the digestive
system (SMR«55, corrected observed*ll.6, 95% CI 31-98), and all other causes
(SMR.355, corrected observed*15, 95% CI 33-90).
SMRs for various specific causes of death using the true number of
observed deaths can be calculated and are presented here. For the total
cohort they are as follows: SMR for all causes of death 1s 84
(observed«269); for malignant neoplasms, the SMR Is 75 (observed«47), for
cancer of the digestive organs and peritoneum, the SMR Is 40 (observed=6);
for skin cancer, the SMR Is 150 (observed«3); for bladder cancer, the SMR 1s
231 (observed=3); for nonmallgnant diseases of the respiratory system, the
SMR Is 25 (observed-4), and for nonmallgnant diseases of the digestive
system, the SMR Is 48 (observed«10).
Analyses were also conducted for those workers classified as termite
control operators, since they were thought to have had the most exposure to
chlordane and heptachlor. SMRs were presented for termite control operators
and for all other applicators for various causes of death. These SMRs were
based on corrected observed values. The SMR for all causes of death for the
termite control operators was 92 and for all other applicators was 78. For
malignant neoplasms, the SMR for both groups was 83. Elevated SMRs were
observed for each group for cancer of the skin (SMR for termite control
operators»148, SMR for all other appllcators-187), and cancer of the bladder
(SMR for termite control operators«215, SMR for all other appl1catorsd87).
None of the above-mentioned SMRs was statistically significant. The authors
stated that the only significant SMRs (p<0.05) observed for termite control
operators were for cancer of the digestive organs (SMR«20, corrected
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observed=l.2) and for cerebrovascular disease (SMR-39, corrected
observed-2.4). The authors also stated that there were no significant find-
ings 1n the "all other applicator" group, nor were there significant differ-
ences In SMRs between the two groups. (The method used to determine sig-
nificant differences between the two groups was not reported.) A nonsig-
nificant Increase In lung cancer was restricted to the "all other appli-
cator" group {SMR-131, corrected observed»16.9). It Is possible to cal-
culate SMRs using the true number of observed deaths for cancer of the skin
and bladder. For skin cancer, the SMR for termite control operators was
120, and for all other applicators was 167. For bladder cancer, the SMR for
termite control operators was 200, and for all other applicators was 250.
Workers were classified according to Intensity of exposure Into three
groups: minimal. Intermediate and highest exposure. None of the causes of
death exhibited an increase In SMR with an Increase In Intensity of expo-
sure. In fact, SMRs based on corrected observed values for lung cancer and
skin cancer tended to decrease with intensity of exposure. For lung cancer
SMRs of 130, 120 and 87 were observed for minimal. Intermediate and highest
exposure, respectively, and for skin cancer SMRs of 190, 187 and 138 were
observed, respectively. The authors did not discuss the statistical sig-
nificance of these findings.
A latency analysis was performed for lung cancer. However, few workers
were followed for >10 years since first employment, and none of the lung
cancer deaths had been followed for that length of time. Positive trends
toward an Increase In lung cancer deaths occurred as the period of latency
Increased.
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There are several limitations to this study. The major limitation Is
the distribution of deaths without death certificates according to the
distribution of those with death certificates. If the age distribution (or
the distribution of any other disease-related variable) of the group without
death certificates had been different from that of the group with death
certificates, the cause-specific death distribution could have been differ-
ent, and could have resulted In Inaccurate calculation of observed numbers.
There was also no quantitative Information available on levels of expo-
sure, and duration of exposure could only be assessed for lung cancer
because of small numbers of observed cases. It Is not possible to assess
risks for chlordane and heptachlor exposure Independently of the risks from
other possible exposures. While the termite control applicators may have
had a greater likelihood of chlordane and heptachlor exposure than other
applicators, their exposure to other pesticides cannot be Ignored.
The authors did not Individually follow up each cohort member. The only
method used to ascertain vital status was a search through SSA records.
Thus, the number of deaths may have been underreported. There was no con-
trol of confounding factors such as smoking and alcohol consumption. This
study provides Inadequate evidence on the carcinogenicity of chlordane and
heptachlor.
Wang and NacMahon (1979b) conducted a retrospective cohort mortality
study that was undertaken 1n two chlordane and heptachlor manufacturing
plants between 1946 and 1976. Personnel records were available for 951
workers who had ever worked at a chlordane production plant In Marshall, IL,
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and for 1425 workers who had ever worked at a heptachlor and endrln plant 1n
Memphis, TN before the spring of 1976. Of these, 1403 subjects were Identi-
fied who met the following criteria: 1) male; 2) who had worked for >3
months; and 3) "adequate" Identifying Information was available. Of the 973
excluded, 7% were excluded because they were female, 64% because they worked
<3 months, and 29% because Identifying Information was not available.
The SSA Identified 104 deaths In the study cohort through the end of
1975. Nine additional deaths, not Identified by SSA, were discovered during
the conduct of a separate study (reference not reported) that Individually
followed up terminated workers from these plants. Death certificates were
obtained from the appropriate states for the 113 deaths (98% of those ascer-
tained) and were coded according to the 8th revision of the ICO by one of
the authors (Wang). Cause-specific SMRs were calculated by means of
Munson's (1974) computer program, using national mortality rates for white
males by age and calendar year In 5-year groups. For the computation of
person-years, the beginning dates were January 1 , 1946 for the Marshall
plant and January 1, 1952 for the Memphis plant, or at the end of 3 months
of employment If that date was later than the appropriate date above. The
authors calculated 95 percent confidence Intervals by an Iterative method
based on m1d-p values.
The authors attempted to correlate the Intensity of exposure with
mortality experience. Complete occupational histories were not available
for each worker, and serum levels of pesticides actively used In 1975 and
1976 did not correlate with a classification of presumed exposure based on
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Job category; however, no data were presented. Thirty-four percent of the
study cohort had <10 years duration of follow-up, 25% had 20-29 years of
follow-up, and 11% had >29 years of follow-up.
The overall SMR for all causes of death was 72 (observed*113, 95% CI
59-86), confirming the healthy worker effect. A deficit was seen In the SMR
for all cancers (SMR=82), but this was not statistically significant. Sta-
tistically nonsignificant deficits were observed for malignant neoplasms:
for cancer of the digestive organs.and peritoneum, the SMR was 82; for lym-
phatic and hematopoietic cancers the SMR was 30; and for all other cancers
combined the SMR was 45. A statistically nonsignificant excess was observed
for lung cancer; the SMR was 134 (p>0.05). The only significantly elevated
SMR was for cerebrovascular disease 
-------
reported) In lung cancer deaths (observed«l, expected=4) among those exposed
for 20 or more years. Conversely, among those employed for 10-19 years and
followed for 10-19 years, there was an excess risk (observed«6,
expected-2.1).
There are limitations to this study. There was no Information available
on levels of exposure or duration of exposure. It Is not possible to assess
risks for chlordane and heptachlor exposure Independently of the risks of
endrln exposure, a pesticide also manufactured at the Memphis plant.
The cohort Included all plant employees. Including those with little
potential for exposure, such as office workers; and therefore, the risk of
cancer may have been underestimated for those directly Involved In chlordane
and heptachlor manufacture. While the period of follow-up was long, the
size of the cohort was very small. Thus, the study had very little power to
detect a real difference 1f one was present.
The authors did not Individually follow up each cohort member. There-
fore, the number of deaths may have been underreported. Excluded from the
cohort were 282 Individuals with missing data, representing -29% of the
final cohort population. The authors provide no data with which to assess
the impact of excluding these workers from the cohort; however, this may be
a random occurrence. If this assumption Is true, then their exclusion poses
no bias to the estimates of risk.
There was no control of confounding variables such as smoking. However,
a significantly low SMR was observed for ischemic heart disease, a disease
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for which smoking Is a well-documented risk factor. The SHR for other
respiratory diseases was also low, though not significantly. This result
suggests that the cohort In question smoked at levels below that of males
nationally, and provides additional evidence that the reported excess
Incidence of lung cancer (SHRel34) may have been an occupatlonally related
Increase. Finally, race was assumed to be white for all study subjects. As
no deaths among nonwhltes were observed, this assumption may be a valid
one. Thus, this study provides Inadequate evidence for the carcinogenicity
of chlordane and heptachlor.
01 tragi 1 a et al. (1981) conducted a retrospective cohort mortality study
of employees at four organochlorIne pesticide manufacturing plants. All
workers (race and sex not specified) who had at least 6 months of employment
In pesticide manufacture prior to December 31, 1964 were Included In the
study population. Vital status was determined for each worker as of
Oecember 31, 1976, through the SSA, State motor vehicle offices, U.S. Postal
Mall Corrective Services, and "other" sources.
Four separate cohorts representing four pesticide plants comprised the
study population. Only one plant had manufactured chlordane and had done so
since 1946. No other pesticides were manufactured at this plant, but other
chemical products manufactured there Included chlorine and dlcyclopenta-
dlene. The cohort at plant 1 consisted of 327 Individuals, representing
8354 person-years of observation. Three percent were lost to follow-up.
Plant 2 had manufactured heptachlor since 1951. Endrln was also produced at
this plant 1n addition to chlorine, chlorendlc anhydride, hexachlorocyclo-
pentadlene and vinyl chloride. The cohort at this plant consisted of 305
01810
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workers (5672 person-years of observation), and 5% were lost to follow-up.
The two remaining plants were not Involved In the production of chlordane or
heptachlor and, thus, results are not discussed here for these two plants.
The chlordane and heptachlor plants had previously been studied by Hang and
MacMahon (1979b), who combined the two plant populations into a single
cohort with a longer period of follow-up In comparison to the follow-up
period reported In this study (01 tragi la et al., 1981).
Death certificates for all known decedents were obtained and coded by a
nosologlst to the ICD-A In effect at the time of death. Those with an
unknown vital status were assumed alive as of December 31, 1976. SMRs were
calculated by using the U.S. white male age-, calendar time- and cause-
specific mortality rates. Statistical significances between the observed
and expected values were tested with the Polsson distribution. Confidence
Intervals were presented for the SMR estimates, but the method of calcula-
tion was not reported by the authors.
Statistically significant (p<0.05) deficits were observed for all causes
of death at the chlordane plant (SHR*68, observed-59, 95% CI 52-87) and at
the heptachlor plant {SHR=66, observed«24, 95% CI 42-98). For deaths
because of malignant neoplasms, the chlordane plant had an SMR of 69, and
the heptachlor plant had an SMR of 91, none being statistically signifi-
cant. At the chlordane plant, risks were elevated for stomach cancer
(SMR-303),- rectal cancer (SMR=178), pancreatic cancer (SMR=110), and
respiratory system cancer (SMR=110). None of these risks was statistically
significant. At the heptachlor plant, excess risk was observed for
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Intestinal cancer (SMR=175), respiratory system cancer (SMRd22), and cancer
of the bladder and urinary system (SMR*606.). These risks were also not
statistically significant.
An analysis by latency was also carried out. SMRs were calculated for
deaths due to all malignant neoplasms according to latency, which was
defined as the number of years from date of first employment. Latency was
categorized Into three time periods: 1) <10 years since first employed,
2) 10-19 years since first employed, and 3) 20 or more years since first
employed. In the chlordane plant, SMRs of 66, 90 and 60 were reported for
all malignant neoplasms for the three latency time periods, respectively.
The individual point estimates were not significant. There were no observed
deaths In the <10-year latency period In the heptachlor plant. The SMR for
all malignant neoplasms for the 10- to 19-year latency period was 91, and
for the 20 or more year period the SMR was 162. These were not sta-
tistically significant; however, there may have been a positive trend of
Increasing risk with Increasing latency at the heptachlor plant.
This study revealed excess risk of cancer at various tissue sites for
all workers at these two plants. While none of the SMRs was statistically
significant, the SMR of 303 (observed*3) for stomach cancer In the chlordane
plant may be Important. However, there are limitations to this study.
No Information on quantitative exposures were provided. It was not
possible to assess the effects of chlordane or heptachlor Independently of
the other toxic chemicals at the two plants, some of which are known to be
carcinogenic. No attempt was made to exclude or' adjust for the effects of
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sex or race, nor was, there an effort to control for other confounding vari-
ables such as smoking or alcohol consumption. However, the size of the
study populations would preclude any real analysis of these variables.
Workers with little or no occupational exposure to these chemicals (I.e.,
office workers, etc.) were Included 1n the cohort. Thus, risks may have
been underestimated for workers Involved In the day-to-day manufacture of
chlordane or heptachlor. The size of the study population was small 1n
spite of follow-up periods of 25 years or more. Thus, the power of this
study to detect a statistically significant result is limited. This study
provides Inadequate evidence to link chlordane or heptachlor exposure to
cancer.
High Risk Subpopulatlons
There are no clear Identifiable high risk subpopulatlons that are
Inherently susceptible to exposure to chlordane, heptachlor or heptachlor
epoxide. Various blood dyscraslas have been associated with exposure to
chlordane and heptachlor (Infante et al., 1978; Furle and Trubowltz, 1976;
Klemmer et al., 1977). Because of the large number of agents associated
with these blood dyscraslas and the small Incidence of these conditions,
Infante et al. (1978) hypothesized that an Idiosyncratic mechanism for
susceptible Individuals may be Involved, but that Identification of members
of this sensitive subpopulatlon would be virtually Impossible.
Several groups may be more affected than the general population because
of high exposure rather than intrinsic susceptlbllty to chlordane, hepta-
chlor or heptachlor epoxide. These high exposure groups Include extermina-
tors; agricultural applicators; workers employed in the manufacture of
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chemicals; Infants nursing on human milk because of maternal excretion of
heptachlor epoxide' and oxychlordane, the major mammalian metabolites of
heptachlor and chlordane, respectively. In mothers' milk (Savage et al.v
1973; Strassraan and Kutz, 1977); the developing fetuses of women exposed to
chlordane because of transplacental transfer (Curley et al., 1969; Wasserman
et al.# 1972, 1974; Zavon et al., 1969); consumers of fish and shellfish
(especially freshwater); persons residing 1n houses after termite extermina-
tion treatment with chlordane and heptachlor.
Summary
Delong and ludwlg (1954) did not observe treatment-related effects In a
human exposed to 1% chlordane vapors for 15 minutes every 3 days for 12
weeks. The clinical case studies of acute exposure to chlordane and hepta-
chlor document a pattern of CNS effects similar to that found In animals.
The course of poisoning frequently Includes Irritability, salivation,
labored respiration, muscle tremors, convulsions and death with or without
an Immediately preceding period of deep depression. These symptoms were
seen at doses as low as 32 mg/kg bw (estimated to be 10 mg/kg bw after
vomiting) In an 18-year-old female (Dadey and Kamraer, 1953) and 104 mg/kg bw
In a 32-year-old female (Derbes et al., 1955). Several blood dyscraslas.
Including four cases of aplastic anemia (Infante et al., 1978; Klemmer et
al., 1977), one case of refractory megaloblastic anemia (Furle and Trubo-
wltz, 1976), one case of acute stem cell leukemia (Infante et al., 1978),
one case of acute lymphoblastic leukemia (Infante et al., 1978) and one case
01810
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of acute myelomonocytlc leukemia (Infante et al., 1978), have been associ-
ated with exposure to chlordane and heptachlor. Infante et al. (197B) also
suggested an association with pre- and/or postnatal exposure to chlordane
and heptachlor.
Three epidemiological studies of workers exposed to chlordane and hepta-
chlor have been reported. One of these studies, conducted 1n chlordane/
heptachlor applicators, was considered Inadequate In sample size and In
duration even though 1t showed Increased morality from bladder cancer
(SHR=277, p<0.05). A second study showed an Increased mortality from lung
cancer (SHR-134), but the Increase was not statistically significant. The
mortality from cerebrovascular disease was statistically significant
{SMR=183, p<0.05). Of the 1043 men Involved In the study, only one liver
cancer was reported. The third study Involved 2141 workers exposed to
organochlorIne pesticides. One of the four plants Involved In pesticide
manufacture produced chlordane and one produced heptachlor. The SHR for
malignant neoplasms was 69 at the chlordane plant and 91 at the heptachlor
plant. There was an excess risk for cancer In various tissues; none was
statistically significant. The last two studies were carried out In
chlordane/heptachlor manufacturing plants.
All of these studies have several limitations. Neither the quantitative
nor length of exposure histories are available for chlordane/heptachlor for
the populations studied. They were also exposed to other pesticides and
chemicals. Adjustments for these other chemical exposures and other con-
founding factors, like smoking and alcohol consumption, were not considered
In any of these studies. All of the study populations were small. In the
01810
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pesticide applicator study. Individual follow-up was not undertaken and the
data were missing on 10.3% of the decedents reported by the Soda! Security
Administration.
Because of these methodological limitations and the limited data. It Is
difficult to establish either a negative or positive association between
chlordane/heptachlor and carcinogenicity. Hence, these studies are con-
sidered Inadequate epidemiologic evidence.
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VII. MECHANISMS OF TOXICITY
Mechanism of Neurotoxicity
As discussed 1n the Effects of Acute Exposure and the Effects of Sub-
chronic and Chronic Exposures Sections in Chapter V, one of the most com-
monly observed effects of chlordane or heptachlor exposure was stimulation
of the CNS. Several Investigators have studied the mechanism for neuro-
toxicity. Hyde and Falkenberg (1976) studied the effect of chlordane on
brain potentials In male Sprague-Dawley rats that were surgically Implanted
with electrodes for EE6 monitoring. Chlordane In a Tween-80-sal1ne vehicle
was administered 1ntraper1toneally at 0.15, 1.75 or 25.0 mg/kg bw/day for 48
days. Control rats received vehicle only. Dose-related changes In neuro-
electrlcal activity (frequency, amplitude and waveform changes) occurred
during exposure, with Increasing Incidence as the duration of exposure
progressed.' No outward signs of toxicity were observed. The EEG changes
Included a shift toward fast beta rhythms (>25 Hz), elevated amplitudes,
reductions of delta (0.5-3 Hz) and theta (4-7 Hz) frequencies. Sinusoidal
waves were replaced by sharp, complex discharges. During the last 6 days of
chlordane Injection, the rats were starved and EEGs monitored. Abnormally
high voltage potentials that suggested lethal patterns were observed. It
was postulated that food deprivation resulted In mobilization of fat depots
of chlordane and/or metabolites, which then became neuroconcentrated. These
EEG disturbances did not return to normal when chlordane treatment was
discontinued, suggesting the persistence of chlordane as a neurotoxin.
St. Omer and Ecoblchon (1971) studied the time course of changes 1n
acetylcholine content of rat brain with respect to the time course of toxic
symptoms Induced by heptachlor. Female Ulstarrats with carotid artery
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cannulatlon were Injected with a single dose of 13.0 mg/kg bw of reference
grade heptachlor In a peanut o1l-1ec1th1n emulsion. Vehicle Injected rats
served as controls. Groups of six rats were sacrificed at 5, 15, 25 and 30
minutes after Injection and brain extracts were prepared for acetylcholine
content determination. Animals not sacrificed after treatment had mild
tremors progressing to severe tremors in 2-26 minutes. Within 10-20 minutes
after treatment, there were episodes of running, rolling and leg-paddling,
followed by mild tonlc-clonlc seizures and Inactivity at 18-26 minutes.
Death of chlordane-treated rats occurred within 2 hours. The average
acetylcholine content of rat brain extracts was -6.5 ^g/g wet tissue at 5
minutes (vehicle control value = 3.3 wg/g and remained constant) and -10.5
ug/g at 15 minutes. Acetylcholine levels declined to -9.5 ng/g at 25
minutes and -9.0 yg/g at 30 minutes after Injection, but remained elevated
above control levels.
In similarly conducted experiments, St. Omer (1971) studied the time
course of changes 1n ammonia and glutamlne content of rat brain. The time
course of toxic symptoms was as previously described (St. Omer and
Eco.blchon, 1971). The ammonia content of the brain Increased significantly
to 2-fold above vehicle control levels at 25 minutes after treatment
(p<0.05). The glutamlne content was unaffected. The Increased ammonia
level coincided temporally with mild seizures In heptachlor treated rats.
It was noted that the level of ammonium Ions In the brain Increase during
states of CMS excitation. Ammonia In the brain Is removed by conversion to
glutamlne. Since, following heptachlor treatment. It appeared that this
conversion did not take place, the accumulation of ammonia without subse-
quent removal Induced the neurotoxicity. St. Omer (1971) concluded that
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heptachlor acted by a mechanism that Interfered with the production or
utilization of ammonia.
The biochemical mechanism of neurotoxicity of heptachlor epoxide was
studied by Yamaguchl et al. (1979) with respect to Inhibition of synaptic
ATPases. Synaptosomal, microsomal, synaptic vesicle and synaptic membrane
fractions were prepared From homogenates of brain from Sprague-Oawley rats.
The sensitivities of various ATPases In these fractions to heptachlor epox-
ide J_n vitro. In decreasing order, were as follows: Ca^*, Hg^f-ATPase
(66.8-76.9% Inhibition) > Mg2,"-ATPase (31.2-48.9% inhibition) > Ca2f-
ATPase (26.5-48X Inhibition) > Na \ K*-ATPase (0-18.« Inhibition). The
0S	A
Inhibition of Ca \ Mg *-ATPase was greatest 1n the synaptic vesicle
fraction (which contained synaptic membrane components). The Inhibition of
2f	2^
Ca , Mg -ATPase by heptachlor epoxide was shown to reduce the binding
2 ^
capacity of Ca *. It was suggested that the resulting Increased level of
Ca^* In the presynaptic region promoted the release of neurotransmitter
(e.g., acetylcholine) contained In the synaptic vesicles, which resulted 1n
the appearance of toxic symptoms.
This phenomenon was further studied (Yamaguchl et al., 1980) using rat
brain synaptosomes and glutamate as the model excitatory neurotransmltter.
The release of transmitter Is controlled by synaptic vesicle concentration
A
of Ca f. In a series of experiments, 1t was demonstrated that heptachlor
2 ^
epoxide stimulated the uptake and Inhibited the release of Ca * by synap-
tosomes, leading to accumulation of Ca^*. Heptachlor epoxide also reduced
the uptake of Ca^f by the endoplasmic reticulum. Thus, Ca^* within the
synaptosomes was more available to synaptic vesicles to trigger release of
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transmitter. The sequestration of Ca2> within the synaptosome was related
A	A
to the observed Inhibition of Ca +, Mg *-ATPase (Yamaguchl et a 1.,
1979). To confirm the In vitro results, heptachlor epoxide was administered
Intraperitoneal^ to rats at 200 mg/kg bw 1n corn oil. Violent convulsions
occurred within 5 hours, at which time the rats were killed and synaptosomes
prepared. The Ca^+, Mg^*-ATPase within synaptosomes was Inhibited and a
4-fold greater concentration of heptachlor epoxide was found In synaptosomes
than 1n whole brain.
Folmar (1978) found that in. vitro rat brain microsomal Nf, K*-ATPase
was significantly Inhibited by chlordane (82%) and heptachlor (72%),
suggesting a role of this ATPase In the neurotoxicity of these Insecticides.
Hichanlsms of Effects on Endocrine and Reproductive Organs
In an attempt to explain the action of chlordane and heptachlor on
ventral prostate homeostasis (e.g., Increased androgen receptor site
content, decreased protein content) observed In rats following subchronlc
pesticide feeding (see Effects of Subchronlc and Chronic Exposures Section
in Chapter V), Shaln et al. (1977) studied 5a-d1hydrotestosterone binding
to androgen receptors l£ vitro. Chlordane and heptachlor were found to be
poor Inhibitors of 5a-d1hydrotestosterone binding to the receptor of
ventral prostate cytoplasmic extracts or tissue mince preparations when com-
pared with the Inhibition by parathlon. It was concluded that 5a-d1hydro-
testosterone binding Inhibition was not a good predictor of the pesticide
effect on ventral prostate homeostasis. An alternative mechanism was not
presented.
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Welch et al. (1971) found that chlordane administered 1ntraper1toneal 1y
to mice at a dose of 25 mg/kg bw once a week for 3 weeks resulted In de-
creased fertility (see Teratogenicity and Other Reproductive Effects Section
In Chapter V). A possible mechanism was Investigated. Microsomes prepared
from ovar1ectom1zed mice that had been pretreated with chlordane Increased
the metabolism of estrogen to polar metabolites by 3-5 times the control
level. In addition, the in vivo uterotropic action of exogenous estrogen
was 88-98% Inhibited In mice pretreated with chlordane. Similar results
were obtained 1n the 1_n vivo and 1_n v1 tro experiments conducted 1n rats with
chlordane (c1s- and trans-1somers) and heptachlor.
Enzyme Induction and Related Mechanisms
Karel and Saxena (1976) observed Increased levels of blood glucose,
serum protein, serum alkaline and serum add phosphatases 1n gerblls that
had been treated Intramuscularly with chlordane (see the Effects of Acute
Exposure and the Effects of Subchrorilc and Chronic Exposures Sections In
Chapter V). These Investigators speculated that the increased level of
serum protein was a result of Increased Incorporation of amino acids Into
protein In the liver, with subsequent release Into the blood stream. The
Increased level of serum alkaline phosphatase may have resulted In part from
Increased activity of osteoblasts and In part by release from necrotic liver
cells. Release of add phosphatase frora degenerating liver cells would
result 1n higher serum levels of this enzyme, as well. The Increase 1n
blood glucose was explained as follows. Chlordane treatment activated the
sympathetic nervous system, resulting 1n adrenal medulla secretion of
adrenaline, and this stimulated anterior pituitary secretion of ACTH. ACTH
activity resulted In production of glucocorticoid hormones, which in turn
enhanced gluconeogenesls.
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Enhanced gluconeogenesls was also observed In liver and kidney cortex of
rats following acute or subchronlc exposure to chlordane, heptachlor and
heptachlor epoxide (Kacew and Slnghal, 1973; Kacew et al., 1973; Slnghal and
Kacew, 1976). The enhancement was evidenced by Increased activities of
pyruvate carboxylase, phosphoenol pyruvate carboxylase, fructose-1,6-d1-
phosphatase and glucose-6-phosphatase, as well as Increased levels of blood
glucose, serum urea and decreased liver glycogen (see the Effects of Acute
Exposure and the Effects of Subchronlc and Chronic Exposures Sections In
Chapter V for dose, routes, durations, etc.). These Investigators Found
that treatment of rats with these pesticides also resulted In significantly
elevated activities of adenyl cyclase and levels of cyclic AMP In the liver
and kidney cortex, suggesting that synthesis of cyclic AMP was enhanced
(Kacew and Slnghal, 1973). Treatment of rats 1ntraper1toneally with cyclic
AMP (2x100 mg/kg bw) resulted In significantly Increased activities of the
four gluconeogenic enzymes. Increased blood glucose and serum urea levels,
and decreased liver glycogen content. Cyclic AMP, therefore, mimicked the
action of pesticides (Slnghal and Kacew, 1976). A mechanism of enhanced
gluconeogenesls was proposed whereby the Insecticides react with an adenyl
cyclase receptor on the plasma membrane, thus stimulating the activity of
the enzyme to synthesize cyclic AMP from ATP. Cyclic AMP, 1n turn, may bind
with a protein kinase receptor, resulting 1n a cyclic AMP Independent
protein kinase that Is translocated to the nucleus. The protein kinase then
catalyzes the phosphorylation of nuclear proteins, which Initiates trans-
cription, resulting In de novo synthesis of messenger RNA that Induces the
synthesis of gluconeogenic enzymes.
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Increased activities of microsomal enzymes (phosphorothloate detoxlca-
tlon, 0-de»ethylase and N-demethylaseJ were observed In rats fed chlordane,
heptachlor or heptachlor epoxide for '3 weeks (KlnoshHa and Kempf, 1970).
Effects on Cellular Respiration
Settlenrtre et al. (1974) reported that heptachlor stimulated the ATPase
of mouse liver mitochondria, but did not stimulate mitochondrial oxidation
of succinate (respiration) 1n the absence of ADP. Respiration was stimu-
lated in the presence of ADP. Heptachlor had no effect on ATP synthesis In
the presence of AOP. It was stated that these effects were contrary to
those observed for uncouplers of oxidative phosphorylation, which are known
to stimulate respiration In the absence of ADP or Inorganic phosphate and
prevent ATP synthesis. An explanation to account for the enhanced respira-
tion with AOP present, that there was an 'Increased Influx of AOP Into the
mitochondria," was rejected on the basis that uptake of 14C-ADP did not
occur. It was suggested that, on the basis of a concentration-related
Increase 1n the light-scattering response of mitochondria by heptachlor, the
Insecticide dramatically altered the mitochondrial membrane, resulting 1n
the observed Increase 1n respiration 1n the presence of AOP. Two possible
mechanisms were proposed: Increased membrane permeability to succinate or
conflrmatlonal changes that Increased the activity of the respiratory chain.
The effects of chlordane, heptachlor and heptachlor epoxide on beef
heart mitochondrial electron transport was studied by Pardlnl et al. (1971).
Chlordane and heptachlor Inhibited the succlnoxldase system to 21.2 and
5.8%, respectively, of control values. HeptachluV epoxide was not Inhibi-
tory. The activity of mitochondrial NADH-ox1dase was inhibited 5.8 and
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10.3%, respectively, of control values. When THPD, which when reduced by
NAOH non-enzymat1cally donates electrons to the electron transport chain at
the step between cytochrome b and cytochrome c, was added, electron trans-
port proceeded, despite the oxidase Inhibition. These results Indicated
that chlordane and heptachlor Inhibition of electron transport occurred "on
the substrate side of cytochrome c,- since the Inhibition of NAOH oxidase
was bypassed by the addition of TNPO.
Svnergl sre/Antaqon1 sni
Harbison (1975) pretreated neonatal Sprague-Oawley rats with Intraperi-
toneal Injections of phenobarbltal before Intraperitoneal injection of
chlordane or heptachlor. The LD^ of heptachlor 1n neonates not pre-
treated with phenobarbltal was 531 mg/kg bw. Phenobarbltal pretreatment
reduced the LD^ to 133 mg/kg bw. The chlordane LD^0 of 1121 mg/kg bw
was reduced to 539 mg/kg bw. Since phenobarbltal Is a known Inducer of
microsomal enzymes, the enhanced toxicity may have resulted from an
Increased capacity of the neonate to blotransform these Insecticides to more
toxic metabolites.
Sperling and Ewlnlke (1969) reported that pretreatment of adult male
rats with an oral dose of 1.8 g turpentine/kg bw/day for 3 days reduced the
oral IDgg of heptachlor from 112 to 70 mg/kg bw. The toxicity of hepta-
chlor epoxide was not affected.
Microsomes prepared from rats pretreated with chlordane intraperitoneal-
ly with 25 mg/kg bw/day for 3 days and challenged with an intraperitoneal
Injection of CCl^ (0.5 mi of 25% solution) had elevated levels of NAOPH-
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cytochrome c reductase when compared with non-pretreated controls, although
the Increase was not statistically significant (Stenger et al., 1975). The
cytochrome P-450 levels were significantly depressed by CC1 ^ challenge
from prechallenge values. Hepatocellular necrosis was extensive followed by
CC14 challenge 1n chlordane pretreated rats. Rats treated only with
CCl^ had less extensive liver damage. Thus, chlordane pretreatment poten-
tiated the toxicity of CCl^, presumably by Inducing microsomal enzymes
that metabolize CC14. That chlordane Induced the microsomal enzymes was
demonstrated by reduced zoxazolamlne paralysis time 1_n v1 vo and Increased
NADPH-cytochrome c reductase and cytochrome P-450 levels 1n microsomes from
non-challenged rats.
Boyd and Taylor (1969) maintained 133 male weanling Wlstar rats on a low
protein diet (3.5% casein) for 28 days. Another group of 122 rats was fed a
normal protein diet (26% casein), and a third group of 141 weanlings was fed
a commercial lab chow (25% protein from fish, milk, soybean and ground
oats). After the feeding period, the rats were fasted overnight and treated
with technical grade chlordane by gavage In a range of eight doses. The
IDjo of chlordane 1n the low-protein diet group was 137*30 mg/kg bw, In
the 26% casein group was 267+44 mg/kg bw and 1n the commercial diet group
was 311 rag/kg bw. Thus, a protein diet protected the rats from chlordane
toxicity.
The effects of protein and the quality of protein on heptachlor toxicity
1n weanling rats was studied 1n a series of experiments (Webb and Miranda.
1973; Miranda and Webb, 1973, 1974; Miranda et al., 1973; Weatherholtz et
al., 1969). When 10% protein diets were fed to weanling rats, heptachlor
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(administered Intraperitoneal1y) was more toxic when the protein was high
quality casein than when the low quality protein, gluten, was used. At 13%
protein, this effect was more pronounced (Webb and Miranda, 1973). Rats
receiving the gluten diet had reduced body weight, low microsomal protein
content and lower activities of heptachlor epoxldase than rats pair-fed the
casein diet. Rats maintained on low protein (5% casein) diets were 3 times
less susceptible to heptachlor toxicity than were rats pair-fed 20 or 40%
case1n-conta1n1ng diets (Weatherholtz et al., 1969). It was suggested that
at low protein, metabolism of heptachlor to heptachlor epoxide was Inhibit-
ed. This hypothesis was tested using an Inhibitor (SKF525-A) or an Inducer
(phenobarbltal) of microsomal enzymes (Miranda et al., 1973; Miranda and
Webb, 1974). These agents were administered to rats that were maintained on
casein or gluten diets before administration of heptachlor or heptachlor
epoxide. The results Indicated that SKF525-A, rather than protecting the
rats from heptachlor, resulted 1n higher mortality In the gluten-fed rats.
Phenobarbltal likewise Increased the toxicity of heptachlor, as expected, 1f
metabolism to heptachlor epoxide was enhanced. In the case1n-fed rats,
SKF525-A had little effect on heptachlor toxicity; phenobarbltal protected
the rats. In similar experiments with heptachlor epoxide, SKF525-A slightly
Increased the toxicity In gluten-fed rats and greatly Increased mortality In
casein-fed rats. Phenobarb!tal completely protected both casein- and
gluten-Fed rats.
Mechanlsn of Carcinogenicity
Maslansky and Williams (1981) found that chlordane and heptachlor were
negative for unscheduled ONA synthesis In rat, mouse and hamster primary
hepatocyte cultures (see Mutagenicity Section 1n Chapter V). They concluded
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that, since the Insecticides were not genotoxlc, the carcinogenicity
Involved an eplgenetlc mechanism, perhaps Involving a promotional effect on
liver cells already predisposed. They based this conclusion on observations
that chlordane and heptachlor Induce tumors only 1n the 11ver and only 1n
mice that readily develop spontaneous liver lesions. Conversely, Becker and
Sell (1979) demonstrated that chlordane Induced primary hepatocellular
carcinoma In a strain of mice (C57BL/6N) that historically and concurrently
did not have spontaneous liver tumors.
S unwary
Several studies on the mechanism of neurotoxicity of chlordane and
heptachlor were reviewed. Hyde and Falkenberg (1976) described EEG changes
In rats duHng subchronlc chlordane exposure. Deprivation of food caused
brain potentials Indicating lethality, perhaps by causing mobilization of
stored chlordane and/or metabolites. Changes 1n acetylcholine (St. Omer and
Ecoblchon, 1971) and ammonia and glutamate (St. Omer, 1971) levels 1n the
rat brain were followed with the time course of symptoms of heptachlor
toxicity 1n rats. Maximum acetylcholine levels occurred Just before the
onset of mild tonlc-clonlc seizures. Increased ammonia content coincided
temporally with mild seizures, while glutamate levels were unaffected by
heptachlor treatment.
Yamaguchl et al. (1979, 1980) found that heptachlor epoxide Inhibited
Ca2+ , Hg^+-ATPase 1n rat brain synaptic vesicles and Increased the level
of Ca2f 1n the presynaptic region; they suggested that this promoted the
release of neurotransmitter. A 4-fold greater concentration of heptachlor
epoxide was found In synaptosomes than In the whole brains of rats that
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developed violent convulsions after a high dose of heptachlor epoxide.
Folmar (1978) found that chlordane and heptachlor Inhibited rat brain micro-
somal Na*, K*-ATPase.
Shaln et al. (1977) concluded that 5<*-d1hydrotestosterone binding
Inhibition experiments were Inadequate to predict the effects of heptachlor
and chlordane on rat ventral prostate homeostasis. Welch et al. (1971)
found that chlordane exposure resulted In decreased fertility of mice.
Chlordane and heptachlor were found to enhance the Jin vitro metabolism of
estrogen and to suppress the uterotropic action of exogenous estrogen ]_n
vivo.
Enhanced gluconeogenesls was explained by an action of chlordane on the
sympathetic nervous system stimulating secretion of adrenalin and ACTHf
which In turn enhanced the production of glucocorticoid hormone (Karel and
Saxena, 1976). Alternately, Slnghal and Kacew (1976) proposed a direct
Interaction of chlordane, heptachlor and heptachlor epoxide with an adenyl
cyclase receptor on the cell membrane, resulting In cyclic AMP synthesis.
Cyclic AMP may react with an Intracellular receptor, producing a protein
kinase that 1s translocated to the nucleus where transcription Is triggered.
The newly synthesized messenger RNA would then Induce gluconeogenic enzymes.
Microsomal enzymes were also observed to be Induced by the Insecticides
(KlnosMta and Kempf, 1970).
Settlemlre et al. (1974) and Pardlnl et al. (1971) described the effects
of heptachlor, heptachlor epoxide and chlordane on cellular respiration.
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PhenobarMtal potentiated the toxicity of chlordane and heptachlor 1n
neonatal rats (Harbison, 1975). Turpentine potentiated the toxicity of
heptachlor, but not heptachlor epoxide In adult rats (Sperling and Ewlnlke,
1969). Chlordane potentiated the toxicity of CC1 ^ 1n rats (Stenger et
al., 1975). A high-protein diet protected rats from chlordane toxicity
(Boyd and Taylor, 1969). A low-protein and a poor-qual1ty-prote1n diet
protected rats from heptachlor toxicity (Webb and Miranda, 1973; Miranda and
Webb, 1974; Miranda et al., 1973; Weatherholtz et al., 1969), Experiments
on the effects of an Inhibitor and an Inducer of microsomal enzymes on the
toxicity of heptachlor In the protein diet groups were equivocal.
Haslansky and Williams (19B1) suggested that chlordane and heptachlor
exert their carcinogenic effects by an eplgenetlc mechanism.
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VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS
Introduction
The quantification of toxlcologlcal effects of a chemical consists of
separate assessments of noncarclnogenlc and carcinogenic health effects.
Chemicals that do not produce carcinogenic effects are believed to have a
threshold dose below which no adverse, noncarclnogenlc health effects occur,
while carcinogens are assumed to act without a threshold.
In the quantification of noncarclnogenlc effects, a Reference Dose
(RfO), [formerly termed the Acceptable Dally Intake (AOI)] 1s calculated.
The RfD Is an estimate {with uncertainty spanning perhaps an order magni-
tude) of a dally exposure to the human population (Including sensitive
subgroups) that Is likely to be without an appreciable risk of deleterious
health effects during a lifetime. The RfD 1s derived from a no-observed-
adverse-effect level (NOAEL), or lowest-observed-adverse-effect level
(LOAEL), Identified from a subchronlc or chronic study, and divided by an
uncertainty factor(s) times a modifying factor. The RfO 1s calculated as
follows:
Selection of the uncertainty factor to be employed In the calculation of
the RfD Is based upon professional Judgment, while considering the entire
data base of toxlcologlcal effects for the chemical. In order to ensure
that uncertainty factors are selected and applied In a consistent manner,
RfD *
(NOAEL or LOAEL)
a
mg/kg bw/day
[Uncertainty Factor(s) x Modifying Factor]
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the U.S. EPA (1966a) employs a modification to the guidelines proposed by
the National Academy of Sciences (NAS, 1977, 1960) as follows:
Standard Uncertainty Factors (UFs)
Use a 10-fold factor when extrapolating from	valid experimental
results from studies using prolonged exposure	to average healthy
humans. This factor Is Intended to account	for the variation
In sensitivity among the members of the human	population. [10H]
Use an additional 10-fold factor when extrapolating from valid
results of long-term studies on experimental animals when
results of studies of human exposure are not available or are
Inadequate. This factor Is Intended to account for the uncer-
tainty 1n extrapolating animal data to the case of humans.
[10A]
Use an additional 10-fold factor when extrapolating from less
than chronic results on experimental animals when there is no
useful long-term human data. This factor Is Intended to
account for the uncertainty In extrapolating from less than
chronic NOAELs to chronic NOAELs. [10S]
Use an additional 10-fold factor when deriving an RfD from a
LOAEL instead of a NOAEL. This factor Is Intended to account
for the uncertainty In extrapolating from LOAELs to NOAELs.
[10L]
Modifying Factor (HF)
Use professional Judgment to determine another uncertainty
factor (MF) that 1s greater than zero and less than or equal to
10. The magnitude of the MF depends upon the professional
assessment of scientific uncertainties of the study and data
base not explicitly treated above, e.g., the completeness of
the overall data base and the number of species tested. The
default value for the MF is 1.
The uncertainty factor used for a specific risk assessment Is based
principally upon scientific Judgment rather than scientific fact and
accounts for possible Intra- and interspecies differences. Additional
considerations not Incorporated In the NAS/ODW guidelines for selection of
an uncertainty factor Include the use of a less than lifetime study for
deriving an RfD, the significance of the adverse health effects and the
counterbalancing of beneficial effects.
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From the RfO, a- Drinking Water Equivalent Level (OWEL) can be calcu-
lated. The OWEL represents a medium specific (I.e., drinking water)
lifetime exposure at which adverse, noncarclnogenlc health effects are not
anticipated to occur. The OWEL assumes 100% exposure from drinking water.
The DWEL provides the noncarclnogenlc health effects basis for establishing
a drinking water standard. For Ingestion data, the OWEL Is derived as
follows:
OWEL = (RfDI x (Body weight In kg) =
Drinking Water Volume In i/day
where:
Body weight - assumed to be 70 kg for an adult
Drinking water volume = assumed to be 2 i/day for an adult
In addition to the RfO and the OWEL, Health Advisories (HAs) for expo-
sures of shorter duration (1-day, 10-day and longer-term) are determined.
The HA values are used as Informal guidance to municipalities and other
organizations when emergency spills or contamination situations occur. The
HAs are calculated using an equation similar to the RfO and OWEL; however,
the NOAELs or LOAELs are Identified from acute or subchronlc studies. The
HAs are derived as follows:
HA . (NOAEL or LOAEL) x (bw) =
(UF) x (	 i/day) 	
Using the above equation, the following drinking water HAs are developed
for noncarclnogenlc effects:
1.	1-day HA for a 10 kg child Ingesting 1 t water per day.
2.	10-day HA for a 10 kg child Ingesting 1 water per day.
3.	Longer-term HA for a 10 kg child Ingesting 1 I water per day.
4.	Longer-term HA for a 70 kg adult Ingesting 2 I water per day.
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The 1-day HA ^calculated for a 10 kg child assumes a single acute
exposure to the chemical and 1s generally derived from a study of <7 days
duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and
Is generally derived from a study of <30 days duration. The longer-term HA
is derived for both the 10 kg child and a 70 kg adult and assumes an
exposure period of -7 years (or 10% of an Individual's lifetime). The
longer-term HA Is generally derived from a study of subchronlc duration
(exposure for 10% of animal's lifetime).
The U.S. EPA categorizes the carcinogenic potential of a chemical, based
on the overall we1ght-of-ev1dence, according to the following scheme:
Group A: Human Carcinogen. Sufficient evidence exists from
epidemiology studies to support a causal association between
exposure to the chemical and human cancer.
Group B: Probable Human Carcinogen. Sufficient evidence of
carcinogenicity In animals with limited (Group B1) or Inade-
quate (Group B2) evidence 1n humans.
Group C: Possible Human Carcinogen. Limited evidence of
carcinogenicity In animals in the absence of human data.
Group 0: Not Classified as to Human Carcinogenicity. Inade-
quate human and animal evidence of carcinogenicity or for which
no data are available.
Group E: Evidence of Noncarclnoqenlcltv for Humans. No
evidence of carcinogenicity In at least two adequate animal
tests In different species or In both adequate epidemiologic
and animal studies.
If toxlcologlcal evidence leads to the classification of the contaminant
as a known, probable or possible human carcinogen, mathematical models are
used to calculate the estimated excess cancer risk associated with the
Ingestion of the contaminant In drinking water. The data used In these
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estimates usually come from lifetime exposure studies using animals. In
order to predict the risk for humans from animal data, animal doses must be
converted to equivalent human doses. This conversion Includes correction
for noncontlnuous exposure, less than lifetime studies and for differences
1n sl2e. The factor that compensates for the size difference Is the cube
root of the ratio of the animal and human body weights. It Is assumed that
the average adult human body weight Is 70 kg and that the average water
consumption of an adult human Is 2 l of water per day.
For contaminants with a carcinogenic potential, chemical levels are
correlated with a carcinogenic risk estimate by employing a cancer potency
(unit risk) value together with the assumption for lifetime exposure from
Ingestion of water. The cancer unit risk Is usually derived from a linear-
ized multistage model with a 95% upper confidence limit providing a low dose
estimate; that 1s, the true risk to humans, while not Identifiable, Is not
likely to exceed the upper limit estimate and. In fact, may be lower.
Excess cancer risk estimates may also be calculated using other models such
as the one-hit, Welbull, loglt and problt. There 1s little basis in the
current understanding of the biological mechanisms Involved In cancer to
suggest that any one of these models Is able to predict risk more accurately
than any other. Because each model 1s based upon differing assumptions, the
estimates derived for each model can differ by several orders of magnitude.
The scientific data base used to calculate and support the setting of
cancer risk rate levels has an Inherent uncertainty that Is due to the
systematic and random errors In scientific measurement. In most cases, only
studies using experimental animals have been performed. Thus, there Is
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uncertainty when the data are extrapolated to humans. When developing
cancer risk rate levels, several other areas of uncertainty exist, such as
the Incomplete knowledge concerning the health effects of contaminants In
drinking water, the Impact of the experimental animal's age, sex and
species, the nature of the target organ system(s) examined and the actual
rate of exposure of the Internal targets In experimental animals or humans.
Dose-response data usually are available only for high levels of exposure
and not for the lower levels of exposure closer to where a standard may be
set. When there Is exposure to more than one contaminant, additional
uncertainty results from a lack of Information about possible synergistic or
antagonistic effects.
Chlordane
Noncarclnoqenlc Effects. There are several reports of accidental or
intentional acute oral exposure of humans to chlordane for which a dose or
an estimation of a dose were obtained. In one case, a 32-year-old woman who
had Ingested 104 mg chlordane/kg body weight died 10 days after exposure
(Derbes et al., 1955). Another case of human exposure Involved an 18-year-
old woman who had Ingested 32 mg chlordane/kg body weight (Dadey and Kammer,
1953). It was estimated that she retained 10 mg/kg bw after vomiting. She
recovered spontaneously. Lensky and Evans (1952) reported a case of a
15-month-old girl, weighing 9 kg, who drank a small amount of a chlordane
suspension. The Investigators believed that the amount was <100 mg of
chlordane -(dose <11.1 mg/kg bw). The child developed tremors, ataxia,
convulsions, rlsus sardonlcus, ophlsthotonos and other CNS disturbances.
Among the symptoms of a 4-year-old girl weighing 11 kg who absorbed an
estimated dose of >0.15 mg chlordane/kg bw were clonic convulsions, loss of
01830
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coordination, hyporeflexla, excitability and sinus tachycardia (Aldrich and
Holmes, 1969). In a similar case, a 20-month-old boy weighing 12.7 kg
swallowed 74% technical grade chlordane (Curley and Garrettson, 1969). His
symptoms Included vomiting, convulsions and seizures. The Investigators did
not calculate a dose. Some of the signs of toxicity observed may have been
associated with the petroleum distillate used In the formulation of liquid
commercial products of chlordane.
Much of the information on single oral exposures of laboratory animals
to chlordane Is concerned with lethality. For rats, reported acute oral
lethal dose range from 83-430 mg/kg bw depending on the vehicle and whether
technical or pure chlordane was administered (Podowskl et al., 1979; Ambrose
et al.. 1953a,b; Gaines, I960; Boyd and Taylor, 1969). Gak et al. (1976)
reported oral LD^s of 390 mg/kg bw for mice and 1720 mg/kg bw for golden
hamsters. Hale and female neonatal Sprague-Oawley rats were less sensitive
to Intraperitoneal administration of chlordane (i.p.	= 1121 mg/kg bw)
than were adult males (1.p. LD^ • 343 mg/kg bw) (Harbison, 1975). Signs
of acute chlordane poisoning Included CNS disturbances such as tremors,
convulsions, Irritability, hyporeflexla, dyspnea, and ataxia, among others
(Lehman, 1951; Gaines, 1960; Boyd and Taylor, 1969; Stohlman et al., 1950;
Arnold et al,f 1977), Histological examination of fatally poisoned rats
revealed nephritis, hepatitis and vascular congestion (Boyd and Taylor,
1969).
However, sublethal doses have also been tested. Ambrose et al.
{1953a,b) observed slight histologic changes in the liver {Intracytoplasmlc
bodies at all dose levels) at doses of 6,25»2!s( mg/kg bw/day by gavage.
01830
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These doses are considered adverse-effect levels (AEl). The next higher
dose, 50 mg/kg bw/day resulted In death of 2 of 5 animals. This dose
represents a frank-effect level (FEL). At exposures of 10-50 mg/kg diet for
14 days In groups of six male Wlstar rats, statistically significant
microsomal en2yme Induction (aniline hydroxylase, amlnopyrlne demethylase
and hexabarbltal oxidase) was observed when compared with controls (Den
Tonkelaar and Van Esch, 1974). This exposure corresponds to doses of
1.0-5.0 mg/kg bw/day assuming that a young rat consumes food at a level of
10% of its body weight per day. Based on the effects observed these doses
might represent NOAELs. These Investigators did not look for other effects.
The NOEL for enzyme Induction was 5 mg/kg diet (0.05 mg/kg bw/day) for
H days.
Subchronlc dietary exposure of mice and rats to technical grade chlor-
dane for 42 days resulted In Increased mortality at 160 and 800 mg/kg diet,
respectively (NCI, 1977a). These doses represent FELs. Histopathologic
examinations of the animals at tnese dietary levels or below were not
performed. Subchronlc feeding of male rats with chlordane for 90 days at a
dose of 19.5 mg/kg bw/day resulted In decreased mean weekly body weight
gains compared with controls and changes In ventral prostate homeostasis
(Increased androgen receptor site content, decreased prostate protein. RNA
and 0NA contents) (Shaln et al., 1977). This latter dose can be considered
an AEL.
Chronic dietary exposure of male and female rats to chlordane at 0-1280
mg/kg diet for 400 days resulted in a variety of adverse changes. Increased
mortality was observed 1n the two higher lose levels. Dietary levels at or
01830
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03/27/87

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above 80 mg/kg diet In both sexes yielded histopathologic liver changes
(vacuolization and enlarged nuclei) that were dose-related. Liver weight
was increased In female rats of the 10 mg/kg diet group (Ambrose et a1.,
1953a.b).
In studies designed to assess the carcinogenicity of chlordane In mice
(IROC, 1973a; NCI, 1977a). such effects as increased mean liver weight,
decreased mean body weight and Increased mortality were observed among
treated groups at dietary levels ranging from 25-63.8 mg/kg diet. In
addition, the Incidences of hepatocellular carcinoma were significantly
Increased above controls In these studies. At 5 mg/kg diet In the IROC
(1973a) study, the only observed effects were Increased mean liver weights
and hepatocytomegaly In females.
In a study by Yonemura et al. (1983) F-344 rats (80/sex/group) were fed
technical chlordane at dietary levels of 0, 1, 5 or 25 ppm for 130 weeks.
Dally dose levels of 0.045, 0.229 and 1.175 mg/kg for the 1, 5 and 25 ppm
treatment groups, respectively, were calculated from food consumption and
body weight data. No effects were observed for hematology, clinical
chemistry and urinalysis endpolnts, and no treatment-related effects were
reported for body weight and mortality. Hepatocellular necrosis was
observed 1n 3, 13, 11 and 27 males (64/group) in the 0, 1. 5 and 25 ppm
groups, respectively. The Increased Incidence was statistically significant
for all treatment groups. Liver adenomas were found In the high-dose males.
The only significant effect In females was hepatocellular swelling In the 25
ppm group.
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Increased llver-Co-body weight ratios were reported for male and female
nice fed chlordane for 2 years at 0.76 ppm (0.09 mg/kg/day), the lowest dose
administered (Inu1 et a 1 ., 1983). liver necrosis was observed at 0.43 and
1.1 mg/kg/day for males only.
Chlordane was not carcinogenic to Osborne-Mendel rats In the NCI (1977a)
bloassay; however, tremors, clinical signs of toxicity, reduced mean body
weight and Increased mortality were observed 1n various male and female
groups treated at 120.8-407.0 mg/kg diet for 80 weeks when compared with
controls. Ingle (1952) described the dose-related effects of dietary levels
of 5-300 mg/kg diet of chlordane 1n male and female Osborne-Mendel rats
treated for 2 years. At 300 mg/kg diet, severe hlstopathologlcal damage to
liver, kidney, heart, adrenals, lung and spleen; Increased mortality;
tremors; and hemorrhaging were observed. These effects were less pronounced
at 150 mg/kg diet. At 30 mg/kg diet, slight liver damage was observed. At
10 mg/kg diet, minor liver damage consisting of an occasional hypertrophic
cell and minor bile duct proliferation was the only effect. No effects on
food consumption, growth rate, mortality, no signs of toxicity and no
histopathologic changes were observed at 5 mg/kg diet. All of the above
doses, with the exception of the last one, represent AELs. The last dose
represents a N0AEL.
An additional report by Vettorazzl (1975) describes a review by a
WHO/FAG scientific panel of an unpublished study In dogs performed by
Wazeter (1967). In this study, the dogs were exposed to diets containing
chlordane at levels of 0-30 mg/kg diet for 2 years. The review panel deter-
mined that an exposure of 3 mg/kg diet was a NOEL and reported that exposure
01830
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to 15 or 30 mg/kg diet resulted In Increased liver weight and histologic
changes In the liver. These latter doses are considered AELs. Assuming
that a dog consumes 2.5% of the body weight In food per day the dietary
level that Is a NOEL 1s equivalent to a dose of 0.075 mg/kg bw/day.
Ingle (1952) found no teratogenic or fetotoxlc effects In neonatal rats
following jn utero exposure to technical grade chlordane from darns that had
been mated In the study discussed above. He did observe toxic symptoms In
pups nursed by dams exposed to 150 and 300 mg/kg dietary levels. Toxic
symptoms also developed In pups not exposed .In utero. but nursed by dams
treated at these levels. Fertility was reduced to -50% of control mice In
female Swiss Webster mice treated Intraperitoneally with three weekly doses
of 25 mg/kg bw technical chlordane (Welch et al., 1971).
Quantification of Noncarc1noqen1c Effects.
Derivation of l-0av and 10-Qav HAs — Satisfactory dose-response data
are not available from which a HA can be derived for 1 and 10 days. How-
ever, In order to have some guidelines available, a 10-day HA for chlordane
may be derived based upon the Ambrose et al. study (1953a,b) In rats. The
toxic effects resulting from dally gastric Intubation of doses 6.25, 12.5,
25.0, 50.0, 100.0 or 200 mg/kg chlordane 1n rats for 15 days were histo-
logical changes In the liver of the treated animals (at all dose levels) and
central nervous system effects at higher dose levels. The minimal hlsto-
pathologlcal changes such as presence of abnormal Intracytoplasmlc bodies of
various diameters were evident at dose levels of 6.25 mg/kg. Therefore,
this dose level may be used In the development of a 10-day HA and a safety
01830
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03/12/87

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where:
factor of 1000 may be applied In the calculation since data are for the
animals rather than human. Accepting 6.25 mg/kg as the minimal effect dose,
the HA Is derived as follows:
1-Day HA * Insufficient data
The 10-day HA for a child can be calculated as follows:
10-day HA = (6-25 mq/kq/day) (10 kq)
(1000) (1 t/day)	^
- 0.063 mg/L
6.25 mg/kg/day « minimal effect level
10 kg - body weight for a child
1000 = uncertainty factor -- Intra- and Interspecies variation
and L0AEL
1 i/day ¦ water consumption per day by a child
It should be noted that this HA for short-term exposure is also appli-
cable for a 1-day HA In view of the metabolism studies of Barnett and
Dorough (1974) and Tashlro and Natsumura (1977).
Derivation of longer-term HA « There are Insufficient toxlcologlcal
data available to calculate a longer-term HA for chlordane.
The National Research Council Report (NRC, 1982), "An Assessment of the
Health Risks of Seven Pesticides Used for Termite Control" was considered
for the derivation of a longer-term HA for chlordane. However, the review
of the limited human studies with long-term exposure did not reveal any
consistent or significant detrimental effect that-mlght be considered for HA
level for chlordane. Details of these human studies are given below.
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Prlncl and Spurbeck (1951) evaluated 34 persons engaged 1n the manufac-
ture of Insecticides, Including chlordane (exposed through skin contact and
Inhalation for 11-36 months). Physical examinations, chest X-rayst urinary
dilution and concentration tests, routine urinalysis, hemoglobin measure-
ments, sedimentation rate, and urinary porphyrin determinations failed to
suggest any abnormalities tn the men. The authors concluded that no adverse
effects were detected 1n men working in a plant with air concentrations of
chlorinated hydrocarbons as high as 10 mg/m". Authors did not specify
that exposure was exclusive to chlordane and, therefore, this study was
considered Inappropriate for a longer-term HA for chlordane.
Alvarez and Hyman (1953) reported a clinical and laboratory study of 24
men 21-49 years old who were exposed to chlordane for 2 months to 5 years
while working In a plant where 1t was manufactured. Each man was given a
complete examination, Including blood chemistry and urine studies. None of
the 24 men had evidence of abnormalities In liver, kidneys, skin, nervous
system and blood-forming organs. However, the authors had observed in seven
men slight Mbrotlc changes 1n the apices of the lungs; one person with a
diabetic condition and two more with hypertension 1n chlordane-exposed
workers. These observations (even though not attributed to chlordane) and
limited numbers of subjects In this study did not Justify Its consideration
for a longer-term HA level.
Assessment of Lifetime Exposure and Derivation of a DWEL. Chlordane
1s classified In Group 02, probable human carcinogen, according to EPA's
welght-of-evldence scheme for the classification of carcinogenic potential.
01830
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The study by Wazeter [(1967), as reported by Vettorazzl, 1975] was
considered the most appropriate to derive the DWEL. However, the results of
the recent chronic rat dietary study by Yonemura et al. (1983) are available
for the derivation of the DWEL. In this study, F344 rats were fed technical
chlordane at dietary levels of 0, 1, 5 or 25 ppm for 130 weeks. Clinical
laboratory studies were performed and organ weights were measured on eight
animals/sex/group at weeks 26 and 52, and on all survivors at week 130.
Gross and microscopic pathology were performed on all tissues. Dally dose
levels of 0.045, 0.229 and 1.175 mg/kg for the 1, 5 and 25 ppm treatment
groups, respectively, were calculated from food consumption and body weight
data. No effects were observed for hematology, clinical chemistry and
urinalysis endpolnts, and no treatment-related effects were reported for
body weight and mortality. Hepatocellular necrosis was observed 1n 3, 13,
11 and 27 males (64/group) In the 0, 1, 5 and 25 ppm groups, respectively.
The Increased Incidence was statistically significant for all treatment
groups. Liver adenomas were found In the high-dose males. The only
significant effect In females was hepatocellular swelling 1n the 25 ppm
group. The L0AEL of 1 ppm diet (0.045 mg/kg/day) was Identified based on
liver necrosis In male rats. Using this L0AEL, the DWEL Is calculated as
follows:
Step 1 - RfD Derivation
RfD = (0-0*5 mq/kq/day) a 0 000045 mg/kg/day - 0.00005 mg/kg/day
(100) (10)
where:
RfO	- Reference Oose: estimate of dally exposure to
the human population that appears to be without
appreciable risk of deleterious noncarclnogenlc
effects over a lifetime of exposure
01830
VIII-14
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0.045 mg/kg/day - LOAEL
100	* uncertainty factor appropriate for use with
data from animal studies
10	« uncertainty factor for use of LOAEL Instead
Of NOAEL
Step 2 - QUEL Derivation
DUEL = (0.00005 mq/kq/day) (70 kg) ^ 0>0oi7 mg/l - 0.002 mg/t - 2 vg/t
(2 l/day)	^	y
where:
70 kg * assumed weight of protected Individual (adult)
2 l/day * assumed volume of water Ingested per day by 70 kg adult
A summary of the data used to calculate the recommended 1-day and 10-day
HAs and the DUEL for chlordane Is provided In Table V1II-1.
Carcinogenic Effects.
Human Studies —
Case Reports. There were 11 case reports Involving CNS effects
where the author looked at the toxic effects of chlordane/heptachlorr eight
case studies Involving blood dyscraslas, and five case studies of neuroblas-
tomas 1n children with pre-/postnatal exposure to chlordane or heptachlor.
The blood dyscraslas in children Included four cases of aplastic anemia and
one case each of refractory megaloblastic anemia, acute lymphoblastic
leukemia, acute stem-cell leukemia, and acute myelomonocytlc leukemia.
Epidemiologic Studies. Three epidemiologic studies of workers
exposed to chlordane and/or heptachlor have been reported. One of these
studies, conducted 1n chlordane/heptachlor applicators, was considered
Inadequate 1n sample size and In duration. However, this study showed
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MBit VII l-l
Siaury of Data Used lo Derive HAs and OUfls for Chlordane
Value of HA or DUEL
Criteria	Anlaal	Duration	fffect	Reference
Dose	Child	Adult
1-day HA
10-day HA
longer-tera HA
QUEL
Increased llfetlac
cancer risk level
for a 70 kg ^<|u^
rat. b.?S
ag/kg/day
rats
IS days
130 weeks
Intracycloplasalc
bodies of various
dlaaeters within
hepatocytes
10AH for liver
necrosis In aale
rats
Hepatic carclnoaas
Insufficient data
0.063 ag/t
Insufficient data
10"*
2.1 Wg/|
10"»
0.27 Mg/t
0.00? ag/t*
10"*
0.0?7 Mg/t
Aabrose et al.,
1953a.b
Voneaura
el al.. 19B3
U.S. EPA. 1980b
'Assualng 100X contribution being froa water

-------
Inadequate In sample size and 1n duration. However, this study showed
Increased mortality from bladder cancer (SMR-277, p<0.05). A second study
showed an Increased mortality from lung cancer (SHR-134), but the Increase
was not statistically significant. The mortality from cerebrovascular

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Animal Studies
Chlordane. Four chlordane carcinogenesis bloassays In mice have
been reported. The strains tested Include C57B1/6N, CQ-1, B6C3F1 and ICR.
In C57B1/6N mice fed 25 or 50 ppm for 18 months, hepatocellular carcinomas
were observed In 27% (16) of the survivors. This mouse strain rarely
develops spontaneous liver lesions. For C0-1 mice fed 5, 25 or 50 ppm for
18 months, liver nodules/hepatocellular carcinomas were observed In the 25
and 50 ppm groups. In B6C3F1 mice fed -*30 and 60 ppm for 80 weeks and then
held for 10 weeks, hepatocellular carcinomas were observed In both males and
females. For ICR mice fed 1, 5 or 12.5 ppm for 24 months, hepatocellular
adenomas and hemangiomas were significantly Increased (p<0.001) In males
receiving 12.5 ppm and nonneoplastic liver lesions were present In males fed
5 ppm and 1n females fed 5 or 12.5 ppm.
Four chlordane carcinogenesis bloassays 1n rats have been reported. The
strains tested Include albino, Osborne-Hendel and Fischer 344. Three of
these studies were considered adequate and one was Inadequate. In albino
rats fed 10, 20, 40, 80, 160, 320, 640 or 1280 ppm for 400 days, there were
no treatment-related tumors. In Osborne-Hendel rats fed 5, 10, 30, 150 or
300 ppm for 2 years, hepatic toxicity was noted at 150 and 300 ppm, but no
liver tumors were noted. In Osborne-Hendel rats fed 203.5 or 407 ppm
(males) and 120.8 or 241.5 ppm (females), respectively, for 80 weeks and
held for an additional 29 weeks, no liver tumors were notedv but thyroid
tumors were significantly Increased. In light of historical data for
Osborne-Hendel rats, the thyroid tumors were not considered to be treatment-
related. In Fischer 344 rats fed 1, 5 or 25 ppm for 130 weeks, there was a
statistically significant Increase In hepatocellular adenomas, which was
considered by the authors as weak evidence for carcinogenicity In males fed
01830	VIII-18	03/27/87

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25 ppm. Hepatocellular swelling was significant In females fed 25 ppm. The
hepatocellular adenomas occurred only In males surviving longer than 104
weeks.
Supporting Evidence —
Mutagenicity. The published literature on mutagenicity testing of
chlordane and heptachlor/heptachlor epoxide Is quite similar. Indeed most
studies report results on both chemicals. Generally, the results have
Indicated that these chemicals are not mutagenic In bacteria or In mammalian
cells In culture and do not Induce DNA repair as measured by unscheduled DNA
synthesis In rodent hepatocytes. While dominant lethal tests In mice have
been negative for both chemicals, the absence of direct cytogenetic tests In
both germinal and somatic cells precludes a conclusion of their potential
for causing- chromosome aberrations.
Structural Relationship. Three compounds, structurally related to
chlordane/heptachlor/heptachlor epoxide, have Induced malignant liver tumors
In animals. Aldrln, dleldrln and chlorendlc acid have produced liver tumors
In mice and chlorendlc add has also produced liver tumors 1n rats.
Carcinogenicity Classification. Based on the accumulated evidence,
chlordane 1s a probable human carcinogen, classified In Group B2 under the
EPA's guidelines for carcinogen risk assessment (U.S. EPA, 1986b). Animal
studies provide sufficient evidence for carcinogenicity: chlordane
Increased the Incidence of liver carcinomas in C57B1/6N, CD-I and B6C3F1
mice; liver adenomas and hemangiomas 1n ICR mice; and liver adenomas In
Fischer 344 rats. Epidemiologic studies provide-Inadequate evidence due to
methodology and data limitations.
01830	VI11-19	03/27/87

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According to the. criteria In the guidelines, the above evidence puts
chlordane 1n Group B2. However, the guidelines allow for the possibility of
downgrading the classification from Group B2 to Group C when the only tumor
response 1s that of mouse liver tumors In strains with high background
rates, or when warranted by a number of other factors. In the case of
chlordane these conditions do not apply, since chlordane caused tumors In
C57B1/6N mice, which do not have a high background rate, and caused tumors
In rats as well. Other pertinent evidence Includes highly significant tumor
responses, up to 11% Increased Incidence over controls. Increased Incidence
In both males and females. Increased Incidence at medium and high doses, a
dose-related Increase 1n the proportion of malignant tumors, and Induction
of tumors by structurally related chemicals. In light of these factors,
downgrading Is clearly not warranted, and chlordane remains In Group B2.
Quantification of Carcinogenic Effects. For some chemicals, several
studies in different animal species, strains and sexes at several doses and
different routes of exposure may be available. A choice must be made as to
which data sets should be used to quantify human risk by low-dose extrapola-
tion. The following procedure was used to make this choice. The animal
studies are evaluated qualitatively to assure that only properly conducted
studies are used. The tumor Incidence data were separated according to
organ sites and tumor types. The data sets used 1n the model are the ones
In which the tumor Incidence Is statistically significantly higher in at
least one test dose level as compared with controls and/or where the tumor
incidence rate shows a significant trend with respect to dose level. Both
biological and statistical considerations have been used to select the most
appropriate data sets.
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Because humans may be as sensitive as the most sensitive animal species,
potency estimates obtained from the most sensitive species tested can be
averaged to estimate potency for the general population. Because some sub-
populations may be more sensitive than the general population, the potency
estimate from the most sensitive sex and strain tested Is also presented.
This approach Is consistent with EPA1s guidelines for carcinogen risk
assessment (U.S. EPA, 1906b).
As previously described, four data sets showed a significant Increase In
hepatocellular carcinomas In treatment groups compared with controls In
mice. These are male and female mice In the IROC study, and male and female
mice 1n the NCI study. Tables VI11-2 through VI11-6 give tumor Incidence
data for these studies.
Estimates of carcinogenic potency can be obtained by fitting the
linearized multistage model to each data set. Table VIII-7 summarizes five
potency estimates obtained 1n this way.
five data sets Involve chlordane: male and female CD-I mice, male and
female B6C3F1 mice, and male F344 rats. The most sensitive sex and strain
tested Is male CD-I mice. From these, the potency 1s estimated at 4.7 per
mg/kg/day.
The most sensitive species tested 1s mice. There are four potency
estimates, ranging from 4.7 down to 0.25 per mg/kg/day, with a geometric
mean of 1.3 per mg/kg/day. This geometric mean from mice 1s consistent with
01830
VIII-21
03/12/87

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TABLE VIII-2
Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas In Female Mice*

Compound:
Analytical grade chlordane

Species, strain, sex:
Mouse, C0-1, female

Body weight:
0.030 kg (assumed)

Length of experiment:
19.5 months

Length of exposure:
18 months

Tumor site and type:
Liver, carcinoma

Route, vehicle:
Oral, diet

Human potency (q-j*):
2.98 per mg/kg/day
Experimental
Average
Equivalent Tumor Incidence
Animal Dose
Animal Dose
Human Dose No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day) No. Examined
0
0
0 0/45
5
0.65
0.052 0/61
25
3.25
0.260 32/50
SO
6.50
0.520 26/37
•Source: IRDC, 1973a
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VIII-22
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TABLE VIII-3
Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas In Male Mice*

Compound:
Analytical grade chlordane

Species, strain, sex:
House, CD-I, male

Body weight:
0.030 kg (assumed)

Length of experiment:
19.5 months

Length of exposure:
18 months

Tumor site and type:
Liver, carcinoma

Route, vehicle:
Oral, diet
•
Human potency (q-j *):
4.74 per mg/kg/day
Experimental
Average
Eaulvalent Tumor Incidence
Animal Dose
Animal Dose
Human Oose No. Responding/
(ppffl)
(mg/kg/day)
(mg/kg/day) No. Examined
0
0
0 3/33
5
0.65
0.052 5/55
25
3.25
0.260 41/52
50
6.50
0.520 32/39
•Source: IRDC, 1973a
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VIII-23
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TABLE VIII—4
Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas 1n Male Mice*
Compound:
Species, strain, sex
Body weight:
Length of experiment
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency (q-j * >:
Technical grade chlordane
Mouse, B6C3F1, male
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
0.76 per mg/kg/day
Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Oose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0
0
2/18
29.9
3.9
0.31
16/48
56.2
7.3
0.58
43/49
•Source: NCI, 1977a
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TABLE VII1-5
Cancer Data Sheet for Derivation of Potency of Chlordane
from Hepatocellular Carcinomas In Female Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency (q-)*):
Experimental
Animal Dose
(ppm)
Average
Animal Dose
(mg/kg/day)
Equivalent
Human Dose
(mg/kg/day)
Tumor Incidence
No. Responding/
No. Examined
0
0
0
0/19
30.1
3.9
0.31
3/47
63.8
8.3
0.66
34/49
•Source: NCI, 1977a
Technical grade chlordane
Houset B6C3F1, female
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
0.25 per mg/kg/day
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03/12/87

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TABLE VIII-6
Cancer Data Sheet for Derivation of Potency of Chlordane
from Liver Adenomas and Carcinomas 1n Male Rats*

Compound:
Technical grade chlordane

Species, strain, sex:
Rat, F344, male


Body weight:
0.35 kg


Length of experiment:
130 weeks


Length of exposure:
130 weeks


Tumor site and type:
Liver, adenoma and
carcinoma

Route, vehicle:
Oral, diet


Human potency (qi*):
1.11 per mg/kg/day

Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Oose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0
0
1/64
1
0.05
0.17
1/64
5
0.25
0.85
4/64
25
1.25
4.25
9/64
~Source: RIASBT, 1983b
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TABLE VIII-7
Human Potency Estimates by Chemical
Chemical
Sex,
Species,
Strain
Tumor Site,
Type
Potency
(mg/kg/day)"1
Reference
Chlordane
male mice
CD-I
liver,
carcinoma
4.74
IRDC, 1973a
Chlordane
female mice
CD-I
11ver,
carcinoma
2.98
IR0C, 1973a
Chlordane
male mice
B6C3F1
11ver,
carcinoma
0.76
NCI, 1977a
Chlordane
female mice
86C3F1
11ver,
carcinoma
0.25
NCI, 1977a
Chlordane
male rats
F344
liver,
adenoma and
carcinoma
1.11
RIASBT, 1983b
Heptachlor
male mice
C3H
11ver,
carcinoma
12.4
Davis, 1965/
Reuber
Heptachlor
female mice
C3H
liver,
carcinoma
14.9
Oavls. 1965/
Reuber
Heptachlor
male mice
B6C3F1
11ver,
carcinoma
2.79
NCI, 1977b
Heptachlor
female mice
B6C3F1
11ver,
carcinoma
0.83
NCI, 1977b
Heptachlor
epoxide
male mice
C3H
liver,
carcinoma
27.7
Davis, 1965/
Reuber
Heptachlor
epoxide
female mice
C3H
11ver,
carcinoma
36.2
Davis, 1965/
Reuber
Heptachlor
epoxide
female mice
CD-I
liver,
carcinoma
1.04
I ROC» 1973b/
Reuber
Heptachlor
epoxide
male mice
CD-I
11ver,
carcinoma
6.48
IRDC, 1973b/
Reuber
Heptachlor
epoxide
female rats
CFN
liver,
carcinoma
6.76
Wltherup et al.,
1959/Reuber
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the potency estimate from rats of 1.1 per rog/kg/day. Because humans may be
as sensitive as the most sensitive animal species, the potency for the
general population 1s estimated at 1.3 per rag/kg/day. These estimates
supersede the potency of 1.61 per mg/kg/day previously calculated by the
U.S. EPA (1980b).
The upper-limit unit risk estimates from the animal data are derived
from a linearized multistage nonthreshold extrapolation model which is
currently programmed as GLOBAL 83. Justification for Us use 1s presented
In EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986b). While
recognizing that alternative statistical modelling approaches exist [e.g..
One-hit, Welbull, Log-Problt and Loglt models* and maximum likelihood
estimates], the range of risks described by using any of these modeling
approaches has little biological significance unless data can be used to
support the selection of one model over another. In the Interest of
approach consistency and of providing an upper bound estimate for the poten-
tial cancer risk, the Agency recommends the use of the linearized multistage
model. EPA considers this model and resulting risk estimate to be an upper
limit value In the sense that the true risk Is unlikely to be higher and may
be lower even zero. An established procedure does not yet exist for making
"most likely" or "best" estimates of risk within the range of uncertainty
derived by the upper and lower limit values.
The unit risk In water, which Is the potency expressed 1n terms of
wg/l drinking water concentrations. Is used to estimate risk when
exposures are expressed as water concentrations. The unit risk 1n water Is
the Increased cancer risk to a person who throughout life drinks water
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contaminated with 1" pg/i of a pollutant. With a linear dose-response
curve, risks at any concentration can be computed by multiplying the unit
risk by the concentration. The unit risk 1s computed by converting a 1
ug/l concentration to a mg/kg/day dose and then multiplying by the
potency. For a 70 kg person who drinks 2 l a day, a water concentration
of 1 yg/l Is equivalent to a dose of:
(1 vg/t) |10~3 mg/ng) (2 l/day)/{70 kg) * 2.9x10** mg/kg/day
Multiplying this dose by the potencies calculated above gives unit risks of
3.7x10"* per vq/i for the general population. The unit risk in water
Is 1.3xl0~4 per Mg/l for the general population and 4.3xl0"4 per
ug/l for sensitive subpopulatlons.
The concentration In water corresponding to an Increased lifetime risk
level of 10"4» 10"5 and 10"4 for a 70 kg human who consumes 2 i/day,
1s calculated to be 2.7t 0.27 and 0.027 yg/l, respectively.
Heptachlor and H«ptach1or Epoxide
Noncardnoqenlc Effects. Unlike the case of technical chlordane,
reports of effects 1n humans swallowing quantifiable amounts of technical
heptachlor were not located 1n the available literature. As In the case of
chlordane, exposure levels In reports of blood dyscrasla associated with
Inhalation and dermal exposure of humans to heptachlor are not quantifiable.
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Huch of the information on single oral exposures of laboratory animals
to heptachlor and heptachlor epoxide Is concerned with lethality. Hepta-
chlor Is more toxic to laboratory animals than chlordane. Acute oral LD^
values In rats for heptachlor range from *0 mg/kg bw for a commercial formu-
lation (Ben-Dyke et al., 1970) to 162 mg/kg bw for female Sherman rats for
technical grade heptachlor (Gaines et al., I960). Other LD5Q values range
from 70-100 mg/kg bw In mice and hamsters (Gak et al., 1976). As In the
case of chlordane, neonatal rats (l.p. LD^ = 531 mg/kg bw) are far less
sensitive to Intraperitoneal Injections of heptachlor than adult males (l.p.
LO^o * 71 mg/kg bw) (Harbison, 1975). For heptachlor epoxide, the acute
oral LD&0 Is -60 mg/kg bw (Sperling and Ewlnlke, 1969; Podowskl et al.,
1979; NAS, 1977). Symptoms of acute Intoxication Include tumors, convul-
sions, paralysis, hyperexcUablHty and Irritability (Gaines, 1960; Lehman,
1951; Hrdlna et al., 1974).
At a single oral dose of 60 mg heptachlor/kg bw or repeated oral doses
of 7 and 12 mg heptachlor/kg bw/day for up to 14 days 1n rats, significantly
Increased levels of serum GPT and serum aldolase (p<0.05-0.001) were associ-
ated with moderate to severe histological liver damage (Krampl, 1971). For
rats that had been administered these doses for 28 days, however, the serum
levels of these enzymes were not statistically significantly different from
controls. The degree of liver damage was less severe than that observed at
7 and 14 days. Rats maintained on diets containing 10 mg heptachlor/kg diet
For 1-7 days had evidence of liver damage and altered liver function:
Increased blood urea. Increased blood glucose, decreased liver glycogen
content. Increased acid and alkaline phosphatase levels when compared with
controls {Enan et al., 1982). Liver, heart and spleen weights were also
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above control weights. Dose-related significant Induction of liver micro-
somal enzymes (aniline hydroxylase, amlnopyrlne demethylase and hexabarbltal
oxidase), at dietary levels of heptachlor (>99%) of 2-50 mg/kg diet, was
observed In rats (Den Tonkelaar and Van Esch, 1974). The no-effect level
was <2 mg/kg diet. However, these Investigators did not look for definitive
adverse effects. Arnold et al. (1977) reported that a single oral dose of
30 mg/kg bw of a 75% heptachlor epoxide and 25% heptachlor mixture resulted
In moderate hypoactlvlty and 25% mortality In mice.
In dietary studies of 42 days duration, no effects on body weight gain,
food consumption or mortality were observed 1n rats at <80 mg/kg technical
heptachlor diet levels or In mice at <40 mg/kg diet levels (NCI, 1977b).
Other parameters were not examined. The dietary concentrations are equiva-
lent to doses of 8 mg/kg bw for rats and 12 mg/kg bw for mice, and are quite
near the dose levels that resulted In liver damage 1n rats 1n the shorter
term studies of Krampl (1971). Shaln et al. (1977) observed decreased mean
weekly body weight gain, reduced food consumption and changes 1n ventral
prostate homeostasis of rats receiving 1.29 mg/kg bw/day of heptachlor.
This dose Is above the 2 mg/kg diet level for 14 days (I.e., 0.2 mg/kg
bw/day) that resulted In enzyme Induction In rats (Oen Tonkelaar and Van
Esch, 1974), but similar to a dose of 10 mg/kg of diet (I.e., 1.0 mg/kg
bw/day) that caused definitive adverse effects 1n rats (Enan et al., 1982).
Klnoshlta and Kempf (1970) reported 1n a meeting abstract that the no-effect
level In rats for enzyme Induction for heptachlor and heptachlor epoxide was
1 mg/kg diet for 91 days. Although details of this study were unavailable
from the abstract, the results support the low-effect level for enzyme
Induction of 2 mg/kg diet reported by Den Tonkelaar and Van Esch (1974).
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Several long-term dietary studies with heptachlor and/or heptachlor
epoxide, which were designed as carcinogenicity studies, provide toxicologic
data. In the NCI (1977b) bloassay 1n mice, no hlstopathologlcal signs of
toxicity were observed at TWA concentrations of heptachlor of 6.1-10 mg/kg
diet for 80 weeks. However, abdominal distention and increased mortality
were observed In the high dose females. The Incidences of hepatocellular
carcinomas were also significant for high dose males and females. Hepta-
chlor was not carcinogenic In rats, but vaginal bleeding also developed In
both groups of treated females (25.7 and 51.3 mg/kg diet).
At dietary levels of 10 mg/kg of heptachlor or heptachlor epoxide In
mice, Reuber (1977a) diagnosed hepatic vein thrombosis and cirrhosis of the
liver, as well as carcinomas, from slides of the Davis (1965) study. In the
IRDC (1973b) study, reviewed by Epstein (1976), a 75% heptachlor epoxide and
25% heptachlor mixture was fed to mice for 18 months. Females and males had
dose-related Increased mean liver weights and hepatocytomegaly at 1, 5 and
10 mg/kg diet. Jolley et al. (1966) found dose-related Increased mortality
In rats fed 5-12.5 mg/kg diet levels of a 75% heptachlor and 25% heptachlor
epoxide mixture for 2 years. WUherup et al. (1955) found non-neoplastlc
lesions In rats at dietary levels >7.0 mg/kg diet for 110 weeks. At <5.0
mg/kg diet, no lesions were observed. Treated males had dose-related
Increased liver weights at levels 1.5-10 mg/kg diet. Although this effect
is not necessarily adverse, Its presence at doses slightly lower than that
which caused tumors and the lack of additional histological analysis dictate
that this be considered an adverse effect. The 1.5 mg/kg diet level was the
lowest concentration of heptachlor tested In any of the chronic studies, and
thus represents the L0AEL.
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Dose-related liver weight Increases, hepatocytomegaly and hepatic cell
vacuolization were observed In rats maintained for 108 weeks on diets con-
taining heptachlor epoxide at 0.5-10 mg/kg diet (Wltherup et al., 1959).
These effects are considered to be adverse. Thus, the 0.5 mg/kg diet level
represents a LOAEL for heptachlor epoxide.
Oogs were administered heptachlor expoxlde In a diet containing various
dose levels for 2 years (U.S. EPA, 1971 IRDC, unpublished report). A level
of 1 ppm In diet caused no adverse effect on parameters measured. Dose-
related changes In biochemical values related to liver function and micro-
scopic changes 1n liver were noted at the higher dose levels (3, 5, 7 and 10
ppm In diet).
No evidence of teratogenicity of heptachlor or heptachlor epoxide was
available. Mestltzova (1967) reported a marked reduction In litter size,
however. In rats that were given heptachlor 1n the diet for several genera-
tions. It was not clear whether the dose was 6 mg/kg bw/day or b mg/kg bw
over the duration of the experiment.
Quantification of Noncarclnoqenlc Effects. No quantifiable human data
on heptachlor or heptachlor epoxide were available. Thus, It Is not
possible to determine If humans are more sensitive to these chemicals than
laboratory animals. In addition, studies on the effects of heptachlor 1n
rabbits were not available to determine Interspecies differences among
animals. Heptachlor and chlordane are structurally similar organochlor1ne
pesticides. Technical and commercial preparations of either are often
contaminated by the other.
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Derivation of 1-Dav HA — A NOEL or a NOAEL could not be defined for
heptachlor epoxide In the 1- to 30-day studies. As reviewed above, however,
a level of 10 mg/kg diet of heptachlor for 14 days 1n rats was the lowest
concentration that resulted 1n definite adverse effects (I.e., evidence of
liver damage and altered liver function (Enan et al., 1902). Although Den
Tonkelaar and Van Esch (1974) reported effects at lower doses (I.e., altered
Hver enzymes), these Investigators did not look for any additional effects
and those observed are not necessarily adverse. The former study Is
preferred over the latter as the basis for a HA, because the former study
offers additional details. A dally transformed dose (d) can be derived from
the former study by assuming a young rat Ingests food equivalent to 10% of
Its body weight/day. The 1-day HA would be calculated from this transformed
dose using a 1000-fold uncertainty factor that represents two 10-fold
factors for both intra- and Interspecies variability to the toxicity of this
chemical In 11eu of chemical specific Information, and an additional 10-fold
factor because the HA Is derived from a LOAEL and not a NOAEL. Thus, a
1-day HA for a child 1s derived as follows:
1-day HA - (1-0 <*'*1 M (10 fcq) „ 0010 ^
(1000) (1 l)
where:
1.0 mg/kg bw - minimal effect level
10 kg 3 assumed body weight of a child
1000 * uncertainty factor -- Inter- and Intraspecles variation
and LOAEL
1 i = assumed water consumption per day by a child
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Derivation of 10-Day HA — Few studies of 30-90 days duration that
were available defined no- or low-effect levels for heptachlor. In one
study that was reported 1n a meeting abstract (Klnoshlta and Kempf, 1970),
microsomal enzyme Induction occurred In rats 1n a dose-related way at
various dietary levels of heptachlor or heptachlor epoxide administered For
91 days. The NOEL for enzyme Induction was 1 mg/kg diet, which Is similar
to the apparent NOEL For enzyme Induction from shorter-term toxicity studies
of <2 mg/kg of diet (Den Tonkelaar and Van Esch, 1974).
These results suggest that the rats were either developing a tolerance
for heptachlor or responding 1n a similar fashion as In shorter-term
toxicity studies, for these reasons, the 10-day HA for a 10 kg child should
be equal to the recontoended 1-day HA In order to protect from transitory
adverse effects on the liver.
For a child: 10-day HA ¦ 0.010 mg/l
Derivation of Lonqer-terw HA — There are Insufficient toxlcologlcal
data available to calculate a longer-term HA for heptachlor or heptachlor
epoxide.
Assessment of Lifetime Exposure and Derivation of a QUEL —
Heptachlor. The Wltherup et al. (1955) study Is the most appro-
priate from which to derive the 0WEL for heptachlor. Investigators studied
the effects of heptachlor on groups of 20 male and 20 female CF rats. The
compound was administered at dietary concentrations of 0, 1.5, 3, 5, 7 or 10
ppm {10 mg/kg/dose) of heptachlor. Mortality among test groups was not
01830
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dose-related. Loss of body weight, decreased food consumption and Increased
liver weights were seen among treated males. Lesions In the liver were
limited to 7 ppm and above and were characteristic of chlorinated hydro-
carbons, I.e., hepatocellular swelling, homogeneity of the cytoplasm and
peripheral arrangements of the cytoplasmic granules of cells of the central
zone of the liver lobules. The NOEL for Increased 11ver-to-body weight for
males only was 3 ppm and the LOEL was 5 ppm. [NOTE: A re-analysis of the
Wltherup et al. (1955) dietary study on the toxicity of heptachor to rats
(by the OPP, RfD Work Group) Indicated that the NOEL of 3 ppm (0.15
mg/kg/day) for increased 11ver-to-body weight for male rats was the most
appropriate for a lifetime HA for heptachlor.] Using this NOEL, the OWEL 1s
derived as follows:
Step 1 - RfD Derivation
RfD (0.15 mq/kq/day)
(100) (3)
¦ 0.0005 mg/kg/day
where:
RfD
= Reference Dose: estimate of dally
exposure to the human population that
appears to be without appreciable
risk of deleterious noncarclnogenlc
effects over a lifetime of exposure
0.15 mg/kg/day (3 ppm) * NOEL
100
= uncertainty factor appropriate for use
with animal studies
3
* additional uncertainty factor to compen-
sate for limited observations and because
the most sensitive toxlcologlcal endpolnt
may not have been determined
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Step 2 - DUEL Derivation
DHEL*0-'0005 mq/kq/dav) (70 kg)
(2 i/day)
= 0.0175 mg/l (rounded to 20 yg/l)
where:
0.0005 mg/kg/day = RfD
70 kg
= weight of protected Individual (adult)
2 t/day
= assumed volume of water Ingested per day by a
70 kg adult
Heptachlor Epoxide. Two studies using dogs are the most appro-
priate from which to derive the DWEL. In the 60-week dog feeding study
(Kettering Labs., 1950) beagle dogs from 23-27 weeks of age were divided
Into five groups (three females and two males) and were given diets contain-
ing 0, 0.5, 2.5, 5 or 7.5 pprn of heptachlor epoxide. Results Included
11ver-to-body weight ratios that were significantly Increased In a
treatment-related fashion. Effects were noted for both males and females at
the 0.5 ppm (0.0125 mg/kg/day) dose level of heptachlor epoxide. No NOEL
was determined for the study. In another 2-generat1on reproduction study
using dogs (U.S. EPA, 1971), animals were administered diets containing
various dose levels of heptachlor epoxide. The dose levels were 0, 1, 3, 5,
7 or 10 ppm of heptachlor epoxide in the diet. This study was designed to
Investigate reproduction parameters associated with heptachlor epoxide
administration. The 0PP and the RfD Work Group considered that the 60-week
dog feeding study providing the L0EL of 0.5 ppm (0.0125 mg/kg/day) 1s the
most appropriate for the derivation of the DWEL. Using this L0EL, the OWEL
1$ derived as follows:
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Step 1 - RfO Derivation
where:
RfD = (°701" mq/kq/daY) = 0.000013 mg/kg/day
(100) (10)
RfO	= Risk Reference Dose: estimate of dally exposure
to the human population that appears to be
without appreciable risk of deleterious
noncardnogenlc effects over a lifetime of
exposure
0.0125 mg/kg/day = lowest-observed-effect level (L0EL)
100	= uncertainty factor appropriate for use with
animal studies
10	= uncertainty factor to compensate for the fact
that a NOEL was not attained
Step 2 - DWEL Derivation
DUEL J0-000013 mg/kg/day) (70 kg) = 0.00044 mg/l (rounded to 0.4 pg/i)
(2 l/day)
where:
0.000013 mg/kg/day « RfD
70 kg	= weight of protected Individual (adult)
2 L/day	= assumed volume of water ingested per day
by 70 kg adult
A summary of the data used to calculate the recommended 1-day, 10-day
and longer-term HAs and the lifetime DWEL for heptachlor/heptachlor epoxide
Is provided In Table VIII-8.
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00
GO
O
TABLF V111-8
Summary of Data Used to Derive HAs and DUELs for Heptachlor/Heptachlor Epoxide
Criteria
Animal Dose
1-day HA
heptachlor
1.0 wg/kg
bw/day
Duration
Effect
Child
Value of HA or QUEL	
Adult
14 days
rats
Lowest level for
evidence of liver
damage and altered
liver function
10 ug/t
(10 tig/day or
1.0 pg/kg bw)
Reference
Enan et al. 196?
10-day HA
heptachlor
As above for the 1 -day HA
OJ
Longer-term HA
Insufficient Data
Lifetime
DUEL for
heptachlor
0.15
mg/kg/day
110 weeks	lowest level for
rats	Increased liver weight
NA
20 wg/l
(100% RSC)'
HI therup et a 1., 1955
o
oo
Lifetime
DUEL for
heptachlor
epoxide
0.0125 mg/kg
bw/day
60 weeks
dogs
LOAEL
NA
*RSC: Percent relative source contribution being from water
NA = Not applicable
0.4 pQ/t
(100% RSC)
KetterIng Labs.. 1958
o

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Carcinogenic Effects.
Hunan Studies --
Case Reports. There were 11 case reports Involving CNS effects
where the author looked at the toxic effects of chlordane/heptachlor, eight
case studies involving blood dyscraslas, and five case studies of neuroblas-
tomas in children with pre-/postnatal exposure to chlordane or heptachlor.
The blood dyscraslas In children Included four cases of aplastic anemia and
one case each of refractory megaloblastic anemia, acute lymphoblastic
leukemia, acute stem-cell leukemia, and acute myelomonocytlc leukemia.
Epidemiologic Studies. Three epidemiologic studies of workers
exposed to chlordane and/or heptachlor have been reported. One of these
studies, conducted In chlordane/heptachlor applicators, was considered
Inadequate In sample size and In duration. However, this study showed
Increased mortality from bladder cancer (SMR-277, p<0.05). A second study
showed an Increased mortality from lung cancer {SMR=134)# but the Increase
was not statistically significant. The mortality from cerebrovascular
disease was statistically significant (SHR*183, p<0.05). Of the 1043 men
Involved In the study, only one liver cancer was reported. The third study
Involved 2141 workers exposed to organochlorlne pesticides. One of the four
plants Involved In pesticide manufacture produced chlordane and one produced
heptachlor. The SMR for malignant neoplasms was 69 at the chlordane plant
and 91 at the heptachlor plant. There was an excess risk for cancer In
various tissues; none was statistically significant. The last two studies
were carried out In chlordane/heptachlor manufacturing plants.
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All of these studies have several limitations. Neither the quantitative
nor length of exposure histories are available for chlordane/heptachlor for
the populations studied. They were also exposed to other pesticides and
chemicals. Adjustments for these other chemical exposures and other con-
founding factors, like smoking and alcohol consumption, were not considered
In any of these studies. All of the study populations were small. In the
pesticide applicator study. Individual follow-up was not undertaken and the
data were missing on 10.3% of the decedents reported by the Social Security
Administration.
Because of these methodological limitations and the limited data. It Is
difficult to establish either a negative or positive association between
chlordane/heptachlor and carcinogenicity. Hence, these studies are consid-
ered Inadequate epidemiologic evidence.
Animal Studies — Three heptachlor/heptachlor epoxide carcinogenesis
bloassays In mice have been reported. The strains studied Include C3H,
B6C3F1 and CD-I mice. In C3H mice fed 10 ppm of both heptachlor and hepta-
chlor epoxide for 2 years, benign liver tumors/hepatocellular carcinomas
were reported In both male and female mice. Hepatocellular carcinomas In
treated groups were generally large and frequently multiple tumors, espe-
cially 1n the epoxide group 1n respect to the controls. For B6C3F1 mice fed
technical grade (containing 22% chlordane) at concentrations of 6.1 or 13.8
ppm (males) or 9 or 18 ppm (females), respectively, for 80 weeks and held
for an additional 10 weeks, hepatocellular carcinomas were significantly
(p<0.001) Increased In both male and female mice. In CD-I mice fed a
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mixture of heptachlor epoxlde/heptachlor (75:25) at concentrations of 1, 5
or 10 ppm for 18 months, nodular hyperplasla/hepatocellular carcinomas were
noted at 5 and 10 ppm In both male and female mice.
Five heptachlor/heptachlor epoxide carcinogenesis bloassays In rats have
been conducted. The strains of rats studied Include Wlstar, Osborne-Mendelt
CD and CFN. In Wlstar rats given five doses of 10 mg/kg bw of heptachlor
and held for 106-110 weeks, no treatment-related tumors were observed. For
Osborne-Mendel rats fed technical grade heptachlor at concentrations of 38.9
or 77.9 (males) or 25.7 or 51.3 (females) ppm for 80 weeks and held for 30
weeks, no liver tumors were noted, although neoplastic nodules were found In
both treated and control rats. In CO rats fed a mixture of heptachlor/
heptachlor epoxide (75:25) at concentrations of 5, 7.5, 10 or 12.5 ppm for 2
years, no liver tumors were noted, although nonneoplastic lesions were noted
1n the livers of rats fed 7.5, 10 or 12.5 ppm. In one study using CFN rats
fed 1.5, 3, 5, 7 or 10 ppm of heptachlor for 110 weeks, the Incidence of
liver tumors was not statistically different 1n treated and control animals.
In a second study using CFN rats fed 0.5, 2.5, 5, 7.5 or 10 ppm of hepta-
chlor epoxide for 108 weeks, treatment-related liver carcinomas were noted
by several pathologists.
Supporting Evidence —
Hutaqcnlcity. The published literature on mutagenicity testing of
chlordane and heptachlor/heptachlor epoxide 1s quite similar, Indeed most
studies report results on both chemicals. Generally, the results have
indicated that these chemicals are not mutagenic In bacteria or In mammalian
cells In culture and do not Induce DNA repair as measured by unscheduled DNA
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synthesis In rodent hepatocytes. While dominant lethal tests In mice have
been negative for both chemicals, the absence of direct cytogenetic tests In
both germinal and somatic cells precludes a conclusion of their potential
for causing chromosome aberrations.
Structural Relationship, Three compounds, structurally related to
chlordane/heptachlor/heptachlor epoxide, have Induced malignant liver tumors
In animals. Aldrln, dleldrln and chlorendlc acid have produced liver tumors
1n mice and chlorendlc acid has also produced liver tumors 1n rats.
Carcinogenicity Classification, Heptachlor/heptachlor epoxide Is a
probable human carcinogen, classified In Group B2 under the EPA's guidelines
for carcinogen risk assessment (U.S. EPA, 1986b). Animal studies provide
sufficient evidence for carcinogenicity: heptachlor/heptachlor epoxide
Increased the incidence of liver carcinomas 1n C3H, CD-I and B6C3F1 mice and
In CFN rats. Epidemiologic studies provide Inadequate evidence due to
methodology and data limitations.
The guidelines consider this evidence sufficient for Group B2, but they
allow downgrading from Group B2 to Group C when the only tumor response Is
that of mouse liver tumors In strains with high background rates, or when
warranted by a number of other factors. The evidence, however, shows highly
significant tumor responses, Increased Incidence In both males and females,
Increased Incidence at medium and high doses, and Induction of tumors by
structurally related chemicals. In light of these factors, downgrading Is
clearly not warranted, and heptachlor/heptachlor epoxide remains In Group B2.
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Quantification of Carcinogenic Effects. For some chemicals, several
studies 1n different'animal species, strains and sexes at several doses and
different routes of exposure may be available. A choice must be made as to
which data sets should be used to quantify human risk by low-dose extrapola-
tion. The following procedure was used to make this choice. The animal
studies are evaluated qualitatively to assure that only properly conducted
studies are used. The tumor Incidence data were separated according to
organ sites and tumor types. The data sets used In the model are the ones
In which the tumor incidence 1s statistically significantly higher In at
least one test dose level as compared with controls and/or where the tumor
Incidence rate shows a significant trend with respect to dose level. Both
biological and statistical considerations have been used to select the most
appropriate data sets.
Because humans may be as sensitive as the most sensitive animal species,
potency estimates obtained from the most sensitive species tested can be
averaged to estimate potency for the general population. Because some sub-
populations may be more sensitive than the general population, the potency
estimate from the most sensitive sex and strain tested 1s also presented.
This approach Is consistent with ERA'S guidelines for carcinogen risk
assessment (U.S. EPA. 1986b).
Eight data sets showed significant Increases In the Incidence of hepato-
cellular carcinomas In treated groups compared with controls. Tables VI11-9
through VI11-16 present the tumor Incidence for these data sets. In rats, a
significant Increase In hepatocellular carcinomas was diagnosed by Reuber.
Incidence data are shown In Table VIII-17. These studies were used to
quantify the carcinogenic risk from heptachlor/heptachlor epoxide exposure.
01830	VIII-44	04/01/87

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TABLE VII1-9
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Hale Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency (q-j *);
Heptachlor
Mouse, C3H, male
0.030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
12.4 per mg/kg/day
Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Oose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0.00
0.000
22/78
10
1.43
0.108
64/87
^Source: Davis, 1965, as diagnosed by Reuber, 1977b
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TABLE VIII-10
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Female Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency {q-| *):
Heptachlor
Mouse, C3H, female
0.030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
14.9 per mg/kg/day
Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Dose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0.00
0.000
2/54
10
1.43
0.108
57/78
•Source: Davis, 1965, as diagnosed by Reuber, 1977b
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TABLE VIII-11
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Hale Mice*

Compound:
Technical grade heptachlor

Species, strain, sex:
Mouse, B6C3F1, male

Body weight:
0.030 kg (assumed)

Length of experiment:
90 weeks

Length of exposure:
80 weeks

Tumor site and type:
Liver, carcinoma

Route, vehicle:
Oral, diet

Human potency (q-|*):
2.79 per mg/kg/day
Experimental
Average
Eaulvalent Tumor Incidence
Animal Dose
Animal Dose
Human Dose No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day) No. E xam1ned
0
0
0 5/19
6.1
0.79
0.063 11/46
13.8
1.79
0.14 34/47
~Source: NCI, 1977b
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TABLE VIII-12
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Female Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency (q-| *):
Technical grade heptachlor
Mouse, B6C3F1, female
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
0.03 per mg/kg/day
Experimental
Animal Dose
(ppm)
Average
Animal Dose
(mg/kg/day)
Equivalent
Human Dose
(mg/kg/day)
Tumor Incidence
No. Responding/
No. Examined
0
0
a
2/10
9.0
1.17
0.094
3/47
18.0
2.34
0.18
30/42
~Source: NCI, 1977b
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TABLE VI1I-13
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas 1n Male Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency (q-|*):
Heptachlor epoxide
Mouse, C3H, male
0*030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
27.7 per mg/kg/day
Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Dose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0.00
0.000
22/78
10
1.43
0.108
73/79
~Source: Oavls, 1965, as diagnosed by Reuber, 1977b
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TABLE VIII-14
Cancer Data Sheet for Derivation of Potency of Heptachlor
from Hepatocellular Carcinomas In Female Mice*
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency {q-| * >:
Heptachlor epoxide
House, C3H, female
0.030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
36.2 per mg/kg/day
Experimental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Dose
Human Oose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0.00
0.000
2/54
10
1.43
0.108
77/81
•Source: Davis, 1965, as diagnosed by Reuber, 1977b
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TABLE VIII-15
Cancer Data Sheet for Derivation of Potency of Heptachlor Epoxide
from Hepatic Carcinomas 1n Female Mice*

Compound:
25:75 mixture of heptachlor/


heptachlor epoxide

Species, strain, sex:
Mouse, C0-1# female

Body weight:
0.030 kg (assumed)

Length of experiment:
19 months, 3 weeks

Length of exposure:
18 months

Tumor site and type:
Liver, carcinoma

Route* vehicle:
Oral, diet

Human potency (q*|*):
1.04 per mg/kg/day
Experimental
Average
Eaulvalent Tumor Incidence
Animal Dose
Animal Dose
Human Dose No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day) No. Examined
0
0
0 6/76
1
0.13
0.01 1/70
5
0.65
0.052 6/65
10
1.30
0.10 30/57
•Source: 1RDC, 1973b, as reevaluated by Reuber
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TABLE VIII-16
Cancer Data Sheet for Derivation of Potency of Heptachlor Epoxide
from Hepatic Carcinomas 1n Male Nice*

Compound:
25.75 mixture of heptachlor/


heptachlor
epoxide

Species, strain, sex:
Mouse, CD-I,
male

Body weight:
0.030 kg (assumed)

Length of experiment:
19 months, 3
weeks

Length of exposure:
18 months


Tumor site and type:
Liver, carcinoma

Route, vehicle:
Oral, diet


Human potency (q*]*):
6.48 per mg/kg/day
Exper1mental
Average
Equivalent
Tumor Incidence
Animal Dose
Animal Dose
Human Dose
No. Responding/
(ppm)
(mg/kg/day)
(mg/kg/day)
No. Examined
0
0
0
0/62
1
0.13
0.010
2/68
5
0.65
0.052
18/68
10
1.30
0.10
52/80
'Source: IRDC
, 1973b, as reevaluated by
Reuber

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TABLE VI11-17
Cancer Data Sheet for Derivation of Potency of Heptachlor Epoxide
from Hepatic Carcinomas 1n Female Rats*
Conpound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure..
Tumor site and type:
Route, vehicle:
Human potency (Qi*):
Heptachlor epoxide
Rat, CFN, female
0.350 kg (assumed)
108 weeks
108 weeks
Liver, carcinoma
Oral, diet
5.76 per mg/kg/day
Experimental
Animal Dose
(ppm)
Average
Animal Dose
(mg/kg/day)
Equivalent
Human Dose
(mg/kg/day)
Tumor Incidence
Mo. Responding/
No. Examined
0
0
0
0/17
0.5
0.025
0.0043
3/22
2.5
0.125
0.021
3/18
5.0
0.250
0.043
7/22
7.5
0.375
0.064
3/21
10.0
0.500
0.085
5/19
~Source: UHherup et al., 1959, as reevaluated by Reuber
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Estimates of carcinogenic potency can be obtained by fitting the
linearized multistage model to each data set. Table VI11-7 summarizes nine
potency estimates obtained 1n this way.
Four data sets Involve heptachlor: male and female C3H mice, and male
and female B6C3F1 mice. The most sensitive sex and strain tested 1s female
C3H mice. From these, the potency 1s estimated at 14.9 per mg/kg/day.
The most sensitive species tested Is mice. There are four potency
estimates, ranging from 14.9 down to 0.83 per mg/kg/day, with a geometric
mean of 4.5 per mg/kg/day. Because humans may be as sensitive as the most
sensitive animal species, the potency for the general population Is
estimated at 4.5 per mg/kg/day. These estimates supersede the potency of
3.37 per mg/kg/day previously calculated by the U.S. EPA (1980a).
The upper-limit unit risk estimates from the animal data are derived
from a linearized multistage nonthreshold extrapolation model which Is
currently programmed as GLOBAL 83. Justification for Its use Is presented
in EPA1s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986b). While
recognizing that alternative statistical modelling approaches exist [e.g..
One-hit, Welbull, Log-Problt and Loglt models, and maximum likelihood
estimates], the range of risks described by using any of these modeling
approaches has little biological significance unless data can be used to
support the selection of one model over another. In the Interest of
approach consistency and of providing an upper bound estimate for the poten-
tial cancer risk, the Agency recomnends the use of the linearized multistage
model. EPA considers this model and resulting risk estimate to be an upper
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limit value In the sense that the true risk Is unlikely to be higher and may
be lower even zero. An established procedure does not yet exist for making
¦most likely' or "best" estimates of risk within the range of uncertainty
derived by the upper and lower limit values.
The unit risk In water Is 1.3x10~4 per yg/i for the general popu-
lation. The concentration In water corresponding to an Increased lifetime
risk level of 10'4, 10~5 and 10"* for a 70 kg human who consumes 2
i/day, 1s calculated to be 7.6, 0.76 and 0.076 yg/t, respectively.
Five data sets Involve heptachlor epoxide: male and female C3H mice,
male and female CD-I mice, and female CFN rats. The most sensitive sex and
strain tested Is female C3H mice. From these the potency 1s estimated at
36.2 per mg/kg/day.
The most sensitive species tested Is mice. There are four potency esti-
mates, ranging from 36.2 down to 1.0 per mg/kg/dayt with a geometric mean of
9.1 per mg/kg/day. This geometric mean from mice 1s consistent with the
potency estimate from rats of 5.8 per mg/kg/day. Because humans may be as
sensitive as the most sensitive animal species, the potency for the general
population Is estimated at 9.1 per mg/kg/day. These estimates supersede the
potency of 57.86 per mg/kg/day previously calculated by the U.S. EPA.
The unit risk 1n water is 2.6x10"* per vq/i for the general popu-
lation. The concentration In water corresponding to an Increased lifetime
risk level of 10~4, 10~5 and 10"* for a 70 kg human who consumes 2
i/day, 1s calculated to be 3.0, 0.30 and 0.038 ug/i, respectively.
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Existing Guidelines, Recomendatlons and Standards
On March 6, 1970, the U.S. EPA (43 FR 12372) cancelled registrations of
most pesticide products containing chlordane and heptachlor as defined in
the notice of Intent to Cancel {39 FR 41298). The exceptions are the "use
of heptachlor or chlordane through subsurface ground Insertion for termite
control and the dipping of roots or tops of nonfood plants."
Water. The U.S. EPA (40 FR 11990) proposed to set Interim Primary
Drinking Water Standards for chlordane at 0.003 mg/l and for heptachlor
and heptachlor epoxide at 0.0001 mg/l for maximum contaminant levels. In
the final U.S. EPA Regulations (40 FR 59566), however, these levels were
deleted because the U.S. EPA was Involved In the suspension and cancellation
proceedings.
The Federal Water Pollution Control Administration (1968) set permiss-
ible surface water criteria for public water supplies at 0.003 mg/l for
chlordane and 0.018 mg/l for heptachlor and heptachlor epoxide. The
criteria for Msh and other aquatic life based on	of 0.002 mg/l for
chlordane and 0.0002 mg/l for heptachlor would be very low; therefore, 1t
Is not recommended that these compounds be used near a marine environment.
The Water Quality Criteria for farmstead use were 0.003 mg/l for chlordane
and 0.018 mg/mt for heptachlor and heptachlor epoxide. Odor thresholds
for chlordane and heptachlor In water were reported by Slgworth (1965) as
0.0005 and.0.02 mg/l, respectively.
U.S. EPA (1980b) determined 4.6 ng/l for chlordane as the water con-
centration corresponding to an Increased lifetime risk of cancer of 10~5.
01830
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This determination was based upon the Incidence of hepatocellular carcinoma
In male CD-I mice following chronic dietary exposure as diagnosed by Reuber
In his re-evaluation of slides from an IROC (1973a) study that was reviewed
by Epstein (1976) and upon a bloconcentrat1on factor of 14,100 In fish.
U.S. EPA (1980a) determined 2.8 ng/t for heptachlor as the water concen-
tration corresponding to an Increased lifetime risk of 10~s. This deter-
mination was based upon the Incidence of hepatocellular carcinoma In male
B6C3F^ mice 1n the NCI (1977b) bloassay.
Food. FA0/WH0 (1978) reconmended a maximum acceptable dally Intake
(AD1) value of 1 yg/kg bw for chlordane. A value of 0.5 yg/kg bw was
recommended for heptachlor (FA0/WH0, 1972).
The registration for all uses of chlordane and heptachlor/heptachlor
epoxide on food crops has been cancelled (43 FR 12372); however, when these
Insecticides were in use, the tolerances for residues 1n or on raw agri-
cultural commodities for chlordane (40 CFR 180.122) and heptachlor and
heptachlor epoxide (40 CRF 180.104) are given below:
Section 180.122 Chlordane; tolerances for residues.
A tolerance of 0.3 part per million Is established for residues
of the insecticide chlordane (1,2,4,5,6,7,8,8-octachloro-2,3-
3a,4,7,7a-hexahydro-4,7-methano1ndene, containing not more than 1
percent of the Intermediate compound hexachlorocyclopentadlene)
In or on each of the following raw agricultural commodities:
Apples, apricots, beans, beets (with or without tops) or beet
greens alone, blackberries, blueberries (huckleberries), boysen-
berrles, broccoli, brussels sprouts, cabbage, carrots, cauli-
flower, celery, cherries, citrus fruits, collards, corn, cucum-
bers, dewberries, eggplants, grapes, kale, kohlrabi, lettuce,
loganberries, melons, nectarines, okra, onions, papayas, peaches,
peanuts, pears, peas, peppers, pineapples, plums (fresh prunes),
potatoes, quinces, radishes (with or without tops) or radish
tops, raspberries, rutabagas (with or without tops) or rutabaga
tops, squash, strawberries, summer squash, sweet potatoes,
tomatoes, turnips (with or without tops) or turnip greens, young-
berries .
01830
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Section 180.104. Heptachlor and heptachlor epoxide; tolerances for
residues.
0.1 part per million In or on cabbage, lettuce, rutabagas, snap
beans.
Zero In or on alfalfa, apples, barley, beets (Including sugar
beets), blackeyed peas, brussels sprouts, carrots, cauliflower,
cherries, clover, com, cottonseed, cowpeas, grain sorghum
(mllo), grapes, grass (pasture and range), kohlrabi, lima beans,
meat, milk, oats, onions, peaches, peanuts, peas, pineapple,
potatoes, radishes, rye, sugarcane, sweet clover, sweet potatoes,
tomatoes, turnips (Including tops}, wheat.
Air. The American Conference of Governmental Industrial Hyg1en1sts
(ACGIH, 1983) has adopted TWA-TLVs of 0.5 mg/raJ for chlordane and hepta-
chlor In workroom air. Likewise, the U.S. Occupational Safety and Health
Administration standard TLV for exposure In the workroom Is 0.5 mg/ma for
chlordane and heptachlor (29 CFR 1910.1000). Corresponding values for
heptachlor are 0.5 mg/m' In the Federal Republic of Germany and 0.01
mg/ma In the USSR (Wlnell, 1975).
/
The U.S. National Research Council (NRC, 1982) has recommended an
interim guideline for airborne chlordane and heptachlor 1n military housing
of 5 and 2 ng/mJ, respectively.
Special Considerations
High Risk Suboopulatlons. High risk subpopulatlons have not been
Identified in the available literature. Cases of blood dyscrasla have been
reported In association with human exposure to chlordane and- heptachlor, as
well as with other pesticides (Infante et al., 1978; Furle and Trubowltz,
1976; Klemmer et al., 1977). Infante et al. (1978) suggested that an Idio-
syncratic mechanism for susceptible, but not readily Identifiable, Indi-
viduals may be Involved.
01830
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Groups that may be or might have been more affected by high exposure
rather than by Intrinsic susceptibility Include exterminators, agricultural
applicators, lawn sprayers and workers Involved 1n the manufacture of pesti-
cides. High exposure may also result In persons who live In areas of high
pesticide use. Breast-fed Infants may be exposed to high levels, since
heptachlor epoxide, a major metabolite of heptachlor, has been detected In
numerous samples of human milk (Kroger, 1972; Rltcey et a!., 1972; Savage et
al., 1973; Bakken and Selp, 1976; Pollshuk et al., 1977b; Jonsson et a 1 -,
1977; Strassman and Kut2, 1977). Oxychlordane, a metabolite of chlordane,
was also detected In human milk (Strassman and Kutz, 1977). Oeveloplng
fetuses may be exposed Ui utero. The transplacental transfer of these
chemicals 1s well documented (Curley et al., 1969; Wasserman et a1.t 1972,
1974; Zavon et al., 1969; Selby et al., 1969; Pollshuk et al., 1977a).
Multiple Pollutant Exposures. Pretreatment of adult male rats with
turpentine potentiated the toxicity of heptachlor, reducing the oral 10^
from 112 to 70 mg/kg bw (Sperling and Ewlnlke, 1969). The toxicity of
heptachlor epoxide was unaffected. Pretreatment of rats with chlordane
resulted 1n enhancement of hepatocellular necrosis Induced by CCl^
(Stenger et al., 1975).
Several cases of mixed exposure with other pesticides were reported to
be associated with blood dyscraslas 1n humans. These Include hemolytic
anemia associated with exposure to chlordane, heptachlor, dleldrln and toxa-
phene (Mulrhead et al., 1959); and aplastic anemias, pancytopenia, thrombo-
cytopenia, leukopenia and agranulocytosis associated with chlordane and
heptachlor, as well as with other pesticides such as lindane and chlorophe-
nothane (Loge, 1965), Dlcamba, Dlazlnon and 2,4-D (Infante et al., 1978).
01830	VIII-59	04/01/87

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IX. REFERENCES
Abbott, D.C., G.6. Collins and R. Gouldlng. 1972. OrganochlorIde pesticide
residues In human fat In the United Kingdom 1969-1971 . Br. Med. J. 2:
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Abbott, O.C., 6.B. Collins, R. Gouldlng and R.A. Hoodless. 1981. Organo-
chlorlne pesticide residues 1n human fat In the United Kingdom 1976-1977.
Br. Med. J. 283(6304); 1425-1428.
ACGIH (American Conference of Governmental Industrial Hyglenlsts). 1983.
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Ahmed, F.E., R.W. Hart and N.J. Lewis. 1977. Pest1c1de-1nduced ONA damage
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Aldrlch, F.O. and J.H. Holmes. 1969. Acute chlordane	Intoxication In a
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Alvarez, W.C. and S. Hyman. 1953. Absence of toxic	manifestations In
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Ambrose, A.M., H.E. Chrlstensen, O.J. Robblns and L.J. Rather. 1953a.
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Ambrose, A.M., H.C. ChMstensen and D.J. Robblns. 1953b. Pharmacological
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Anonymous. 1962. Registry on blood dyscraslas: Report to council. J. Am.
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Arnold, O.W., G.L. Kennedy, Jr., M.L. KepHnger, J.C. Calandra and C.J.
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Arthur, R.D., J.D. Cain and B.F. Barrentlne. 1975. The effect of atmos-
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Atlas, E., R. Foster and C.S. G1rm. 1982. Air-sea exchange of high molecu-
lar weight organic pollutants: Laboratory studies. Environ. Scl. Techno!.
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Bakken, A.F. and M. Se1p. 1976. Insecticides In human breast milk. Acta
Paedlatr. Scand. 65: 535-539.
Balba, H.N. and J.G. Saha. 1978. Studies on the distribution, excretion
and metabolism of alpha and gamma Isomers of [L4C] chlordane In rabbits.
J. Environ. Scl. Health. B13(3): 211-233.
Barnes, R. 1967. Poisoning by the Insecticide chlordane. Med. J. Austr.
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Barnett, J.R. and H.M. Dorough. 1974. Metabolism of chlordane In rats. J.
Agrlc. Food Chem. 22: 612-619.
Barquet, A., C. Morgade and C.D. Pfaffenberger. 1981. Determination of
organochlorlne pesticides and metabolites 1n drinking water, human blood
serum and adipose tissue. J. Toxicol. Environ. Health. 7(3-4): 469-479.
BCPC (British Crop Protection Council). 1977. Pesticide Manual, 5th ed.f
H. Martin and C.R. Worthing, Ed. BCPC. p. 93, 294.
Becker, F.F. and S. Sell. 1979. Alpha-fetoproteln levels and hepatic
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Ben-Dyke, R.( D.M. Sanderson and D.N. Noakes. 1970. Acute toxicity data
for pesticides. Wlldl. Rev. Pestle. Control. 9: 119-127.
Benes, V. and R. Sram. 1969. Mutagenic activity of some pesticides 1n
Orosophlla melanoqaster. Ind. Med. 30: 50-52.
Biros, F.J. and H.F. Enos. 1973. Oxychlordane residues In human adipose
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Blevlns, R.O. and T.E. Sholes. 1978. Response of HeLa cells to selected
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Boyd, E.M. and F.I. Taylor. 1969. The acute oral toxicity of chlordane 1n
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5. REPORT D AT £ .
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