United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/600/6-87/004
December 1986
Research and Development
Carcinogenicity
Assessment of
Chlordane and
Heptachlor/
Heptachlor Epoxide
-------
-------
EPA-600/6-87/004
December 1986
CARCINOGENICITY ASSESSMENT OF
CHLORDANE AND HEPTACHLOR/HEPTACHLOR EPOXIDE
Carcinogen Assessment Group
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
-------
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for
use, i
-------
CONTENTS
Tables
Figures !!!!!'" V11
Preface ' " * * x.
Abstract . x!.
*** VTT
Authors, Contributors, and Reviewers * .;,
** A 1 I |
1. CONCLUSIONS ljf
2. INTRODUCTION 2_1
3. GENERAL BACKGROUND INFORMATION 3-1
3.1. CHEMICAL AND PHYSICAL PROPERTIES 3_!
3.1.1. Identification .....' 3_1
3.1.2. Synonyms !!!.'**" 3.3
3.2. USES
3.3. ROUTES'AND'PATTERNS OF'EXPOSURE ." .* .' .* .' .' .* .' .* * .' * * .* .' flj
4. HAZARD IDENTIFICATION 4_1
4.1. METABOLISM AND PHARMACOKINETICS 4_!
4,1.1. Absorption 4_j
4.1.1.1. Chlordane .... 41
4.1.1.2. Heptachlor .....'.'. 4.3
4.1.2. Tissue Distribution 4.4
4.1.2.1. Chlordane 44
4.1.2.2. Heptachlor .'.*.'.'.'.' 4!?
4.1.3. Metabolism 4_g
4.1.3.1. Chlordane 4_g
4.1.3.2. Heptachlor ! ! ! 1 ! 4-11
4.1.4. Excretion ............... 4-15
4.1.4.1. Chlordane 4_15
4.1.4.2. Heptachlor .'.".'." 4-17
4.1.5. Species Differences 4_17
4.2. TOXIC EFFECTS 4_20
iii
-------
CONTENTS (continued)
4.2.1. Human Studies .................... ~
4.2.2. Laboratory Animal Studies ........ ...... '"^
4.2.2.1. Chlordane ................. 4-24
4.2.2.2. Heptachlor .............. . *~CI
4-??
4.3. MUTAGENICITY ................... ......
4-??
4.3.1. Chlordane ....... » ....... ....... .7
4.3.2. Heptachlor ..... ..... ........... *""
4.4. CARCINOGENICITY ....................... 4"34
4_?4
4.4.1. Animal Studies ....................
4.4.1.1. Chlordane ................. J"34
4.4.1.2. Heptachlor .......... ....... ^"Di
4.4.2. Epidemiologic Studies ................ 4"63
4.4.2.1. Infante et al . (1978) . . . ........ 4-63
4.4.2.2. Wang and MacMahon (1979a) ......... 4-64
4.4.2.3. Wang and MacMahon (1979b) ......... 4-°°
4.4.2.4. Ditraglia et al . (1981) ........ 4-/1
4.5. STRUCTURE-ACTIVITY RELATIONSHIPS ........ ....... 4'74
4-74
4.5.1. Aldrin ........................
4.5.1.1. Evidence for Carcinogenicity - Human
Exposure (IARC, 1982) ........... 4~74
4.5.1.2. Evidence for Carcinogenicity - Animal
Studies (IARC, 1982) .......... 4~76
4.5.1.3. Evidence for Activity in Short-Term
Tests (IARC, 1982) ............. 4~76
4.5.2. Chlorendic Acid ......... .......... 4~76
4.5.2.1. Evidence for Carcinogenicity - Human
Exposure (NTP, 1985). ........... 4~76
4 5.2.2. Evidence for Carcinogenicity - Animal
Studies (NTP, 1985) ....... ..... 4~76
4.5.2.3. Evidence for Activity in Short-Term
Tests (NTP, 1985) .......... 4-77
4.5.3. Dieldrin. ...... .. ..... ..... ' ' 4"78
4.5.3.1. Evidence for Carcinogenicity - Human
Exposure (IARC, 1982) ...... ..... 4-78
-------
CONTENTS (continued)
5.
4.5.3.2. Evidence for Carcinogenicity - Animal
Studies (IARC, 1982) 4-78
4.5.3.3. Evidence for Activity in Short-Term
Tests (IARC, 1982) 4-78
4.5.4. Endrin 4-79
. ,£
4.5.4.1. Evidence for Carcinogenicity - Human
Exposure (IARC, 1974) .... 4-79
4.5.4.2. Evidence for Carcinogenicity - Animal
Studies (IARC, 1982) 4-79
4.5.4.3. Evidence for Activity in Short-Term
Tests (NTP, 1985) 4-79
4.5.5. Endosulfan 4-79
4.5.5.1. Evidence for Carcinogenicity - Human
Exposure (NCI, 1978) 4-79
4.5.5.2. Evidence for Carcinogenicity - Animal
Studies (NCI, 1978) 4-80
4.5.5.3. Evidence for Activity in Short-Term
Tests (NTP, 1985) 4-80
4.5.6. Hexachlorocyclopentadiene 4-80
4.5.6.1. Evidence for Carcinogenicity - Human ,ji
Exposure (U.S. EPA, 1984b) 4-80
4.5.6.2. Evidence for Carcinogenicity - Animal
Studies (U.S. EPA, 1984b) 4-81
4.5.6.3. Evidence for Activity in Short-Term
Tests (U.S. EPA, 1984b) 4-81
4.5.7. Isodrin . 4-81
4.5.7.1. Evidence for Carcinogenicity - Human
Exposure (HSDB, 1985) 4-81
4.5.7.2. Evidence for Carcinogenicity - Animal
Studies (HSDB, 1985) 4-81
4.5.7.3. Evidence for Activity in Short-Term
Tests (HSDB, 1985). . . 4-81
RISK ESTIMATION FROM ANIMAL DATA. . 5-1
5,1. SELECTION OF DATA 5_1
5.1.1. Chlordane. . 5-1
5.1.2. Heptachlor/Heptachlor Epoxide 5-6
5.2. CHOICE OF EXTRAPOLATION MODELS 5-6
-------
CONTENTS (continued)
5.3. INTERSPECIES DOSE CONVERSION. 5-17
5.4. ESTIMATION OF CARCINOGENIC POTENCY 5-18
5.4.1. Chlordane 5-18
5.4.2. Heptachlor 5-21
5.4.3. Heptachlor Epoxide 5-21
6. SUMMARY . 6-1
6.1. QUALITATIVE DATA 6-1
6.1.1. Human Studies 6-1
6.1.1.1. Case Reports 6-1
6.1.1.2. Epidemiologic Studies .... 6-1
6.1.2. Animal Studies 6-2
6.1.2.1. Chlordane .... 6-2
6.1.2.2. Heptachlor/Heptachlor Epoxide ....... 6-3
6.1.3. Supporting Evidence .... 6-4
6.1.3.1. Mutagenicity 6-4
6..1.3.2. Structural Relationship , 6-5
6.2. QUANTITATIVE ANALYSIS .... 6-5
6.2.1. Chlordane 6-5
6.2.2. Heptachlor .... 6-6
6.2.3. Heptachlor Epoxide . 6-6
7. REFERENCES 7~l
APPENDIX A: THE MUTAGENICITY ASSESSMENT OF CHLORDANE -
ADDENDUM TO THE REGISTRATION STANDARD A-l
APPENDIX B: THE MUTAGENICITY ASSESSMENT OF HEPTACHLOR/HEPTACHLOR
EPOXIDE - ADDENDUM TO THE REGISTRATION STANDARD B-l
-------
TABLES
1. Chemical and physical properties of chlordane,
heptachlor, and heptachlor epoxide 3-2
2. Species differences in the metabolism of
three chemicals 4-19
3. Elimination of a single oral dose of 14C-trichloroethylene
in Osborne-Mendel rats and B6C3F1 mice A 'J?-21
4. Elimination of a single oral dose of 14c-trichloroethylene . :
in Alderley Park Wistar rats and Swiss-Webster mice 4-22
5. Summary of mouse dietary carcinogenicity tests for chlordane . . . 4-35
6. Summary of rat dietary carcinogenicity tests for chlordane .... 4-36
7. Incidence of liver lesions and tumors in CD-I mice following
dietary administration of chlordane . 4-39
8. Incidence of hepatocellular carcinoma in mice following
chronic dietary administration of chlordane 4-40
9. Neoplastic and/or toxic lesions of the liver in ICR mice
fed chlordane . . . . 4.43
10. Liver tumors and nonneoplastic lesions in Fischer 344
rats fed chlordane ,4-47
11. Liver lesions in Fischer 344 rats fed chlordane for 26
or 52 weeks 4-49
12. Liver neoplasms in male Fischer 344 rats fed chlordane 4-49
13. Summary of mouse dietary carcinogenicity tests for heptachlor
and heptachlor epoxide L4'r52
14. Summary of rat dietary carcinogenicity tests for heptachlor and
heptachlor epoxide . . '4-53
15. Incidences of liver lesions in C3H mice treated with heptachlor
or heptachlor epoxide 4-54
16. Incidences of hepatocellular carcinoma in C3H mice treated
with heptachlor or heptachlor epoxide 4-54
17. Incidence of hepatocellular carcinoma in B6C3F1 mice following
chronic dietary exposure to heptachlor/chlordane mixture .... 4-56
vii
-------
TABLES (continued)
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Incidence of nodular hyperplasia in CD-I mice exposed to
heptachlor/heptachlor epoxide (25:75) mixture .
Incidence of hepatic carcinoma in CD-I mice following
chronic dietary exposure to heptachlor epoxide (25:75)
Incidences of neoplastic nodules in Osborne-Mendel rats
following chronic dietary exposure to heptachlor .
Incidence of hepatic carcinoma and neoplastic nodules in CFN rats
following chronic dietary exposure to heptachlor epoxide
Cancer data sheet for derivation of potency of chlordane from
hepatocellular carcinomas in female mice (IRDC)
Cancer data sheet for derivation of potency of chlordane from
hepatocellular carcinomas in male mice (IRDC)
Cancer data sheet for derivation of potency of chlordane from
Cancer data sheet for derivation of potency of chlordane from
hepatocell ul ar carcinomas in female mice (NCI)
Cancer data sheet for derivation of potency of chlordane from
liver adenomas and carcinomas in male rats (RIASBT)
Cancer data sheet for derivation of potency of heptachlor from
hepatocellular carcinomas in male mice (Davis/Reuber)
Cancer data sheet for derivation of potency of heptachlor from
hepatocellular carcinomas in female mice (Davis/Reuber). .....
Cancer data sheet for derivation of potency of heptachlor from
hepatocellular carcinomas in male mice (NCI)
Cancer data sheet for derivation of potency of heptachlor from
Cancer data sheet for derivation of potency of heptachlor epoxide
from hepatocell ul ar carcinomas in female mice (Davis/Reuber). . . .
Cancer data sheet for derivation of potency of heptachlor epoxide
from hepatocellular carcinomas in male mice (Davis/Reuber) ....
Cancer data sheet for derivation of potency of heptachlor epoxide
from hepatic carcinomas in female rats (IRDC/Reuber)
viii
4-58
4-58
4-60
4-62
5-2
5-3
5-4
5-5
5-7
5-8
5-9
5-10
5-11
5-12
5-13
5-14
-------
TABLES (continued)
34. Cancer data sheet for derivation of potency of heptachlor epoxide
from hepatic carcinomas in male mice (IRDC/Reuber) ...
35. Cancer data sheet for derivation of potency of heptachlor epoxide
from hepatic carcinomas in female mice (Witherup/Reuber)
5-15
5-16
36. Human potency estimates by chemical
5-19
-------
FIGURES
1. The structures of chlordane and heptachlor . . . .
2. Proposed metabolic pathways of chlordane in rats .
3. Proposed metabolic pathway of heptachlor in rats .
4. '.Chemicals selected for structure-activity analysis
4-2
4-12,
4-14
4-75
-------
PREFACE
The Carcinogen Assessment Group within the Office of Health and Environ-
mental Assessment has prepared this carcinogenicity assessment at the request
of the Hazard Evaluation Division within the Office of Pesticides and Toxic
Substances. The assessment contains a qualitative and quantitative evaluation
of the carcinogenicity data available as of November 1985.
XI
-------
ABSTRACT
Evidence pertaining to the carcinogenicity of chlordane and heptachlor/
heptachlor epoxide is reviewed and evaluated. This report covers studies
completed before 1985. Case reports and epidemiologic studies of pesticide
applicators and pesticide manufacturing,workers are reviewed, but because of
methodologic limitations, these studies establish neither a positive nor a
negative association between cancer and chlordane or heptachlor/heptachlor
epoxide exposure. A number of independent studies of laboratory animals,
however, demonstrates that chlordane and heptachlor/heptachlor epoxide cause
liver cancer in mice and rats. Based on the accumulated evidence, chlordane
and heptachlor/heptachlor epoxide are classified as probable human carcinogens,
Group B2 using EPA's Guidelines for Carcinogen Risk Assessment. The carcino-
genic potency of chlordane and heptachlor/heptachlor epoxide is estimated by
fitting mathematical models to the laboratory animal data. These estimates
indicate that chlordane and heptachlor/heptachlor epoxide are rather potent
carcinogens, ranking in the second quartile of potential carcinogens evaluated
by EPA's Carcinogen Assessment Group. A separate mutagenicity assessment of
chlordane and heptachlor/heptachlor epoxide is attached as an appendix to this
report. The report also includes an extensive list of references pertinent
to the carcinogenicity of chlordane and heptachlor/heptachlor epoxide.
-------
AUTHORS, CONTRIBUTORS, AND REVIEWERS
The Carcinogen Assessment Group (CAG) within EPA's Office of Health and
Environmental Assessment is responsible for the preparation of this carcino-
genicity assessment. The document was written by Dynamac Corporation, Ro'ck^i^
ville, Maryland, under EPA contract number 68-02-4131, and was reviewed by tfhe
staff of the CAG. The CAG provided overall direction and coordination for
document production (Dharm V. Singh, Project Officer). .
PRINCIPAL AUTHORS
Finis L. Cavender
Brion T. Cook
Norbert P,. Page
Dynamac Corporation
Rockville, MD
CONTRIBUTING AUTHORS
Vincent James Cogliano
Carcinogen Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Aparna M. Koppikar
Carcinogen Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
D.E.B. Potter
Dynamac Corporation
Rockville, MD
Dharm V. Singh
Carcinogen Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
xiii
-------
Lawrence R. Valcovic
Reproductive Effects Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
REVIEWERS
;_The following individuals reviewed earlier drafts of this document and
provided valuable comments:
Michael A. Berry
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, NC
Jerry N. Blancato
Exposure Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
James W. Holder
Carcinogen Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
W. Bruce Peirano
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Cincinnati, OH
Dharm V. Singh
Carcinogen Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
xiv
-------
1. CONCLUSIONS
Based on the accumulated evidence, chlordane is a probable human carcino-
gen, classified in Group B2 under the EPA's Guidelines for Carcinogen Risk
Assessment. Animal studies provide sufficient evidence for carcinogenic-ityiv
chlordane increased the incidence of liver carcinomas in C57B1/6N, CD-I,--'and
B6C3F1 mice; liver adenomas and hemangiomas in ICR mice; and liver adenomas-In
Fischer 344 rats. Epidemic!ogic studies provide inadequate evidence due to
methodology and data limitations.
According to the criteria in the guidelines, the above evidence puts
chlordane in Group B2. However, the guidelines allow for the possibility of
downgrading the classification 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. In the case of chlordane '"r
./'i
these conditions do not apply, since chlordane caused tumors in C57B1/6N mice--
which do not have a high background rateand caused tumors in rats as wellv-
Other pertinent evidence includes highly significant tumor responses, up to-77
percent increased incidence over controls, increased incidence in both males
and females, increased incidence at medium and high doses, a dose-related in-
crease in the proportion of malignant tumors, and induction of tumors by struc-
turally related chemicals. In light of these factors, downgrading is clearly
not warranted, and chlordane remains in Group B2.
For chlordane, the carcinogenic potency, averaging estimates from the most
sensitive species tested, is 1.3 per mg/kg/day. The potency using the most
sensitive sex and strain is 4.7 per mg/kg/day. These are plausible upper
bounds for the increased cancer risk from chlordane, meaning that the true risk
is not likely to exceed these estimates and may be lower. The molecular potency
1-1
-------
index for chlordane is 5.2 x 102 per mmol/kg/day. This places chlordane in the
second (upper middle) quartile of suspect carcinogens ranked by the Carcinogen
Assessment Group (CAG).
Heptachlor/heptachlor epoxide is a probable human carcinogen, classified
in Group B2 under the 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 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 tunor responses, increased incidence in both males and females,
increased incidence at medium and high doses, and induction of tumors by struc-
turally related chemicals. In light of these factors, downgrading is clearly
not warranted, and heptachlor/heptachlor epoxide remains in Group B2.
For heptachlor, the carcinogenic potency, averaging estimates from the
most sensitive species tested, is 4.5 per mg/kg/day. The potency using the
most sensitive sex and strain is 14.9 per mg/kg/day. These are plausible upper
bounds for the increased cancer risk from heptachlor, meaning that the true
risk is not likely to exceed these estimates and may be lower. The molecular
potency index for heptachlor is 1.7 x 103 per mmol/kg/day. This places hepta-
chlor in the second quartile of suspect carcinogens ranked by the CAG.
For heptachlor epoxide, the carcinogenic potency, averaging estimates from
the most sensitive species tested, is 9.1 per mg/kg/day. The potency using the
*!,
most sensitive sex and strain is 36.2 per mg/kg/day. These are plausible upper
1-2
-------
bounds for the increased cancer risk from heptachlor epoxide, meaning that the
true risk is not likely to exceed these estimates and may be lower. The molec-
ular potency index for heptachlor epoxide is 3.5 x 103 per mmol/kg/day. This
places heptachlor epoxide in the most potent quartile of suspect carcinogens
ranked by the CAG.
1-3
-------
-------
2. INTRODUCTION
In preparing this assessment, the CAG has considered the Drinking Water
Criteria Document for Heptachlor, Heptachlor Epoxide, and Chlordane recently
drafted by the Environmental Criteria and Assessment Office (U.S. EPA, 1985a).
Much of the prior material presented in the 1977 Risk Assessment of Chlordane
and Heptachlor, prepared by the CAG (U.S. EPA, 1977) has also been incor-
porated into this current assessment. As guidance in preparing this assess-
ment, the EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986)
were followed.
2-1
-------
-------
3. GENERAL BACKGROUND INFORMATION
Chlordane and heptachlor are structurally related chlorinated hydrocarbon
insecticides that have been and/or are being used to control termites and pests
on field crops. They are synthesized by the condensation of two cyclopenta-
diene rings (hexachlorocyclopentadiene and cyclopentadiene) to form chlordane.
In the second step (chlorination), the reaction conditions can be adjusted to
give either technical chlordane or technical heptachlor.
Technical grade chlordane is a light-yellow-colored liquid consisting of
approximately equal parts cis- and trans-isomers, along.with chlordene isomers,
heptachlor, and their impurities. Technical grade heptachlor is a white crys-
talline solid containing approximately 73 percent heptachlor and 22 percent
transchlordane. Heptachlor epoxide is a degradation product of heptachlor and
has an estimated vapor pressure of 3 x 10~4 mmHg at 25°C.
3.1. CHEMICAL AND PHYSICAL PROPERTIES
Those properties most relevant to carcinogenesis and human exposure are
listed in Table 1.
3.1.1. Identification
Chlordane
Molecular
Formula:
Molecular
Weight:
CAS No.:
Heptachlor
Cl
Heptachlor Epoxide
409.76
57-74-9
Cl Cl H
C10H5C17
373.32
76-44-8
C10H5C170
389.32
1024-57-3
3-1
-------
LU
Q
X
g
LU
3
rn
o
t
0.
LU
rc
o
2=
o;
0
rn
^r"
»
D.
LU
ar
n
iii
^y*
-
o.
o
z
_j
o
t 4
LU
""I
Ml1!
O
t 1
LU
CO
O1
0
o
LO
rH
IO
r-H
1
0
r-H
CO
3T
PS
pE
LO
r-H
1
t-H
4-5
O
O
LO
^-
t-H
LO
CO
t-H
c
^
CM
[ *
03
O
O
LO
r*.
t-H
4-3
c:
r
O
CL
CO
C
r
r-«t
l«
o
CO
{
03
1
o
o
LO
CM
t^
03
O>
rc
£E
^.
1
0
r-H
t^
O)
J^
03
^
C
r
_J
Ol
E
o
o
LO
LO CM
O
O
CO
f
£_
^-*. CO
c- E
Q) 0
E v>
O !-
in i
r in
1 C
in 03
r- t-
O 4-5
- -*~ -
CJ 0
O 0
t~- LO
O 0
t-H t-H
1 1
IO .3-
O 0
rH i-H
[ %
C
i
o
CL
01
C
r
40
r
cu
s:
*-*
£
tl
3
O.
c-
3
O
c:
VX
CI
3
CT>
LO
t-H
|
p^.
LO
r-H
CJ
O
LO
CM
,| ^
03
co
IO
r-H
|
cn
LO
r- 1
>^
i ^
r
>
03
C_
cn
o
r
1-
r
O
CO
CL
co
X
CO
o
o
LO
CM
[ ^
03
Ol
~r*
^
^
^j-
1
O
rH
X
CO
CJ
o
LO
CM
1 ^
03
0)
C
gn
E
LO
1
O
t-H
X
t-H
CO
£_
3
in
CO
c_
CL
£_
o
CL
03
co
0
1 ^
03
t-
O)
03
s
£=
r
_J
^*^,
o>
E
vo
LO
O
o
c_
03
C
r
CJ
O
LO
CM
1 ^
03
_I
C71
^L
CTt
..^
1 ^
r"
r-
pi-
e~\
3
O
CO
1 ^
03
>>
c:
03
E
c
r
I-M
O3
0
r
f
g
O
a>
c_
0
«t-
tn
4->
c:
CO
>
r
O
in
o
r
C
03
a>
t-
o
_£_
4-i
r- 0
3 T-
t_ 40 W>
O CO 03 4->
0} CM O 03 i
4-> O
O3 LO -i T3 O3
--^ £_ T3 03 O
CO 4-5 C r- O
3. .. 0 Q. £_
W r T- -O
LO o u "re j=
o
c a>
03 rc
co g
H-^.
CL CDO
E to
i «3- r~-
i a~i
CM TD
vo LO c .
O rH 03
=* II
HO
LO _J 0
1 ~-, E LO
LO o> CLCM
to E CL
4^
CM rH rH 03
T3
c: tm
nsco rc
E
E--. E
a. a>
CL E 0
VO
r-l LO 1
1 CO
1 LO -O
to LO c
* rH 03
* II
II CJ
LO _J O
1 ~-~ E LO
r^ a» CLCM
CO E Q.
4-^
CO i-H t-H 03
-o
" ^ d CO
<_ osco rc
a> - E E
E t- E *>. E
O O> CL O>
in « E CL E 0
r- .G o x: to
1 4-> (/) 4-* r^- tO 1^-.
V> CL-I Q. l~~.
C O) 1 O) CT> -O
03 "O tn "O LO ID C
t- T- r- 1 03
4-» E 0 E II
~^ *~* II 0
i 1 rH 10
£_ t_ ---. E LO
J= E -C E CM CO CLCM
O O CO E CL
CO t- CO t- 4-5
CM 1- CO
O3 i O CO C- C
> O3 E O O3 O
LU .C M- _l CL O
03
LO
CO
O^
II
A
^£
a.
LU
CO
ro
E
o
c_
"O M-
O)
T3 -O
I QJ
> 4J
o a.
t_ 03
°- 3
4^
O
C
LU
03 O
4-5 a;
03 rr>
a o
03 CO
3-2
-------
3.1.2. Synonyms
Each chemical has a number of synonyms and trade names (U.S. EPA, 1985a).
1) Chlordane
l,2,4,5,6,7,8,8-octachlor2,3,3a,4,7,7a^hexahydro-4,7-methano
-IH-indene
l,2>4,5,6,7,8,8-octachloro-4J7-methano-3a,4,7,7a-tetra-hydroindane
l,2,4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4s7-methanindan
Dichlorodene Chi or Kil
Octachlor Corodane
Topiclor 20 Ortho-Klor
Velsicol 1068 Synklor
Dpwchlor Belt
Kypchlor Niran
2) Heptachlor
l,4,5,6,7,8,8-heptachlor-3a,4,7,7a-tetrahydro-4,7-methanoindene
3-chloro-chlordene
3,4,5,6,7,8,8a-heptachlorodicyclopentadiene
Heptagran Velsicol 104
Rhodiachlor E3314
Drinox Heptamul
3) Heptachlor epoxide
2,3,4,5,6,7,7-heptachloro-la,lb,5,5a56,6a-hexahydro-2,5-
methano-2H-indene(l,2-b)oxirene
l,4,5,6,7,8,8-heptachloro-2,3-epoxy-3a,4,7,7a-tetrahydro-
4,7-methanoindan
Epoxyheptachlor
Velsicol 53-CS-17
ENT 25
584
3-3
-------
3.2. USES
Chlordane and heptachlor, both contact insecticides, have generally had
the same uses in the past, primarily in a wide variety of soil applications
(U.S. EPA, 1977; U.S. EPA, 1985a, b). The main uses for chlordane have been
in the control of cutworms, ants, root weevils, rose beetles, grasshoppers,
grubs, and termites. It had been used mainly in agriculture (25 percent), home
use such as turf treatment, garden, and household use (25 percent), and soil
treatment around buildings (50 percent). Technical grade chlordane is a varia-
ble mixture of chemicals that typically consists of 24 percent trans-chlordane,
21.5 percent chlordene isomers, 19 percent cis-chlordane, 10 percent heptachlor,
7 percent nonachlor, and 18.5 percent other miscellaneous impurities.
Heptachlor has some fumigant action and has been used primarily to con-
trol termites and insects that attack field crops. Heptachlor has been used
in agriculture (about 70 percent) and in termite control (about 28 percent).
Technical heptachlor consists of approximately 73 percent heptachlor, 22 per-
cent trans-chlordane, and 5 percent nonachlor. Heptachlor epoxide is not
commercially available in the United States, but it is a degradation product
of heptachlor (U.S. EPA, 1985a, b).
3.3. ROUTES AND PATTERNS OF EXPOSURE
Both chlordane and heptachlor are chemically stable and persist in the
soil, although heptachlor is slightly less persistent than chlordane in soils.
Heptachlor is oxidized in soil to heptachlor epoxide, which then is more
persistent than chlordane (U.S. EPA, 1977). Residues of chlordane have been
detected for up to 10 years after a single application.
Because of their long history of use in a wide variety of applications,
residues of chlordane, heptachlor, their oxidation products (oxychlordane and
heptachlor epoxide), and impurities (mainly nonachlor) are found in foods,
3-4
-------
soils, sediments, wildlife, and in air near application sites.
Heptachlor in soils, water, and plants is metabolized to heptachlor epox-
ide and to 1-hydroxychlordene epoxide; chlordane and nonachlor are apparently
not metabolized in these systems. In animals, heptachlor is metabolized to
heptachlor epoxide, which is well retained in lipid tissue. Chlordane in
animals is metabolized to oxychlordane, which is also stored with no further
metabolism. Nonachlor is apparently not metabolized and is accumulated by
animals.
Food residues have been measured in the FDA Total Diet Program as well
as in the USDA and FDA Food Surveillance Program. The latter programs are
designed to sample foods shipped in interstate commerce. In the total diet
study, chlordane and oxychlordane were found only at the lower limit of detec-
tion (0.003 ppm in the total diet), if at all, but in the surveillance survey
residues with a somewhat higher detection limit were found in root vegetables,
meat, and fish in 7 to 17 percent of the samples. Heptachlor was rarely detec-
ted, and heptachlor epoxide was found sporadically in dairy products, meat,
fish, and poultry at the lower detection limit of 0.003 ppm in the total diet
survey. However, in the Food Surveillance Program, heptachlor epoxide residues
greater than 0.03 ppm were found in 19 percent of red meat samples, 17 percent
of poultry, and 14 percent of dairy products. Based on these data, it is
estimated that human consumption of heptachlor epoxide is on the order of
1.0 Mg/day for adults in marketed foods of widespread distribution in the
United States. Oxychlordane, heptachlor, chlordane, and nonachlor appear
sporadically in foods. Chlordane and nonachlor are also found in freshwater
fish. Consumption by sport fishermen may be as high as 2.2 yg/day.
During 1970 and 1972, two national ambient air monitoring surveys sampled
pesticides at a number of sites across the United States. Heptachlor, chlor-
3-5
-------
dane, heptachlor epoxide, and oxychlordane (in order of decreasing frequency)
were detected. Chlordane and heptachlor were detected in a study of suburban
areas in 1976. The average air concentration of heptachlor in the community
with the highest reading was 6.3 ng/m3. Heptachlor, and to a lesser extent
chlordane and heptachlor epoxide, are widespread in ambient air, with typical
mean concentrations on the order of 0.5 ng/m3. The amounts of chlordane and
heptachlor in air above treated fields were reported to be as high as 16 ng/m3,
even 3 weeks after treatment. Chlordane concentrations as high as 72 ppm have
been found in house dust for up to 4 years after fumigation treatment.
The data available for residues in human tissue are more extensive and
reliable than the data for food residues. The principal source of this data
has been the National Human Monitoring Program conducted between 1970 and 1974
(U.S. EPA, 1977). Heptachlor epoxide was found in 97 percent of approximately
7,000 samples of human tissue tested. Similarly, oxychlordane was found in
approximately 97 percent of 5,000 samples, and about 98 percent of 755 samples
showed residues of trans-nonachlor. The geometric mean levels of heptachlor,
oxychlordane, and nonachlor are about 0.085, 0.11, and 0.11 ppm, respectively.
There is some evidence that chlordane may undergo sensitized photolysis
in aquatic media. The direct photolysis of chlordane in ambient aquatic media
without a sensitizer; oxidation processes involving singlet oxygen and peroxy/
radical; hydrolysis; and biodegradation in ambient aquatic media are not likely
to be significant fate-determining processes. The three processes that are
likely to determine the fate of this compound in aquatic media are evaporation,
sorption, and bioaccumulation. The evaporative half-life of this compound from
1 m depth of water was estimated to be 28 to 33 hours. Thus, although the evap-
oration from aquatic media may not be rapid, it could be significant in the ab-
sence of any other faster processes. Chlordane is expected to be significantly
3-6
-------
sorbed from the aquatic phase to suspended particles and sediment, which may
..play a significant role in determining its aquatic fate. ;
Heptachlor may undergo significant photolysis in ambient aquatic media,
since laboratory studies have demonstrated photolytic decomposition in 1 week
and complete decomposition in 2 weeks. The singlet oxygen reaction with hep-
tachlor in aquatic media may be significant, with the half-life of heptachlor
for this reaction estimated to be about 1 day. The hydrolysis of heptachlor in
aquatic media is also an important process, with an estimated hydrolytic half-
life in the range of 1 to 3 days. The volatilization half-life of heptachlor
from aquatic media is estimated to be 2 to 10 days from pond, river, and lake
water. Although most estimates indicate that the evaporative half-life of
heptachlor in aquatic media appears to be longer than that of chlordane (2 to
10 days vs. 1 to 2 days), Huang (1970) reported that the evaporation rate of
heptachlor from aquatic media is faster than for chlordane. Heptachlor may be
significantly sorbed onto suspended particles and sediments present in aquatic
media, which may play a significant role in determining the fate of heptachlor
in aquatic media. The biodegradation rate of heptachlor in aquatic media is
slower than the rate of hydrolysis.
The fate of heptachlor epoxide in aquatic media has not been comprehen-
sively studied. Photolysis, oxidation, or hydrolysis are not expected to be
significant fate-determining processes for heptachlor epoxide in aquatic media.
Biodegradation also does not appear to be a significant process. The two pro-
cesses that are likely to determine the fate of heptachlor epoxide in aquatic
media are volatilization and sorption. The rate of volatilization of hepta-,,
chlor epoxide from aquatic media is much slower than for heptachlor. However,
it is difficult to make a reasonable estimate of the evaporative half-life for
this compound because of the lack of appropriate data. The sorption of this
3-7
-------
compound on suspended particulate matter or sediment may be a moderately signi-
ficant process (U.S. EPA, 1985a).
In summary, exposure to chlordane and heptachlor can occur to a variety
of human populations through direct use of the materials or indirectly through
their entry into environmental pathways. Potentially exposed populations con-
sist of workers in manufacturing and formulating plants; field and home appli-
cators of the pesticides; and the general population through atmospheric,
aquatic, or food residues.
3-8
-------
4. HAZARD IDENTIFICATION
4.1. METABOLISM AND PHARMACOKINETICS
Chlordane and heptachlor are related bicyclic chlorinated hydrocarbons.
In fact, heptachlor is a minor metabolite of chlordane. Chlordane has two
isomers, cis and trans, and the chemical structures of chlordane and heptacblor
are given in Figure 1. As there are virtually no data on the metabolism and
pharmacokinetics of chlordane and heptachlor in humans, the review in this..sec-
tion consists of animal studies only.
4.1.1. Absorption
4.1.1.1. ChlordaneQuantitative data on the absorption of chlordane from the
gastrointestinal tract of laboratory animals were not available. However, the
systemic toxicity of the insecticide to laboratory animals following oral,
dermal, and inhalation exposure indicates that some absorption does take place.
That chlordane is absorbed can also be inferred from excretion data. Chlordane
consists of a mixture of components, including the cis- and trans-isomers. A
high purity (98+ percent) formulation of the insecticide, designated HCS-3260,
contains cis- and trans-chlordane in 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 body
weight (bw) by gavage, or a single oral dose of each 14C-labeled isomer at 0.2
mg/kg bw (Barnett and Dorough, 1974). Elimination of radioactivity in the
urine over 7 days was 6 percent for females and 2 percent for males following
14C-HCS-3260 treatment. In other studies, following treatment with cis- and
trans-chlordane-14C, female rats eliminated 8.5 and 5 percent of the admini-
stered radioactivity, respectively. These results indicate that at least 2
to 8.5 percent of the administered chlordane dose was absorbed by the gastro-
intestinal tract of rats. Based upon a study in which a male rabbit was given
4-1
-------
Cl
Cl
cis-chlordane
trans-chlordane
Figure 1. The structures of chlordane and heptachlor,
4-2
-------
14C-HCS-3260 in a dietary concentration of 25 ppm (25 mg/kg diet) for 2 days,
33 percent of the radioactivity was excreted in the urine and 21 percent in
the feces 24 hours after dosing (Barnett and Dorough, 1974). Thus, it would
appear that at least 33 percent of the chlordane was absorbed by the rabbit as
compared with only 2 to 8.5 percent for the rat after oral administration.
Pulmonary absorption of an unspecified amount of 14C-chlordane (11,500
dpm/yg) in 20 pL ethanol administered as an aerosol intratracheally to female
Sprague-Dawley rats was measured by Nye and Dorough (1976). No intact 14C-
chlordane was detected in exhaled air. A peak blood concentration of radio-
activity of approximately 4 percent of the applied dose was reached in <5 min-
utes.
Ambrose et al. (1953b) reported in 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 in ethyl alcohol, indicating greater absorption with
the lipophilic vehicle. *2.-
4.1.1.2. HeptachlorLimited information was found on the absorption of hep-ta-
chlor following ingestion by animals. However, the systemic toxicity of hepta-
chlor following oral, dermal, or inhalation exposure is an indication of the
absorption of the insecticide. The U.S. EPA (1980b) reviewed an abstract of a
Soviet study (Mizyukova and Kurchatov, 1970) and reported that heptachlor ad-
ministered intragastrically in a single oral dose of 120 mg/kg bw to rats was
detected in blood within 0.5 to 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 in open-air cages so that they were exposed to
the ambient air of Stoneville, MS, an area where insecticides had been heavily
used. Control groups of male and female rabbits (10 each) were housed in a
4-3
-------
room at Mississippi State University, a low pesticide use area. The average
" level of heptachlor epoxide (heptachlor was either not measured or not detec-
ted) in Stoneville air was 1.86 ng/m3; the air at Mississippi State was not
sampled. Heptachlor epoxide residue levels in adipose tissue of the test
rabbits averaged 0.039 ppm, as 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 Stoneville area.
4.1.2. Tissue Distribution
4.1.2.1. ChlordaneThe tissue distribution of 14C-HCS-3260, cis- or trans-
chlordane-14C, and the metabolite oxychlordane in male and female rats following
treatment with single oral doses was compared by Barnett and Dorough (1974).
At 1 day following doses of 0.05 to 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 to 0.08 ppm), while the concentrations of
radioactivity in fat were somewhat higher (the average for all treatments was
approximately 0.47 ppm). Male and female rats treated with 0.1 mg 14C-HC-3260/
kg bw had higher tissue residue levels of radioactive equivalents in 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 radio-
activity in fat than male rats after treatment with any preparation. At 7 days
after dosing with 1.0 mg 14CrHCS-3260/kg bw, radioactivity in all tissues
declined; radioactivity in fat declined to 2.0 ppm for females and 1.19 ppm for
males. Slightly more radioactivity was present in rat tissues following oral
doses of trans- as compared with cis-chlordane-l^C.
When 14C-HCS-3260 was administered to male rats in the diet at 5 ppm (5
mg/kg diet) for 56 days, the tissue distribution of radioactive equivalents
was 0.42, 0.91, 0.55, 0.68, and 14.73 ppm for muscle, brain, kidney, liver,
4-4
-------
and fat, respectively (Barnett and Dorough, 1974). After discontinuing treat-
ment for 28 and 56 days, the concentration of radioactivity in 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 radio-
activity in all tissues resulting from the absorption of transrather than from
cis-chlordane-14C occurred in female rats treated with 25 ppm (25 mg/kg diet).
Analysis of the nature of the radioactivity revealed that approximately 30 to
60 percent of the radiocarbon was associated with oxychlordane.
Ambrose et al. (1953a) found that the peri renal fat of male rats contained
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 either 50 to 220 ppm
(50 to 200 mg/kg diet) cis- or trans-chlordane, 100 ppm (100 mg/kg diet) of
fixed ratios of the isomers from 9:1 trans:cis to 1:9, 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 approximately 6, 10, and 23 pg/g lipid of transchlordane,
respectively. Much greater concentrations of oxychlordane than trans-chlordane
were stored in adipose tissue by females (approximately 104, 202, and 471 pg/g
lipid) and males (approximately 5, 15, and 22 ug/g lipid) after being fed
trans-chlordane. The feeding of cis-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 approximately 7 times more
oxychlordane than the parent compound in adipose tissue. The results of feed-
4-5
-------
ing ratios of cis- 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 cis-, trans-, or technical
grade chlordane at 0 to 150 ppm (0 to 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; and
pigs were fed diets containing the cis- or trans-isomer at 300 ppm (300 mg/kg
diet) for 90 days. The respective levels of oxychlordane in the fat of these
species were 0.2 to 150, 3 to 24, and 36 to 90 ppm, respectively.
Rabbits received trans-chlordane-14C daily per os in doses of 14.3 mg/
rabbit/day for 10 weeks (Poonawalla and Korte, 1971). Two weeks after treat-
ment was discontinued, low levels of radioactivity were detected in kidney
(0.05 percent of administered dose), liver (0.52 percent), heart (0.09 per-
cent), lung (0.04 percent), spleen (0.03 percent), testes (0.03 percent), and
brain (0.04 percent) tissue. Higher levels were found in adipose tissue (2.59
percent in abdominal fat, 1.53 percent in subcutaneous fat) and in muscle (5.68
percent). Barnett and Dorough (1974) found that the tissues of a rabbit fed
14C-HCS-3260 (25 mg/kg diet for 2 days) contained 14C-oxychlordane and 14C-
dichlorochlordene. .,,.._.
The distribution of radioactivity in the tissues of rabbits administered
cis- or trans-chlordane-14C orally in four doses, one capsule given every 4 days
containing 100 mg per capsule, were as follows: fat > kidney > muscle > liver
> brain (cis-); kidney > fat > liver > muscle > brain (trans-) (Balba and Saha,
1978). The majority of the radioactivity in the tissues was associated with
oxychlordane, regardless of which isomer was administered.
The mobilization of chlordane or its metabolites from adipose tissue of
4-6
-------
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 chlor-
dane 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 percent of administered
radioactivity/tissue) in selected tissues of rats following the previously
described intratracheal administration of 14C-chlordane (Nye and Dorough,
1976) was 23.9 percent in lung, 19.6 percent in liver, 0.3 percent in kidney,
and 0.1 percent in the bladder and its contents.
4.1.2.2. HeptachlorAn abstract of a Soviet study (Mizyukova and Kurchatov,
1970) reported that following a single intragastric dose of 120 mg/rat hepta-
chlor 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 in blood, liver, and fat and persisted in the
adipose tissue for 3-6 months.
Radomski and Davidow (1953) studied the tissue distribution of heptachlbr
epoxide in rats and dogs following oral administration of heptachlor. Nine
rats of either sex received dietary concentrations of heptachlor at 30 to 35
ppm (30 to 35 mg/kg diet) for 2 months. Heptachlor epoxide levels in the fat
of six female rats averaged 384 pg/g tissue, whereas fat in male rats con-
tained an average of only 43 pg/g tissue. Much lower levels of .heptachlor
epoxide were detected in liver (0.4 to 33 pg/g), kidney (0 to 2l'pg/g), and
muscle (0 to 27 pg/g) tissue. None was detected in brain tissue. A similar
pattern of distribution was observed in three female dogs that received 1 mg
heptachlor/kg bw in capsules daily for 12 to 18 months. Fat contained an aver-
age heptachlor epoxide concentration of 636 pg/g; liver, 36 pg/g; kidney,
4-7
-------
7.5 yg/g; and muscle, 13 yg/g. Again, no heptachlor epoxide was detected
in the brain. Small amounts of unchanged heptachlor were detected in the fat
of dogs when higher levels of the insecticide were administered. Administra-
tion of 1 mg heptachlor/kg bw to four dogs resulted in no detectable heptachlor
in fat after 26 weeks, while three of eight dogs, surviving 6 to 18 weeks, dosed
with 3 mg/kg bw daily had <1 yg heptachlor/g fat tissue. A daily dose of 5 mg/
kg bw resulted in an average concentration of heptachlor in fat of 6.5 yg/g. ,
This dose was fatal to the dogs within 2 to 11 weeks.
Radomski and Davidow (1953) also determined the rate of accumulation and
disappearance of heptachlor epoxide in 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 approximately 225 yg/g fat in 8 weeks, as compared with only 50 yg/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 Radomski and Davidow (1953) failed to detect heptachlor epoxide
in rat and dog brain tissue after heptachlor treatment, Yamaguchi et al*
(1979), using a gas chromatographic (GC) method, detected 3.15 ppm heptachlor
epoxide in brain tissue of rats 5 hours after an intraperitoneal injection of
200 mg heptachlor/kg bw.
4.1.3. Metaboli sm
4.1.3.1. ChlordaneThe metabolism of chlordane has been well studied in rats
and rabbits in vivo and in vitro. Oxychlordane was a metabolite of both cis-
and trans-chlordane (Street and Blau, 1972). A metabolic pathway, based upon
4-8
-------
in vitro studies with rat liver homogenates, was proposed in which either iso-
mer is dehydrogenated to 1,2-dichlorochlordene with subsequent epoxidation to
oxychlordane. In these experiments, trans-chlordane was converted to oxychlor-
dane at a sevenfold greater rate than was the cis-isomer.
Barnett and Dorough (1974) isolated seven radioactive metabolites (in tf
addition to the respective unchanged parent compounds) in the feces of rats
that had been administered cis- or trans-chlordane-14C or the 3:1 mixture of
cis- 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 cis- or trans-chlordane-14C for 14 days). These
metabolites, analyzed by thin-layer chromatography (TLC) and gas-liquid chro,-
matography (GLC), were tentatively identified as hydroxychlordane; chlordene^
chlorohydrin; monochloro and dihydroxy derivatives of chlordane; cis- and/or
trans-dihydroxychlordane derivatives; a trihydroxylated chlordene; and a con^
jugated form of a hydroxylated chlordane metabolite. No oxychlordane or diV;
chlorochlordene was detected in feces; however, oral administration of oxy-,
chlordane resulted in fecal excretion of unchanged oxychlordane. The nature of
urinary metabolites was essentially the same as fecal metabolites in rats fed
HCS-3260 in dietary concentrations of 25 mg/kg diet; however, oxychlordane was
also present. The 24-hour feces of a rabbit fed 14C-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. --
In rabbits, Balba and Saha (1978) identified the following urinary metab-
olites of cis-chlordane: l-hydroxy-2-chlorochlordene, trans-chlordene chloro-
hydrin, and 1-hydroxychlordene. Urinary metabolites in rabbits of trans-chlor-
4-9
-------
dane were l-hydroxy-2-chlorochlordene, 1,2-dichlorochlordene, trans-chlordene
chlorohydrin, and 3-hydroxychlordane. The metabolism of cis- and trans-chlor-
dane-14C was also studied in rats in vivo and in vitro by Tashiro and Matsumura
(1977). Male rats maintained on diets containing cis- or trans-chlordane-14C
in a concentration of 100 ppm (100 mg/kg diet) for 4 weeks excreted 13 metab-
olites from cis- and 14 metabolites from trans-chlordane in the feces. The
fecal metabolites of both isomers, identified by TLC and GLC, included hepta-
chlor, 1,2-dichlorochlordene, oxychlordane, l-hydroxy-2-chlorochlordene, 1-
hydroxy-2-chloro-2,3-epoxychlordene, chlordene chlorohydrin, monohydroxy di-
hydrochlordene, 1,2-dihydroxychlordene, and trihydroxydihydrochlordene. These
metabolites were found in different proportions, depending upon the administered
Isomer. The only urinary metabolite identified from both cis- and trans-chlor-
dane was a glucuronide conjugate of 1-hydroxydihydrochlordene. The in vitro
incubation of the isomers with rat liver microsomes and cofactors resulted in
the same metabolites qualitatively.
Brimfield et al. (1978) also studied the in vitro metabolism of the pure
cis- and trans-isomers of chlordane by microsomal preparations from Sprague-
Dawley rats fed the individual isomers in dietary concentrations of 100 ppm
(100 mg/kg diet) for 9 days. Incubation of cis-chlordane-induced enzymes with
cis-chlordane resulted in the following metabolites, identified by GC-MS:
cis-chlordane, dichlorochlordene, oxychlordane, dihydroheptachlor, hydrochlor-
dene, and l-chloro-2-hydroxydihydrochlordene. Similar products from trans-
chlordane were identified; however, heptachlor rather than dihydroheptachlor
was formed, as was hydroxychlordane. A metabolic pathway, based upon reduc-
tive dechlorination via dihydroheptachlor to dihydrochlordene, was proposed.
According to this scheme, these molecules can also be hydrolyzed, desaturated,
and epoxidized. In the Brimfield et al. (1978) proposal, oxychlordane is an
4-10
-------
end point, whereas in the scheme proposed by Tashiro and Matsumura (1977),
oxychlordane undergoes further hydroxylation and reduction. Many of the pro-
posed intermediates in the Brimfield scheme have not actually been found in
this or other studies (Brimfield et al., 1978).
Therefore, using the in vivo data by Barnett and Dorough (1974) and
Tashiro and Matsumura (1977) as the primary references along with supporting
in vitro data, the metabolic pathways proposed by Tashiro and Matsumura (1977)
best fit the data reported. Chlordane is dehydrogenated to dichlorochlordene,
which is epoxidized at carbons 2 and 3 and hydroxylated at carbon 1 (Route A).
An additional route whereby chlordane is hydroxylated at carbon 1 to yield :
1-hydroxydihydrochlordene was also proposed (Route B). Heptachlor is a minor
metabolite of both cis- and trans-chlordane. These pathways are presented in
Figure 2.
4.1.3.2. HeptachlorDavidow and Radomski (1953) first identified heptachlor
epoxide in the adipose tissue of dogs that had been treated with daily oral
doses of 1-3 mg heptachlor/kg,bw in corn oil. They concluded that the metab-
olite arose by the epoxidization of heptachlor. Subsequently, these investi-
gators (Radomski and Davidow, 1953) also isolated heptachlor epoxide, identical
to the compound found in dogs, from the adipose tissue of male and female rats
maintained on diets containing 30 to 35 ppm (30 to 35 mg/kg diet) heptachlor.
In an abstract of a Soviet study, Ermakov (1977) reported that heptachlor,
administered to rats and rabbits in single oral doses of 28-50 mg/kg bw, was
metabolized by reactions involving hydrolysis, hydroxylation, epoxidation,
dehydrogenation, and molecular rearrangement, but details as to intermediate
structures were not given in the abstract.
The most extensive investigation of the metabolic fate of heptachlor in
rats was performed by Tashiro and Matsumura (1978) in vivo and in vitro. Over
4-11
-------
Cl
Heptachlor
/* I MOST ACTIVE
N ROUTE
i MAJOR ROUTE
- MINOR ROUTE
ci
Cls-Chlordane
I I
Cl Cl
Trans-Chlordane
ROUTE B
!
ROUTE A
-Cl
OH
1-Hydro xydihydrochlorene
UDPGA
1-Hydroxy-2-Chlorochlordene
Cl ^f ^ "Cl
Cl OH
1-Hydroxy-2-Chloro-
2,3-Epoxychlordene
Cl
Cl 0-C6H906
Qlucuronide Conjugate
Cl
OH
ci
Cl OH
1.2-D!hydroxyd!hydrochlorden8
cr "Y" "^ OH
Cl
Trihydroxylated Metabolite
Figure 2. Proposed metabolic pathways of chlordane in rats,
SOURCE: Tashiro and Matsumura, 1977.
4-12
-------
a 10-day period following a single oral unspecified dose of l^C-heptachlor
(position of label not specified) in corn oil, rats excreted >50 percent of the
administered radioactivity in feces, and <5 percent in urine. The relative
abundance of fecal metabolites, expressed as percent of ^-compounds, were as
follows: unchanged heptachlor, 26.2 percent; heptachlor epoxide, 13.1 percent;
1-hydroxychlordene, 19.5 percent; l-hydroxy-2,3-epoxychlordene, 17.5 percent;
1,2-dihydroxydihydrochlordene, 3.5 percent; and two unnamed metabolites, one of
which accounted for 19 percent of the radioactivity, the other for <0.1 percent.
The latter metabolite was designated as H-2 by these authors, and its structure
is identical to that proposed by Matsumura and Nelson (1971) 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 percent of the total 14C-com-
pounds: heptachlor, 4.4 percent; heptachlor epoxide, 85.8 percent; 1-hydroxy-
chlordene, 2.9 percent; 1-hydroxyepoxychlordene, 3.0 percent; 1,2-dihydroxydi-
hydrochlordene, 0.9 percent; and an unknown, 3.0 percent (Tashiro and Matsmura,
1978). No H-2 fecal metabolite was detected. The identity of the metabolites
was confirmed by TLC analysis and comparison with authentic standards of fecal
metabolites from rats fed diets containing 100 ppm (100 mg/kg diet) heptachlor
for 4 weeks.
Matsumura and Nelson (1971) administered heptachlor epoxide to four rats
in dietary concentrations of 10 ppm (10 mg/kg diet) for 30 days. The authors
estimated that each rat consumed 5 mg of heptachlor epoxide over the test
period and excreted 950 yg of a fecal metabolite (see Figure 3) and 66 ug of
heptachlor epoxide in the feces.
Brooks et al. (1968, 1970) investigated the in vitro metabolism of hepta-
chlor epoxide by pig liver microsomes. The product, formed upon incubation at
4-13
-------
Cl Cl
Heptachlor epoxide
Cl Cl
Heptachlor
Unnamed fecal metabolite
H-2
(2,3-epoxychlordene)
OH
1-Hydroxychlordene
Cl OH
1-Hydroxy-2,3-epoxychlordene
OH
Cl OH
1,2-Dihydroxydihydrochlordene
Figure 3. Proposed metabolic pathway of heptachlor in rats,
SOURCE: Tashiro and Matsumura, 1978.
4-14
-------
45°C for 60 hours, was identified as'heptathlon epoxide diol:
Incubation of heptachlor epoxide with rabbit microsomes also resulted in the
formation of heptachlor epoxide diol as well as another unidentified product.
The in vivo product, 2,3-epoxychlordene, was not formed or identified in
these in vitro preparations.
Therefore, the metabolic pathway of heptachlor in rats, as proposed by
Tashiro and Matsumura (1978), is presented in Figure 3.
4.1.4. Excretion
4.1.4.1. ChlordaneIn the experiments of Barnett and Dorough (1974), >90 per-
cent of the administered radioactivity was excreted over 7 days by rats that
were given single oral doses (0.2 mg/kg diet) of cis- or trans-chlordane-^4C or
l^C-HGS-3260, Females excreted 6 percent of the radioactivity in the urine,
while males excreted only 2 percent. At higher doses (0.5 or 1.0 mg/kg diet)
of HCS-3260, the pattern of elimination was essentially the same. Females
excreted slightly, but not significantly, more of the cis- than the trans-
isomer. When 14C-HCS-3260 was fed in the diet for 56 days, fecal elimination
as measured by radioactivity was 70 percent for the 1 mg/kg diet level, 75 per-
cent for the 5 mg/kg diet level, and 80 percent for the 25 mg/kg diet level,
indicating possible decreased absorption; the investigators made no mention of
possible biliary excretion. Elimination of cis-chlordane (75 percent) was
greater than trans-chlordane (65 percent) following 14 days of dietary levels
of the isomers at 25 mg/kg diet.
4-15
-------
Tashiro and Matsumura (1977) reported similar results for elimination from
.rats treated with single oral doses of cis- (5.4 mg/kg bw) and trans-chlordane-
14C r(9«7 mg/kg bw) in corn oil. The total 7-day elimination of radioactivity
from cis- and trans-chlordane was 85 and 66 percent of the administered dose,
respectively. The 24-hour total excretion was 59 percent for cis- and 27 per-
cent for trans-chlordane.
Rabbits excreted 18 percent of an orally administered single dose of 200
mg/kg bw of chlordane in the urine collected over 16 days when organic chlorine
contents were measured (Stohlman et al., 1950). The peak urinary elimination
of organic chlorine occurred within 2 days and amounted to 9 percent of the
dose.
Poonawalla and Korte (1971) observed appreciable urinary excretion of
radioactivity by rabbits that received 14.3 mg/rabbit of trans-chlordane-14C
daily for 10 weeks. At the end of this period, approximately 70 percent of
the daily dose had been eliminated: 22.7 percent was excreted in the feces,
30 percent of which was unchanged trans-chlordane, and 47 percent was excreted
as urinary metabolites. In agreement with these results, excretion of radio-
activity by one rabbit 24 hours after termination of feeding 25 ppm (25 mg/kg
diet) of HCS-3260-14C for 2 days was 21 percent in feces and 33 percent in
urine (Barnett and Dorough, 1974).
Balba and Saha (1978) also observed appreciable urinary excretion of
radioactivity of cis- or trans-chlordane-14C by rabbits that were treated with
either isomer orally at a dose of 100 mg/rabbit in a capsule every 4 days up
to 400 mg/rabbit; however, in this study, urinary excretion did not exceed
fecal excretion. For the cis-isomer, 48.5 percent and 28.4 percent of the
radioactivity were eliminated in the feces and urine, respectively. For the
trans-isomer, fecal and urinary excretion were 46.1 percent and 35.8 percent,
4-16
-------
respectively.
When an unspecified amount oftehlordane-l^C in 20 yL of ethanol was ad-
ministered intratracheally to female Sprague-Dawley rats, elimination of radjio-
activity was primarily in the feces (Nye and Dorough, 1976). After a lag fl
period of 2 days during which <20 percent of the dose was eliminated, fecal, ,
excretion rose to approximately 50 percent by day 4 and 56 percent by day 6.
Urinary excretion over 6 days amounted to 12 percent of the dose. No radio-
activity was detected in the expired air of these animals. ,.._,-
4.1.4.2. HeptachlorTashiro and Matsumura (1978) reported that rats excreted
>50 percent of the administered single oral unspecified dose of radioactivity
from -^C-heptachlor in the feces over 10 days. Urinary excretion of radio-
activity accounted for <5 percent of the administered amount.
In an abstract of a Soviet study (Ermakov, 1977), it was reported that 16
to 40 percent of the orally administered dose of heptachlor (28 to 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. As reported in an abstract of another Soviet study (Mizyukova and ,
Kurchatov,- 1970), and as reviewed by U.S. EPA (1980b), heptachlor and its .-.-
metabolites were excreted mainly in the feces of rats within 5 days of an
intragastric dose of 120 mg/kg bw. ,
Each of four male albino rats excreted an average of 950 ng of the fecal
metabolite of 99 percent pure heptachlor epoxide during 30 days of maintenance
on a diet containing 10 ppm (10 mg/kg diet) heptachlor epoxide (each rat con-
sumed approximately 5 mg over 30 days) (Matsumura and Nelson, 1971). Approxi-
mately 66 pg of unchanged heptachlor epoxide was also excreted in the feces,?
4.1.5. Species Differences , ,,
Some 30 to 40 percent of the 186 NCI chemicals tested in both rats and mice
4-17
-------
were found to be positive in one species and negative in the other (Gold et
a!., 1984). Of these, approximately the same number were rat-positive and
mouse-negative as were mouse-positive and rat-negative. However, a review of
the bioassays of chlorinated hydrocarbons reveals a preponderance of mouse-
positive and rat-negative studies. As such, are chlorinated hydrocarbons more
toxic in mice than rats; initiators in mice but not in rats; or are there other
differences that lead to the apparent discrepancy between rats and mice for
carcinogenicity studies of chlorinated hydrocarbons?
In the selection of the laboratory animal species for a carcinogenesis bio-
assay, rats and mice have generally been used because of their size, lifespan,
and the fact that these species are reliable predictors of human carcinogens
for at least some chemicals. As the need to extrapolate from animals to humans
has increased, along with the pressure to calculate the risk of exposure, the
selection of species and the dosages continues to be discussed. As late as
1976, when the NCI guidelines were published (Sontag et al., 1976), considerable
discussion of the dosage centered on the "maximum tolerated," and only the no-
tion of the importance of metabolism data in relation to target concentrations
was provided. Recently, Haseman (1985) focused on dose selection for NTP
studies and restated the possible importance of pharmacokinetic and metabolism
studies. Much of the discussion of metabolism data centers on qualitative
similarities to humans and the possible consequences of metabolic overload.
In actuality, very little metabolism data are available in both rats and
mice for any class of chemicals, including chlorinated hydrocarbons. The
metabolism of chlordane and heptachlor has been studied in rats but not in
mice; however, there is considerable evidence that significant species differ-
ences exist with regard to other substances. Such differences could be direct-
ly related to the carcinogenic response in rats and mice.
4-18
-------
There have been a number of reports that emphasize metabolic differences
among laboratory animal species (Brodie, 1962; Forrest and Aber, 1968; NAS,
1969; Oehme, 1970; Hanley et al., 1970; Conney et al., 1974; Zapp, 1977). 9c
Table 2 shows the large range of metabolic differences that can occur among
animal species and humans.
TABLE 2. SPECIES DIFFERENCES IN THE METABOLISM OF THREE CHEMICALS
Species
Mouse
Rat
Guinea pig
Rabbit
Dog
Human
Metabolic
Hexabarbital
19
140
a
60
260
360
half -life (min)
Antipyrene
11
141
110
63
107
600
Aniline
35
71
45
35
167
aData not available.
SOURCE: Adapted from Conney et al., 1974.
Similarly, a 200-fold difference was noted among species to elicit a phar-
macologic response of ICI 33,828 on the pituitary, while the maximum plasma
concentration was identical in all species tested, including humans. In addi-
tion, consideration of the rate and pattern of metabolism is of great impor-
tance (NAS, 1969). These types of data have recently led investigators to '
place greater emphasis on pharmacokinetic and metabolism data (Neal, 1983; NTP,
1984; Bernstein et al., 1985).
The use of pharmacokinetic data in estimating the human inhaled dose from
oral data for laboratory animals has been reported (Cavender and Salasin, 1982).
4-19
-------
Although considerable data for rats were available, very little data for mice
were found for chlorinated hydrocarbons. Recently, Prout et al. (1985) pub-
lished data for rats and mice for trichloroethylene, and Tables 3 and 4 present
pharmacokinetic data for two strains of rats and two strains of mice. Of par-
ticular importance is the difference in urinary excretion and exhaled parent
compound. The mouse apparently has a greater capacity for metabolizing tri-
chloroethylene, which may lead to higher concentrations of important interme-
diates or metabolites than are possible in the rat. In this case, the parent
compound is exhaled over time in the rat but not in the mouse. For compounds
that are readily stored in fat depots, they may be metabolized over days and
weeks. These data emphasize the fact that, although the relative proportion of
metabolites is similar for the two species, there are considerable differences
in the quantitative data, especially at the high dosages. Unfortunately, sim-
ilar data do not exist for chlordane, heptachlor, or heptachlor epoxide. How-
ever, these data do emphasize the fact that the pharmacokinetic and metabolic
differences may be quite different for the two species and may relate to the
"sensitivity" of mice and/or the "resistance" of rats to these chlorinated
hydrocarbons. \
4.2. TOXIC EFFECTS
The toxic effects of chlordane/heptachlor were not addressed in a previ-
ous report of the Carcinogen Assessment Group (U.S. EPA, 1977). Thus, the key
studies are reviewed here.
4.2.1. Human Studies
Data describing effects in humans range from information collected through
clinical case studies and human monitoring data to epidemiologic studies. The
three effects seen most frequently in clinical case studies are central nervous
system (CNS) effects, blood dyscrasias, and neuroblastomas. Eleven case studies
4-20
-------
1 1 1
o
1 1
rH
U-
CO
to
CQ
o
"*
co
«=c
LU
§
LU
LU
z
o;
0
CO
co
o
z
*""*
LU
LU
_J
3T
LU
O
Of.
O
_J
0
r 1
1
1 1
u_
o
LU
CO
0
o
_1
^
o
LU
j_j:|
CD
|_^
co
LI-
CD
z
o
^H
1 1
t 1
LU
CO
LU
CO
-o
CO
.p
CO
u
X
CU
co
CO
O
o
,_
IO
o
.O.
v>
as
o
fO
CJ
CU
en
c
IO
o
c
CM
O
U
CU
o
LL.
c
'£
'
CU t-
p- ^-^
(O
co
CO
0.
CO
en
CO**^
o en
=TrH
O CO
+ 1+1
CO CTl
rH CO
P-. rH
O rH
+ 1+1
r** CTi
CTl rH
o co
+ 1+1
LO LO
in in
1 1
co P-.
rH <3-
+ 1+1
.CO CO
tO LO
tO LO
CM
O
CU
00
4-> 3
IO O
cm s
o
rH
00 CTl
rH rH
+ 1+1
LO rH
r*» P~»
CTi CTl
«3- 0
O rH
+ 1+1
«* 00
CO «*
0 O
o o
CM 1
0 0
+ 1+1
P^ CO
CM CM
CTl 00
0 0
+ 1+1
00 P--
CM rH
OO "*
0 0
+ 1+1
1 1 1 1
co «*
CM
1
CM
CU
V)
!-> 3
as O
on s:
rH CO
LO rH
+ 1+1
f-» O
CM tO
rH LO
0 O
+ 1+1
tO CTl
CM VO
OO O
O rH
+ 1+1
rH rH
CM CM
rH
^- 00
rH «*
+ 1+1
CM CM
rH CTl
^- in
^
CM
O
CO
I/)
4-> 3
OS O
oc s:
o
o
LO
00 «=!
CO CM
+ 1+1
rH tO
CO r«!
CTl CTl
LO CO
0 0
+ 1+1
00 «*
co «a-
O 0
o o
CM CM
O 0
+ 1+1
rH O
rH CM
to-*
O 0
+ 1+1
CM «*
CM CM
oo to
O CM
+ 1+1
^r in
CM «*
CM
1
CM
CO
OO
0 O
+ 1+1
rH O
CM to
in in
O CTi
+ 1+1
P^ 0
rH CO
rH
00 OO
0 0
+ 1+1
CM OO
CTl rH
CM «*
CM
O
o>
00
!-> 3
ie o
o
0
o
rH
in CM
0 rH
+ 1+1
P^ CM
LO LO
CTl CTl
«d- CM
O CTl
+ 1+1
3- to
CM 00
o o
O 0
CO ^t-
0 O
+ 1+1
CO to
0 rH
to CM
O rH
+ 1+1
*! O
rH CM
CM rH
O rH
+ 1+1
CTl to
t 1 ^~
CM
P-.
1
CM
CU
oo
-(-> 3
IO O
en 2:
to CM
CM «5f
+ 1+1
CO to
1 CO
1 rH
rH «^-
0 0
+ 1+1
o o
rH IO
to o
O CM
+ 1+1
CM CO
CM LO
1 1
CO 1
CO CO
+ 1+1
CM 1
CO CO
rH «3-
CM
o
CO
4-> 3
as O
Qi 2:
o
o
o
CM
CTi to
LO CM
+ 1+1
O CTi
CO rH
CTl CTl
O LO
Q 0
+ 1+1
co co
i 1 CO
O 0
0 0
i 1 rH
0 Q
+ 1+1
«d- oo
0 rH
LO P~
0 0
+ 1+1
CM «=J-
rH CO
rH tO
0 0
+ 1+1
oo oo
o co
CM
P--
1
CM
CO
00
4J 3
as O
oi. E:
%
LO
co
t_ CTl
£^ rH
CM *
! Q 1
I CO i
. 4-> as
1 )->
10 c/> CO
C 00.
IO fO 4-^
i CO O 3
E t- 0
as t-.
co a. o a.
t_ =3
10 O C E
t_ -i- O
c en c_
5 en M-
o t_ c:
j± co -i- -a
00 Q. C CO
«/> C CO
a 3
T3 LU
..£_!- O
LU 3 to C£.
1 O CO =5
o LL. on o
Z(O J3 CO
4-21
-------
1
re
o
H-
.£)
CO
CO
re
o
t_
re
o
H- « . .
UJ.
UJ -O
_J - .": 0)
>* - cn
3= C
H- re
UJ - J=
o UJ u
c: o T5 c
1 *V t %
J £ r'
a: o>
1-4 UJ O
H- co o>
1 CO
O UJ CD
«3* 3» CO CM
rH 1 0 O
U. CO
O * ^
UJ CO '
CO
o a
i *^
'«-
H- -J . ! .
2: uj
rH O
5 ~
' re
a
et-
UI
f
CO
MSC
» re
VJ
ai
o
QJ
OL
CO
ai cn
co -i<:
o ~^
a cn
^^
in 10
CD 0
+ 1+1
0 O
rH rH
«* rH
rH rH
+ 1+ 1
=* ^f
J^*« rH
rH
cn CM
CM <0
+ 1+1
in CM
in co
rH rH
C3 *3t"
in in
+ 1+1
to in
"3- (O
in in
rH
0)
CO
4-> 3
re o
o
rH
O O
o o
«=f CM
o o
+ 1+1
cn co
rH CM
r-- co
«3- O
+ 1+1
CM 00
r- rH
=f 10
0 O
+ 1+1
O^ ^~I
CM CM
CM
0}
V)
4-* 3
re o
cn s:
CO 10
<3- rH
+ 1+1
O^ T i
*
CM <0
o cn
rH
co co
rH O
+ 1+1
rH CO
o in
rH
1 1
1 1
CM CM
0 O
+ 1+1
CM OO
o o
cn CM
0 0
+ 1+1
«* 10
rH O
CM rH
0 0
+ 1+1
co 10
o o
CO
a>
CO
+-> 3
re o
CM CO
«* in
+ 1+1
cn in
cn CM
*^" rH
o co
rH O
+ 1+1
r-» in
CO l»-
CO CO
rH «*
+ 1+1
CO rH
CO CM
rH
o co
«3- in
+ 1+1
cn =r 3
re o
i cn
rH «d-
+ 1+1
CO CM
«
in cn
cn cn
«3- CM
rH rH
+ 1+1
<* o
in «*
CM
1 1
1 + 1
0
^J-
CO CO
o o
+ 1+1
10 cn
0 0
r** to
0 0
+ 1+1
CO rH
O rH
i"H *srf"
0 0
+ 1+1
«=r r-
O 0
CO
a>
CO
-t-> 3
re o
CXL E:
i.
.c
CM
a r--.
oo
^
f- 1 re
CO CO
C CO
re re
CO 0
E c-
re
co a. o
t. 3
re o c:
C- -r-
c cn
3 cn
O t- c
J= 0) -r-
co o. c
co co re
CO i E
3 re co
"re -i- *"
> C CO
re 3
o
. . C_ -r-
UJ 3 CO
I 0 CO
O U_ Qi
"zz re .a
m
CO
cn
rH
n
,__
re
j %
a>
^_j
3
O
t_
a.
e
o
i.
M-
o
CO
a.
re
-a
UJ
o
a:
o
CO
-------
describing CNS effects following oral, dermal, or inhalation exposure to tech-
nical grade chlordane are described in the literature. Six of these case
studies involve oral exposure to chlordane. Effects ranged from irritability,
salivation, labored respiration, muscle tremors, and convulsions to death.
Derbes et al. (1955) described the death of a 32-year-old woman who ingested
104 mg/kg of chlordane in a dust formulation. Findings at autopsy included
cerebral congestion and edema and lower nephron nephrosis. Convulsions were
exhibited before death. Convulsions and vomiting accompanied other oral inges-
tions of chlordane (Dadey and Kammer, 1953; Lensky and Evans, 1952; Aldrich and
Holmes, 1969; and Curley and Garrettson, 1969). The other five reports involv-
ing CNS effects involve dermal or a combination of dermal and inhalation expo-
sure. Although dose level could not be quantified for these exposures, two
resulted in death. Other CNS effects include convulsions, lethargy, and diz-
ziness (Kutz et al., 1983; Derbes et al., 1955; Furie and Trubowitz, 1976;
Klemmer et al., 1977; and Barnes, 1967).
Eight cases of blood dyscrasias have been reported following exposure to
chlordane or heptachlor: four aplastic anemias (Infante et al., 1978; Klemmer
et al., 1977), one refractory megaloblastic anemia (Furie and Trubowitz, 1976),
one acute stem cell leukemia (Infante et al., 1978), one acute lymphoblastic
leukemia (Infante et al., 1978), and one acute myelomonocytic leukemia (Infante
et al., 1978). Muirhead et al. (1959) and Loge (1965) reported 5 and 12 cases
of aplastic anemia, respectively, associated with either chlordane; a combina-
tion of chlordane and heptachlor; or chlordane, heptachlor, and other chemicals.
The American Medical Association's Council on Drugs (Anonymous, 1962) stated
that a "specific cause-effect relationship exists" between exposure to chlor-
dane and resulting blood dyscrasias.
Infante et al. (1978) described five cases of neuroblastoma in children
4-23
-------
with a pre- and/or postnatal history of exposure to technical grade chlordane.
This is consistent with documented transplacental transfer of chlordane in
humans (Curley et al., 1969; Wasserman et al., 1972, 1974; Zavon et al., 1969).
Savage et al. (1973, 1981) and Strassman and Kutz (1977), in results from
human monitoring studies, reported levels of heptachlor epoxide and oxychlor-
dane, metabolites of heptachlor and chlordane, respectively. Savage et al.
(1981) found detectable levels of heptachlor epoxide in 67 percent of 1,436 hu-
man milk samples from nursing mothers at 163 hospitals stratified by geographic
location. Oxychlordane was reported in 74 percent of the samples. Samples
collected from the southeastern United States tended to have a higher percent-
age of samples with detectable levels of each metabolite than other regions of
the country. Human milk samples of 54 women in Hawaii were collected during
1979 and 1980 (Takei et al., 1983). Oxychlordane was detected in 100 percent
of the samples, with a mean of 0.068 ppm and a maximum value of 0.55 ppm.
Heptachlor epoxide was also detected in all samples, with a mean of 0.036 ppm
and a maximum value of 0.068 ppm.
Data from the EPA's National Human Monitoring Program show that levels of
Oxychlordane and heptachlor epoxide in human adipose tissue have remained con-
stant over the years 1970 to 1983 (U.S. EPA, 1985a). The geometric mean of,
Oxychlordane from 1972 to 1983 (excluding 1982) was 0.11 ppm with a maximum
mean of 0.09 in 1981. Levels of heptachlor epoxide ranged from a geometric
mean of 0.07 ppm in 1972 and 1977-79 to 0.09 ppm in 1970-71, 1973, 1981, and
1983, with a mean of 0.08 ppm for all years (excluding 1982).
4.2.2. Laboratory Animal Studies
4.2.2.1. ChlordaneSymptoms of acute chlordane intoxication include CNS stim-
ulation, as evidenced by irritability, tremors, and convulsions (U.S. EPA,
1985a). When chlordane was given orally to rats at a dose of 25 mg/kg bw/day
4-24
-------
for 15 days, it had no toxic effects, whereas 50 mg/kg bw or more resulted in
toxic effects and death; cumulative toxic effects were observed (Ambrose et
al., 1953a). The toxic effects of chlordane in rats include CNS stimulation,
stomach ulcers, inflammation of the intestine, nephritis, hepatitis, an in'-"
crease in liver weight, coma, and death (Boyd and Taylor, 1969).
In a review by Fouts (1970), chlordane induced hepatic drug-metabolizing
enzymes in all species examined. Estradiol-17B and estrone metabolism were'
also stimulated by chlordane pretreatment in mice and rats, respectively w
(Welch'et al., 1971).
Osborne-Mendel rats were fed diets containing chlordane for 80 weeks cmd
held until week 109. Decreased body weight gain was noted in males receiving
407 ppm (approximately 20.3 mg/kg bw) in the diet and in females receiving 242
ppm (approximately 12.1 mg/kg bw). Tremors were noted in the females at week
44, and the animals that survived to termination were in poor condition (NCI,
1977a). -
B6C3F1 mice were fed diets containing chlordane for 80 weeks and held un-
til weeks 90-91. No body weight effects were noted, but tremors were observed
in female mice at week 20. Alopecia was noted in male and female mice, and a
hunched appearance was noted in a few mice. Abdominal distension was observed
in all groups but was predominant in the females. Increased mortality inci-'
dence was noted for male mice fed 29.9 ppm (approximately 1.5 mg/kg bw) (px'
0.02) and 56.2 ppm (approximately 2.8 mg/kg bw) (p <_ 0.01). No mortality dif-
ferences were noted for female mice (NCI, 1977a).
In an unpublished study by Velsicol Chemical Corporation (1984), Wistar4
rats and cynomolgus monkeys were exposed to 0, 0.1, 1.0, or 10 mg/m3 for 90
days. Some of the low-exposure group rats were held for 17 days after exposure
as a recovery group. No effects were noted on mortality, body weight, food''
4-25
-------
consumption, ophthalmoscopy, pulmonary function, rectal temperature, hema-
tology, or urinalysis for either rats or monkeys. Blood levels of calcium,
cholesterol, and glutamine dehydrogenase and liver cytochrome P-450 levels were
increased in male and/or female rats. Increased organ weights were noted for
male and/or female rats for the brain, liver, kidneys, and thyroids, while
there was a decrease in adrenal weight in female rats. For monkeys, liver and
thyroid weights were increased but not statistically significantly different
from control group values. For microscopic pathology, only adaptive liver
changes were significantly different for rats and monkeys. Male rats also ex-
hibited an increase in the height of the follicular epithelium of the thyroid.
Based on these results, the NOEL for chlordane in rats and monkeys was 0.1
mg/m3 and the LOEL was 1.0 mg/m3.
In an earlier unpublished study by Velsicol Chemical Corporation (1983b),
ICR mice were fed 0, 1, 5, or 12.5 ppm chlordane (approximately 0, 0.15, 0.71,
or 1.79 mg/kg bw) in the diet for 2 years. In mice receiving 5 or 12.5 ppm,
liver changes were noted, including'increased levels of serum glutamic-oxalo-
acetic transaminase (S60T) and serum glutamic-pyruvic transaminase (SGPT);
hepatocellular swelling and necrosis; and increased liver weight in both male
and female mice. The NOEL for chlordane was 1 ppm and the LOEL was 5 ppm.
In another unpublished study by Velsicol Chemical Corporation (1983a),
Fischer 344 rats were fed diets containing 0, 1, 5, or 25 ppm chlordane
(approximately 0, 0.05, 0.25, or 1.25 mg/kg bw) for 130 weeks. There was a
significant increase in liver weight for females receiving 5 or 25 ppm at weeks
26 and 52, but not at week 130. Liver weight was significantly increased in
males receiving 5 or 25 ppm at week 130, but not at weeks 26 or 52. An in-
creased incidence of hepatocellular swelling was noted in males receiving 1, 5,
or 25 ppm. Based on these results, the NOEL for female rats was 1 ppm but was
4-26
-------
not established for male rats. The LOEL for female rats was 5 ppm and for,male
rats was 1 ppm.
' . '*_*..".:
. .An additional unpublished chronic study was performed in dogs by Wazeter
(1967,, cited in Vettorazzi, 1975). The dogs were maintained for 2 years on ,,,
diets containing chlordane at levels of 0, 0.3, 3, 15, or 30 ppm (approximately
0, 0.008, 0.075, 0.375, or 0.75 mg/kg bw). The effects observed were increased
liver weight and histologic changes in the liver. A scientific review panel
for WHO/FAO examined this study and concluded that no effects were observed, at
exposures of 3 mg/kg diet or less. No other data ,on the experimental design or
results of this study were found.
Mice maintained for 4 months on a diet containing 100 ppm chlordane
(approximately 14.3 mg/kg bw) had decreased viability of offspring. (Deichmann,.
and Kepiinger, 1966). Chlordane decreased fertility in male and female rats
(Ambrose et al., 1953a) and in female mice (Welch et al., 1971). Ambrose et,,
al. (1953b) reported reduced survival of offspring for rats fed 640 and 1280 ,
ppm chlordane (approximately 32 and 64 mg/kg bw).
4.2.2.2. HeptachlorSymptoms of acute heptachlor intoxication include trem-
- :' if :
ors, convulsions, paralysis, and hypothermia (U.S. EPA, 1985a). Daily oral
administration,, of 2 or 5 mg/kg bw heptachlor for 78-86 days to pigs, sheep, ,and
rats induced hepatic, necrosis and synthesis of smooth endoplasmic reticulum.
Rats were the most sensitive species (Halacka et al., 1975). Chronic admini-
stration of intramuscular injections in rats with daily doses of 3 and 15 mg/kg
bw. heptachlor or 1 and 5 mg/kg bw heptachlor epoxide decreased liver size but.
had no. effect on other tissues. Certain hepatic and renal gluconeogenic en-.
zymes were stimulated (Kacew et al., 1973).
Shain et al. (1977) found that feeding of heptachlor (99.8 percent) to
male Sprague-Dawley rats for 90 days at an average dose of 1.29 mg/kg bw/day-
4-27
-------
significantly reduced the food consumption and mean body weight gain in a sub-
group of 12 randomly selected rats. In another subgroup of 22 rats, body
weight was not reduced and no changes were observed in testicular or ventral
prostate. Histologic examination of the rats was not performed, since the
study was designed to determine the effect of heptachlor on prostate homeosta-
sis. Rats were castrated 24 hours before sacrifice. Cytoplasmic, but not
nuclear, androgen receptor site content of the ventral prostate was signifi-
cantly increased. Ventral prostate protein content was reduced to 13 percent
of control levels, and cell number was reduced to 18 percent of control.
In an unpublished report by IRDC (1973b), a 25:75 mixture of heptachlor:
heptachlor epoxide was administered to groups of 100 male and 100 female CD-I
mice in the diet at 0, 1, 5, and 10 ppm (approximately 0, 0.14, 0.71, and 1.43
mg/kg bw) for 18 months. An interim sacrifice of 10 mice/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 consumption was
observed. Mean 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 sacrifice, was
29 percent for males and 30 percent for females at the highest dose level and
51-66 percent for all other groups, including controls. Increased incidence
of hepatocytomegaly in 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 (p < 1 x lO'9). Reevaluation of histologic slides by
six other pathologists resulted in several different interpretations; however,
the overall result was a decreased incidence of hyperplasia in favor of a
greater incidence of carcinoma. Further discussion of this study is presented
4-28
-------
in the Cardnogenicity section.
Davis (1965) administered 0 and 10 ppm (approximately 0 and 1.43 mg/kg bw)
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 percent)
as well as control (34 percent) mice. For heptachlor epoxide, survival was:'9.5
percent. A twofold increase over controls in the incidence of hepatic hyper-
pi asi a and of benign tumors was observed for both compounds, although reevalu-
ation of the slides by four other pathologists changed the interpretation in
favor of a greater number of hepatomas.
Reuber (1977a, 1978), using slides from the study of Davis (1965), found
hepatic vein thrombosis and cirrhosis among the heptachlor and heptachlor
epoxide (10 ppm in the diet) treated mice. These conditions were not observed
in any of the 127 control slides available for review. For heptachlor-treated
mice, 13 percent (10 percent of males, 15 percent of females) had hepatic vein
thrombosis and 6 percent had venous occlusion with recent liver infarcts.
Thrombosis of the cardiac atrium was also present in some mice with hepatic .
vein thrombosis. The incidence of cirrhosis was 2/86 treated males and 5/77
treated females. For heptachlor epoxide, 10 percent of treated mice (7 percent
of males, 11 percent of females) had hepatic vein thrombosis and 9 percent had
venous occlusion. Cardiac atrium thrombosis was present in 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) bioassay of the carcinogenicity of heptachlor, groups
of 50 male and 50 female Osborne-Mendel rats and groups of 50 B6C3F1 mice of
each sex were maintained on diets containing technical grade heptachlor for
80 weeks plus 21 and 10 weeks of observation for rats and mice, respectively.
Controls for the rats consisted of 10 matched and 60 pooled untreated rats
4-29
-------
(controls from concurrent and recent bioassays of other related compounds) per
sex. For male mice, 20 matched and 100 pooled controls were used, arid for
female mice, 10 matched and 80 pooled controls were used. Male rats were fed
38.9 or 77.9 ppm (approximately 1.95 or 3.90 mg/kg bw) and female rats were fed
25.7 or 51.3 ppm (approximately 1.29 or 2.57 mg/kg bw) while male mice were fed
6.1 or 13.8 ppm (approximately 0.87 or 1.97 mg/kg bw) and female mice were fed
9.0 or 18.0 ppm (approximately 1.29 or 2.57 mg/kg bw).
For rats, mean body weights of high-dose males and females were consis-
tently 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 bleeding developed
after 80 weeks. A dose-related but not statistically significant increase in
mortality was observed for treated males. The increase was statistically sig-
nificant for 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 incidences
of follicular and C-cell hyperplasia in thyroid glands of treated and control
groups were observed to be non-treatment-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 were prevalent in high-dose
females. Adverse clinical signs developed in mice of all groups. Male mortal-
ity was unaffected; however, there was a significant dose-related increase in
mortality for treated female groups. Hepatocytomegaly and diffuse hyperplasia
occurred infrequently in individuals of all groups.
Witherup et al. (1955) studied the effects of heptachlor on groups of 20
4-30
-------
male and 20 female CF rats administered dietary concentrations of 0 to 10 ppm
(approximately 0 to 1.43 mg/kg bw) for 110 weeks. Mortality among test groups
was not dose-related. Loss of body weight, decreased food consumption, and
increased liver weight were noted among treated males but not among females.
These signs were marked for males fed 10 ppm. Liver lesions described as
"chlorinated hydrocarbon" type and considered to be nonneoplastic were observed
in 17 percent of the males and 50 percent of the females at 10 ppm, and in 38
percent of the males and 17 percent of the females at 7 ppm. At dose level,s at
or below 5 ppm diet, none of these lesions was observed.
In a later study, Witherup et al. (1959) studied the effects of heptachlor
epoxide on groups of 25 male and 25 female CFN rats administered the compound
in the diet for 108 weeks. The dietary concentrations ranged from 0.5 to 10
ppm (approximately 0.07 to 1.43 mg/kg bw). Control rats received heptachlor
epoxide-free diets. No differences were observed with respect to food consump-
tion or growth rate. There was a dose-related increase in liver weight in
females. Hepatic cell vacuolization occurred in treated males. Hepatocyto-
megaly, degeneration, and regeneration in unspecified groups were reported.
Mortality was higher than the control level for all treated groups, but a
dose-response relationship was not observed.
Jolley et al. (1966) administered a 75:25 mixture of heptachlor:hepta-
chlor epoxide to groups of 25 female CD rats in the diet at concentrations of
5 to 12.5 ppm (approximately 0.25 to 0.625 mg/kg bw) for 2 years. A group of
54 rats received diets free of insecticide. Mortality was increased in a
somewhat dose-related way. Comprehensive histologic evaluations were per-
formed. 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 con-
4-31
-------
trol levels at 7.5, 10.0, and 12.5 ppm.
Mestitzova (1967) found that heptachlor (98.1 percent) administered in
the diet at a dose at 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 admini-
stered daily or whether it represented a total dose consumed over 1 year. In
the former case, the dose would be quite high, roughly corresponding to a
concentration of 120 mg/kg diet, assuming that a rat consumes 5 percent daily
of its body weight as food. In the latter case, the dose would be quite low,
resulting in a daily average of approximately 0.016 mg/kg bw/day.
Injection of 1.5 mg/egg heptachlor resulted in a 12 percent reduction in
hatchability but no abnormal chicks (Smith et al., 1970). However, it was
toxic to sea urchins and many abnormal embryos were induced (Bresch and Arendt,
1977).
4.3. MUTAGENICITY
Evaluations of the published mutagenicity studies on chlordane and hepta-
chlor have recently been completed by the Office of Pesticide Programs (OPP).
The OPP memoranda of November 1986 containing descriptions of the studies and
evaluations are appended to this document. Presented below are the overall
interpretations of the data extracted from the OPP chlordane and heptachlor
reports, respectively.
4.3.1. Chlordane
A total of 25 published articles containing information on 34 assays test-
ing technical- or commercial-grade chlordane were reviewed. Approximately two-
thirds of the studies were judged unacceptable or inconclusive because of study
deficiencies or inadequate reporting of critical information required for inde-
pendent verification of results. While the remaining nine studies were adequate
4-32
-------
to satisfy minimal data requirements for the genetic end points routinely ?
requested by OPP (gene mutation, chromosome damage, and DNA repair), they do
not provide a fully comprehensive data set upon which to definitively assess
the mutagenic potential of chlordane. The following general conclusions on the
mutagenicity of chlordane are warranted:
1. Neither technical- nor commercial-grade chlordane appears to be muta-
genic in bacterial assays.
2. Although negative responses have been obtained in some less well-; ;
established mammalian cell systems, chlordane and/or one of its isomers
have induced positive results without activation in other mammalian
cell mutation systems. However, in these positive studies testing in
the presence of S-9 activation was not conducted. Hence, the question
of whether chlordane induces gene mutations in mammalian cells still
needs to be clarified.
3. Chlordane was negative in mouse dominant lethal assays and in an in
vitro mammalian cell cytogenetics assay; however, there are no data
for in vivo somatic cytogenetic end points.
4. Chlordane was reported negative in in vitro DNA repair assays using
mouse, rat, and hamster hepatocytes.
5. Positive effects of chlordane have been reported for sister chromatid
exchange (SCE) in fish and human cells in culture and for gene con-
version in yeast. All of these assays have had limited utility in
genetic toxicology, and the paucity of data for more traditional
assays precludes the incorporation of these findings into an overall
classification of the mutagenetic potential of chlordane.
4.3.2. Heptachlor
Eighteen published articles containing information on 32 assays were
4-33
-------
reviewed for genotoxic effects of heptachlor/heptachlor epoxide. Again, the
majority of the assay reports were judged inadequate or inconclusive for reasons
mentioned previously for chlordane. The results of these assays, however, were
not significantly different from the results in the 10 assays judged to be
acceptable. Following are the general conclusions on the mutagenicity of hep-
tachlor.
1. Neither heptachlor nor heptachlor epoxide appears to induce gene
mutations in bacteria or mammalian cells in culture.
2. Both heptachlor and heptachlor epoxide were negative in mouse dominant
lethal assays. While one study did report an increase in resorptions
and "abnormal mitoses" in a three-generation reproduction test, this
was only reported as an abstract, and was therefore judged inconclu-
sive. The absence of data for either in vitro or in vivo direct
cytogenetic effects precludes a definitive statement about the clasto-
genic potential of heptachlor.
3. Heptachlor was negative in in vitro DNA repair assays using mouse,
rat, and hamster hepatocytes, and in bacterial DNA damage tests using
repair-deficient strains.
4.4. CARCINOGENICITY
4.4.1. Animal Studies
4.4.1.1. ChiordaneChiordane has been studied in four mouse and four rat
long-term carcinogenesis bioassays. Tables 5 and 6 present a summary of the
experimental design and tumor results. Each study is described in more detail
in the following sections.
4.4.1.1.1. Studies with Mice.
4.4.1.1.1.1. Becker and Sell (1979). A 90:10 mixture of chlordane/hepta-
chlor was fed to an unspecified number of male C57B1/6N mice at concentrations
4-34
-------
UJ
Q
Oi
O
31
O
C£
0
u_
co
l
co
UJ
1
>
1
I 1
z.
UJ
CD
O
4^
t 1
{J
a:
-
o:
i
UJ
i i
Q
UJ
CO
~~)
O
^"
u_
o
>-
s:
CO
LO
UJ
CO
g
OJ
0
c
O) OJ
t- 0
oj c_
<4- 3
OJ O
o: co
CO
] ^
i
3
CO
OJ
s_
o
E
3
1
M- C
0 0
^^ !
C OJ 4->
O £- 03
i- 3 >
4-> CO t_
03 O OJ
£_ Q. CO
Z3 X -Q
O OJ O
OJ . .
co E
O Q.
Q 0.
*k_^'
X
QJ
CO
"""7
o
c
r
03
S-
j ^
CO
A
1
O)
CO
T3
C
03
£_
QJ
v^ ^y^
U r~-
OJ O1
CO .-H
CO
03
E
O
c:
r- l_ £_
, O OJ O
O3 *"" 4-3 3
O 4-5 O 4-3
e O Q f~
03 O Z. O
^~
3 *"* c"
i IO O
1 1 I CO !-
0)^- £_ 4->
O O 03
48 ^.^ E
03 CM > O
Q. £_ If-
QJ C 3 C
n: co «r-
o
E
oo
t-i
E
Q-
Q-
CD
LO
t_
O
LO
C\J
-o
O)
<^
o
0)
Q-
CO
±1
O
z.
z.
to
"^^
r^
CD
1
LO
0
!*-
O
OJ -O
E OJ
f~~ 4-5
4-5 g~
0)
~O t/)
c:
O 03
c oj
r~ ~O
J3
LO
CO
>tn
03 ^H
CO
i »
en «<
1-H ^
UJ
<\
o
Q CO
Qi
i i !3
T3
C 1 4-> . -
03 O 03 £-
4-> OJ
LO O3 CO J3
CM O. 03 3
OJ E OJ
4-> -C O Qi
03 C -
«s *r«-
to^^ o E
OJ O £- Q-
i Q 03 Q-
3 a: o
"O ^~^ C^
O C- LO
C 03
E i T3
£- Q. 3 C
a> a., 03
> 1
i CD OJ LO
! LO O CM
o
E
oo
r-H
£_
o
** E
LO Q-
CM Q-
«O
LO LO
1 '
oQ
SI
CD
0
t-)
Q
^ 4- *3R
03
r~.
p.^
cy*
r 1
n
i i .
CJ
CO CO
03 a;
E r
O 03
c E
- OJ
0 4-
03 T5
O f~
03
C_
03 CO
< OJ
3 t
t 03
r E
0)
0 -C
03 ja
o.
OJ c
:c -r- c
o
+j
(O
>
\^ vx f
5 co 5 OJ
3 CO
0 r 0 -Q
00 Q-r-H 0
1 1
CM 00
CO CO
LO 10
og ,03
CT> t 1
e
CT> O
CM CO
u_
oS
^
CD
LO
t-H
U_
CO
e ^
IO
03
CO
OO
O">
t <
«
1
co
<
i i
OL
CO LO
03
O rH
C
a> c
"O **"
03
CO
C (Q
03 E
O
3 '^~
r CD
i C
OJ 03
0 E
4-> JC
03
CL-0
OJ C
a: 03
o
E
<*
CM
£_ £
0 Q.
Q.
V,
LO LO
e
"CM
r-l i 1
U_
08
^ '
O
oo
a:
o
i i
t_
OJ
> LO
r- CO
r 4-> OJ
03 c
t_ 03
O CO E '
E OJ
3 r T3
4-J 03 C
c E 03
O OJ
c 4- E
a.
"CO.
CO -r-
OJ LO E
i CO . CL
E O i I
i- LO
E to -a
Q- OJ C 4->
a.! 03 03
-------
O>
o
O> O>
t- o
O) t-
M- 3
0> O
o; to
Ul
0£
o
oc
o
Oi
o
u.
CO
o
5
Ul
CD
o
O 3 (O
r- tO >
+J O t_
(00.0)
<- X tO
3 o> ja
o o
tO CL
o o.
Q-
X
O)
to
CO
to
o as
(- 00
CM
LO
O>
Dl
O) O O)
+J -I- >
(O to O
c: > (O
CO -r-
E i E
4-> O-
(O > Q.
0) to
t- S_ O
+j o co
O 3 +J
to
%
a
o
CD
ot
"CM O.
o oo
"CO
>O CM
O VO ^-1
CM r-l
OO«3
"O
0)
to
U CL I
I- 0.4-J
x cz
O O 01
»-> O E co
00 4J t-.
^: (O o
oi-a o) E
r- c: t- 3
£_
>>
CM
> E
00.
oo a.
o o
LO O
r-
a>
o
CO
CL
3 O
O T-
C- +->
en c co
oo
oo
CO
co
cu
i >> o. > i o .e
*~ 4-> O C r
>, t_ O) O) T- (O
4J O CO tO CO i E
r-EO>O)OJ2tOO)
r JS E-O Sco E"O
OT-OJOOO3C
I x:«»- c: i Q.+J to
c
o
5 = 3 *
o i cr> t-
oo O-CM o>
co
c i c o.
(O E (O O-
uo o-co LO
. . .
oo r^ o 1-1
o o CM * i
CM «d- i-l CM
CO
to
E co co
o c a> jc
c > o o>
O) CO !- O !-
+J C E
, -O «3 t«-
O) i i t- to
O CT> O >|T-
+j -i- o> -r-
Q- c: co
O
oo
t-
O
* a.
LOO.
l CM
LO
I
oo
o
CM
a>
o
c
a>
c
c_
o
CO
o
4-36
o
LO
O)
a
O)
o>
c:
c_
o
ja
CO
o
o
co
oo
o>
o
CO
-------
of 25 or 50 ppm (approximately 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 experiment,
27 percent (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 avail-
able. Elevated levels of alpha-fetoprotein were associated with animals with
primary hepatocellular carcinomas. Cells from the carcinomas grew when trans-
planted, 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 premalignant lesions.
In a companion study, acetylaminofluorene was administered to the same
strain of mice (C57B1/6N) at 0.045 or 0.03 percent diet. After 61 weeks, seven
(18 percent) of the survivors had liver carcinomas, and three showed growth at
2.4 to 4 months when transplanted. On a comparison basis, chlordane was con-
sidered to be a more potent carcinogen than acetylaminofluorene. Another key
finding in this study was that chlordane induced hepatocellular carcinomas in a
strain of mice that does not spontaneously develop hepatocellular carcinomas.
4.4.1.1.1.2. The International Research and Development Corporation
(IRDC. 1973a). IRDC, under contract to the Velsicol Chemical Corporation,
administered analytical grade chlordane in the diet at concentrations of 0, 5,
4-37
-------
25, or 50 ppra (approximately 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 compoundrelated 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 autoly-
sis. 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
IRDC (1973a), was dose-related at the two highest dose levels (p < 0.01). For
males, the incidences were: 1/47 (2 percent), 34/52 (65 percent), and 38/50
(76 percent) at 0, 25, and 50 ppm, respectively, whereas for females, the
incidences were 0/57 (0 percent), 32/51 (63 percent), and 36/51 (71 percent),
respectively. The number of hepatornas 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 (U.S. EPA, 1985b) reexamined the IRDC slides and found highly
significant (p < 1 x 10"9) incidences of hepatic carcinoma rather than hyper-
plastic nodules at the 25 and 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 in close agreement with the diagno-
sis. Table 7 presents a breakdown of liver lesions as diagnosed by Reuber.
The incidences of hepatic carcinoma, as determined by Reuber, are presented in
Table 8, along with those found in the NCI study to be discussed next.
4.4.1.1.1.3. National Cancer Institute (1977a). In the NCI bioassay,
groups of 50 male and 50 female B6C3F1 mice were fed chlordane consisting of
4-38
-------
TABLE 7. INCIDENCE OF LIVER LESIONS AND TUMORS IN CD-I MICE FOLLOWING DIETARY
ADMINISTRATION OF CHLORDANE
Dose
(ppm)
0
5
25
50
Sex
M
F
r>
M
F
M
F
No. of mice
examined
33
45
55
61
52
50
39
37
No. of mice exhibiting liver
H
20(61)
26(58)
34(62)
32(52)
7(13)
13(26)
7(18)
11(30)
N
1(3)
0
6(11)
1(2)
3(6)
4(8)
0
0
SC
0
0
3(5)
0
9(17)
7(14)
4(10)
2(5)
lesions and tumorsa
LC
3(9)
0
2(4)
0
32(62)
25(50)
28(72)
24(65)
TC
3(9)hw'
5(9)
0
41(79)
32(64)
32(82)
26(70)
aH = hyperplasia; N = nodules; SC = small carcinomas (including hyperplastic
nodules with focal carcinomas); LC = large carcinomas >_ 5 mm; TC = total
carcinomas.
bTwo liver sarcomas diagnosed in the 5 ppm female group.
SOURCE: IRDC, 1973a; breakdown of liver lesions diagnosed by Reuber (U.S. EPA,
1985b).
4-39
-------
TABLE 8. INCIDENCE OF HEPATOCELLULAR CARCINOMA IN MICE FOLLOWING
CHRONIC DIETARY ADMINISTRATION OF CHLORDANE
No. of tumor-bearing mice/no, ofmice examined
(% Positive)
Dose
(ppm)
Male
Female
Strain CD-la
0
5
25
50
Strain B6C3Flb
3/33 (9%)
5/55 (9%)
41/52 (79%)
32/39 (82%)
' 0/45 (0%)
0/61 (0%)
32/50 (64%)
26/37 (70%)
0 (pooled)
0 (matched)
29.9
30.1
56.2
63.8
17/92 (18%)
2/18 (11%)
16/48 (33%)
NA
43/49 (88%)
NA
3/78 (4%)
0/19 (0%)
NAC
3/47 (6%)
NA
34/49 (69%)
aIRDC, 1973a; tumor incidence as determined by Reuber. Tumor incidences
significantly different from controls for 25 and 50 ppm in the diet
(p < 1 X 10-9) (U.S. EPA, 1985b).
bNCI (1977a); dose-related trend significant at p < 0.0001.
CNA - Not applicable.
4-40
-------
71.7 percent cis-chlordane, 23.1 percent trans-chlordane, 0.3 percent hepta-
chlor, 0.6 percent nonachlor, 1.1 percent hexachlorocyclopentadiene, and 0.25
percent chlordene isomers in the diet for 80 weeks, at two dosesa predicted
maximum tolerated dose (MTD) and 1/2 MTD. This was followed by a 10-week ob^,
! ^ -*fa ;
servation period. As upward or downward adjustments were made in dose levels,
the doses are expressed as time-weighted average (TWA) concentrations. Ttie;f!\IA
concentrations for male mice at the high dose and low dose were 29.9 and 56.2
ppm (approximately 4.27 and 8.03 mg/kg bw), respectively, and 30.1 and 63.8 ppm
(approximately 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 ijn body
weight gain among groups. Tremors were observed in 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 (Table 8). -I ;-;
4.4.1.1.1.4. Research Institute for Animal Science in Biochemistry -and
Toxicology, Japan (RIASBT, 1983a). In this study, conducted for the Velsicol
Chemical Corporation (1983b), technical grade chlordane (distribution of iso-
mers not specified) was fed to groups of 80 male and 80 female ICR mice at
levels of 0, 1, 5, or 12.5 ppm (approximately 0, 0.14, 0.71, of 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 sac-
rificed at 52 weeks. There was no 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
4-41
-------
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
liver-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 9. There was no increase in hepatic tumors in female mice. Other
than for the liver tumors in male mice, there were no significant differences
in tumors at other sites related to chlordane exposure.
In addition to liver tumors, there were remarkable increases in 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) in 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 in
the livers of males, with a less distinct trend in females.
4.4.1.1.2. Studies with Rats.
4.4.1.1.2.1. Ambrose et al. (1953a, b). In this study, technical grade
chlordane was fed to groups of three to five male and female albino rats for
400 days at dietary levels of 0, 10, 20, 40, 80, 160, 320, 640, or 1280 ppm
(approximately 0, 0.5, 1.0, 2.0, 4.0, 8.0, 32.0, or 64.0 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
4-42
-------
UJ
2:
Q
a:
0
_j
n:
°
Q
UJ
U_
LLJ
o
1
SI
a;
o
i i
^
i^
i i
cv
UJ
>
H- (
_J
UJ
n:
I
u_
o
co
o
1 H
CO
UJ
_J
o
i i
X
0
1
a:
o
Q
^f
O
i i
i
co
i
0-
0
UJ
z:
Cn
UJ
1
CG
q-
O
O
re
i 1 i 1 CD Cn Cn O
LO CM
re
co o I-H . -=d- en LO
CM i-l
i-H O CM CM CM 00
I-H O O CO T-H «i-
I 1
re re re f^ re
oo en ^~ en en en
CM i 1 LO i-H
' .
re .a
LO 1 OO OO CO CO
T 1 LO CM
(
co co t i oo co r-
r-H
CO CO «=* LO CM tO
r 1
1
en
re re c:
E E -
O O r
C C r
01 co co c c:
-a -o S o o
re re co -i !
)-> -p
- ' -c_ . 't_ £_ re re
re re re t_ t-
r r i CO CO
3 3 re 3 c c:
r i E +->t_ C 4->T3 >> O
re rere re rec +-> £-
Q. Q-O E CLre 4-> O
a> co co cu re co
3C Z 3r 3C U_ 'Z.
' f"*t
3fn-'"
'f
T-H
o
o
0
V
a.
- -. ';
CO
3
r
re «*"-^ . " ;
o
r
O O : .
£_.
4-> V
C
0 CL .
O ^ o
CO
E CO OO
O 3 CTl
£- , , <
t- re
> **
i ^ - 'f
c i ' o
CO O !-
£_ C- +J
co +-> re
> o c_>
r- (-
4J 4-
c re
re 4-» o
0 C -r-
r- 0) E
<<- i- CO
r- CO ^:
CM- O
CD 4 ^
r *r- r~-
co -a o
tjt
^> **^ "I-"
i i to
r 4-^ 'r~~
re c co
o re >
r- O
»-> -
t/i<»-
- !- UJ
-M C O
re CD a;
^ !- =3
oo co c5
re .a co
4-43
-------
enlarged livers and other liver changes were found in male and female rats fed
chlordane at 21 80 ppm, and pathologic changes in the liver was occasionally
found in male rats fed 40 ppm. No treatment-related increase in tumors was
found. The study duration (400 days) is considered too short and the number
of animals too small for this to be a valid carcinogenicity study.
4.4.1.1.2.2. Ingle (1952). Six groups of 20 male and 20 female Osborne-
Mendel rats were fed chlordane for up to 2 years at dietary dose levels of 5,
10, 30, 150, or 300 ppm (approximately 0.25, 0.5, 1.5, 7.5, or 15.0 mg/kg bw).
Harked toxicity 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,
myocardium, 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 histopatho-
logic changes in the liver, kidneys, lungs, pancreas, testes, ovaries, heart,
or spleen were noted at 5 ppm. No treatment-related tumor incidence was found.
4.4.1.1.2.3. National Cancer Institute (1977a). In the NCI carcinogeni-
city study, groups of 50 male and 50 female Osborne-Mendel rats were fed chlor-
dane (94.8 percent pure) in the diet for 80 weeks, at two dose levelsa pre-
dicted MTD and 1/2 MTD. This was followed by a 29-week observation period.
As upward or downward adjustments were made in dose levels, the doses are
expressed as TWA concentrations. The TWA concentrations for male rats at the
low dose and high dose were 203.5 and 407 ppm (approximately 10.2 and 20.4
mg/kg bw), respectively, for males, and 120.8 and 241.5 ppm (approximately 6.04
and 12.08 mg/kg bw), respectively, for females. Ten rats of each sex served as
matched controls, and 60 rats of each sex served as pooled controls. Complete
4-44
-------
necropsies and histologic examinations were performed, except in 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, that
became progressively worse as the study continued. Among female rats, there
was a highly significant (p = 0.003) dose-related increase in mortality. Mor-
tality was not significantly increased for male rats. Only two hepatocellular
carcinomas were observed, one in a low-dose male and one among the pooled con-
trols. A significant (p < 0.05) increase in neoplastic nodules of the liver
was seen in the low-dose females but not in the high-dose females or in 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 (follicular
cell 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/
or were known to be variable in populations of control rats.
In a more recent review of tumors in Osborne-Mendel rats in the NCI stu-
dies (over 900 of each sex), Goodman et al. (1980) presented data indicating
7.1 percent follicular cell tumors in control males and 3.4 percent in control
females. These data provide additional support for NCI's decision to discount
the importance of 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 in the high-dose
male group (7/44) as compared to 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,
4-45
-------
as they had occurred spontaneously throughout the bioassay program. All other
tumors were common for this strain of rat, and were not treatment-related.
4.4.1.1.2.4. The Research Institute for Animal Science in Biochemistry
and Toxicology, Japan (RIASBT, 1983b). In this study, conducted for the Vel-
sicol Chemical Corporation, chlordane (distribution of isomers not specified)
was fed to groups of 80 male and 80 female Fischer 344 rats at levels of 0, 1,
5, or 25 ppm (approximately 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 subsets
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 in 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 in mice (provided by Velsicol) 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 liver.
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 10 presents
the tumor and nontumor lesions of the liver. There was a significant increase
in adenomas of the liver in males receiving 25 ppm as compared to controls, but
4-46
-------
re
UJ
z
o
Qi
O
n:
o
O
UJ
U-
oo
«=C
o;
00
a:
UJ
0
oo
i i
LJU.
Z
1 1
GO
z
o
1 f
oo
UJ
_J
o
h 1
I
GO
<£.
1
Q-
O
UJ
^y
o
o
z
oo
on
o
1
i*s
UJ
i t
_i
o
UJ
CO
«=c
1-
"^ X
E
Q.
O.
~_-
Ol
to
o
Q
**»
CU
E
u.
LO
CM
LO
i 1
O
^-^
£
CL
Q.
O)
to
O
Q
CO
CU
re
LO
CM
LO
1
O
^*
LO
^~
IJD
^~
vo
^^»
IO
^J-
to
^^
to
^^
to
^«
tO
-o
CU
c
i
pS
re
X
CU
t/>
CU
3
to
(/)
^
[ ^
M-
o
o
z
f*l
O O CO i-H CM i 1
CO
O O CO i I Ol i-<
f 1
CM O CM O VO O
t 1 i-H
o o r~ o o LO
OJ
JD -Q J3
CTl CM CM I--- CM r-H
«* CM CM i-H
co I-H ^c I-H cn co
I 1 t 1 1 1
o o o
^H CO LO OO LO I-*
i 1 i-H i-l
^H O LO OO tO
re c !-
e .1- to
0 0 >> t-
c: i t_ +j re
CU CU O !-» i
T3 S o> re 3
re v> c M- i
P_
C- t_ £- £_ C CU
re re re re o u re
i re i i i ! o >
3 E 3 3 3+J 4->tO
^-* o ^ r-~ r re re re
i E r r i t- O.r
CU >> CU CU CU CU CU *"*
O J= O O OC J=£-
O O O O O CU CU
-|J c: ^ 4-> 4-> CD i Q.
re cu re re re cu re ^)
Q- CO Q. Q. Q.-O O J=
CU CU CU CU CU O
3i s: IE a: DC u.
.
o
0)
T3
3
r ^"^*
O ^H *-%'
C 0 LO
i- O 0
'
POO
o
C V V
cu a. o.
c. *^~* re
re co
cu cu co
CJ 3 3 CT>
LO r r i-H
re re
-o > > >
c c:
re ^ i o
00 !-
10 t- t- 4J
CM 4-3 4-3 re
c c: t_
to O O O
J>i O O Q.
flj £_
CU E E O
S 0 0 0
£_ £_
4-> 4- , >, -r-
I i tO
tO 4-> 4-> i
i c c cu
re re re ^*
E o o
r >r r0
C ai a;
-E -i- !- =3
H- 00 CO O
re ja o oo
4-47
-------
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 mammary gland in females receiving
1 ppm as compared to controls but no significant increase at 5 or 25 ppm.
Nonneoplastic lesions occurred frequently. There was a dose-related in-
"i ' ' " ' , > '
crease in the incidence of hepatocellular swelling and necrosis in male rats.
When compared to controls, the incidence of hepatocellular swelling was signi- ,
ficantly increased in all dosed males, and the incidence of hepatocellular
necrosis was significantly increased in males receiving 1 and 25 ppm. The
incidence of hepatocellular swelling was significantly higher in females re-
ceiving 25 ppm than in controls. There was also an increase in focal hepato-
cellular hyperplasia in males receiving 25 ppm, but the increase was not sig-
nificantly different compared to controls. Most of these lesions of the liver
occurred after 78 weeks of the study. A slight increase in nonneoplastic liver
lesions was seen in the 26- and 52-week sacrifice groups (Table 11).
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 versus 1/64 in controls). The historical
incidence of this tumor in F344/CRJ rats for the testing laboratory was 2.5
percent in males and 2.3 percent in females. The control incidence in this
study was 1.6 percent.
An independent review of the liver histopathology, conducted by Dr. Gary
M. 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 12). It was noted that only
two slices of liver, one from the median lobe and one from the left lobe, were
4-48
-------
TABLE 11. LIVER LESIONS IN FISCHER 344 RATS FED CHLORDANE FOR 26 OR 52 WEEKS3
Lesion 0
26 weeks
Hepatocel lular fatty 0
degeneration
Focal necrosis 0
Bile duct proliferation 0
52 weeks
Hepatocel lular fatty 0
degeneration
Hepatocel lular swelling 0
Focal necrosis 0
Bile duct proliferation 0
Small granuloma 0
aLivers from eight rats/group were
Males/Dose (ppm)
1 5 25
001
0 00
0 0 1
201
002
0 11
000
0 00
examined.
Females/Dose
015
0 0 1
- >.
010
1 00
000
0 1 0
0 1 0
001
0 0 1
(ppm)
25
I-' , y'i
0
' 3t.t
0
... . 1
0
3
'"1
0
-'£
- ' ! !-
SOURCE: Velsicol Chemical Corporation (1983a).
TABLE 12. LIVER NEOPLASMS
Neopl asm
Adenoma
Carcinoma
Total neoplasms
IN MALE FISCHER 344 RATS
Dose (ppm)
015
lb 1 4
lb 0 0
2 1 4
FED CHLORDANE3
25
8k '
2-b
10C
. :
;- -i-.-
^Pathology by Gary M. Williams, M.D., dated March 9, 1984.
"One animal had both an adenoma and carcinoma.
cSignificantly different from control value (p£0.05).
SOURCE: Velsicol Chemical Corporation (1983a).
4-49
-------
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 in the liver of both males and females were increased
in dosed animals compared to controls. The principal changes consisted of hepa-
tocellular 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 in
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 hepatocell-
ular tumors when fed at a level of 25 ppm in the diet to male F344 rats for 130
weeks. There was also a significant increase in nonneoplastic lesions of the
liver in both male and female rats; namely, an increased incidence of hepato-
cellular swelling in males receiving 1, 5, or 25 ppm and in females receiving
25 ppm. In addition, there was a significant increase in hepatocellular necro-
sis in males receiving 1 or 25 ppm, but no corresponding 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 ppm and 25 ppm, respectively;
the LOEL in males is 1 ppm.
4.4.1.1.3. Other Species.
4.4.1.1.3.1. Wazeter (1967). In this unpublished study, Wazeter, as cited
4-50
-------
in Vettorazzi (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 (approximately 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) is considered inadequate
for a carcinogenicity assay in dogs.
4.4.1.2. HeptachlorHeptachlor has been studied in three mouse and five rat
long-term carcinogenesis bioassays; Tables 13 and 14 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 in more
detail in the following sections.
' .- : '-I'"'. ".
4.4.1.2.1. Studies with Mice.
4.4.1.2.1.1. The Food and Drug Administration (Davis, 1965)The carcino-
genicity of heptachlor and heptachlor epoxide (purity not specified) was studied
using groups of 100 male and 100 female C3H mice fed a diet mixture containing
0 or 10 ppm (approximately 1.43 mg/kg bw) for 2 years. Survival was generally
low: 50 percent of the controls, 30 percent of the heptachlor-treated, and
9.5 percent of the heptachlor epoxide-treated mice survived the 2-year period;
early deaths were due to tumors. FDA pathologists found a two-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 15).
A revaluation was performed by Reuber (1977b), resulting in a change in
diagnosis for many benign tumors to liver carcinomas. Hepatic carcinomas in 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
16.
4-51
-------
Lf>
LJLJ
0>
O
OI
t_
cu
O)
CO
CT>
CO Q-
O LU
o
o
o;
O)
o
co LLJ
o>
O>
oo
OO
o
eC (O O)
Q E r
U- O
> i 'io
(13
CQ ^: -
O)
a>
(0 O
O.J3
0)
O) !-
O)
COO)
r- jQ Qi
a. i
d) 4-> fO
LU
C OJ 4->
O t- CO
I- 3 >
4J tO C_
CO O OJ
C- Q. to
3 X J3
Q CD O
3 !-
i -!-»
350)
to
o o .o
oo «-< o
CO
>
o t-
00 JQ
r-t O
E oo
LU
LU
CO
o
c: Q.
co
oo
I
oo
o u.
c. i
CO E
CO
LO Q-
«O
co
-------
UJ
o
H-H
o
UJ
Oi
0
o
1
^
UJ
T"
Q
fit
oi
o
1
^<
1
Q-
UJ
3:
^yf
O
u_
oo
r~ "
oo
UJ
>_
1
1 1
o
i i
UJ
o
i t
QZ
o
>_
(V
=C
1 1 1
I-H
0
1
t^
oc
u_
0
>_
C£
SI
S
OO
.
1 1
UJ
_]
co
+-) 00 i_
(TJ O CU
I- CL 01
3 X -Q.
a QJ o
O)
to
O
Q
X
CO
9
O
C
(r-
03
£_
j i
oo
r-^ r-^ «=C
t * O3 o3 D-
. ,__ UJ
, 03 4-> 4->
ro O) O)
ja 40 OO
4_j t~- Old) C7> Q- O> Q- «
0 £ .= >,=£ = E =
^_ ^H t_O<£> .)->! LO-P'i-LOOO
O5 CTi C_ 5 QJ "~3 CTi (U IS O"> Cl) 13 C7l C7l
0 r-l Z i^- rH V:^-'ii-l ^ r-l i-l
<1) t-
CO O)
n v-Q O3 « > OO
CO +J tO C O) '-^ !- 03
"O ^O3 ^.03 t- CT> r~* E
a> o o o c o
+jto co E<" Er CT-CC
o3t- 3 -i-t-T-T-
r^- O *> r '^~ 4-^ O > 4-* 4-^ CD O
CU E Ol 3 *» *H~ O O3 T3 4-* d) ^
t_3 t_"o-atoco ctoja c a>4->coCL)03U
cc 3 OE ai-oaio i S-TS
OJ.I- 4->O.'O'-3 COt-C tO-r- CU OC
E -r O) CT>4-) O3CUO3 (13 to C-(/)c:o3
4_> Q.) f | ^ [ % QJ > QJ CU 1 ^- *^ **~^
O3co CUtoo3i -aw *->-£ '-i QJO cot-
OJ03 >o3QJO-i-QJ Or O. O E lOE4->aJOJ
t_a> !- 1 t_ t_ o r c: a. crt-o. o 3 j= i JD
[ % t ^M r^ ] % | * t fl3 »r QJ "^ OJ f*^ T3 4-> O^ 3 3
O O C >> E E LO > *^ TD CU
oc o cu c: o -C cu oo. o-i o ocr ooi
Z-r- 2:CT-04J,4-> 03 IO 03 ^t ^
r > 0 > 5 S
r CO C- r-l ^d ^: i- S-
030JQJ StO^QJ >> O OO
^O t^ t/) i 3 ^0 ^^ t: J
^5 ^^ ^D v^ ^^ ^^ f^ jQ ^SJ T"! t"")
LO T3 O M- 3 OO Q.CO O
.1.
S u. 00. « E E
, . II i 1 LO O. > « Q.
CDIO -OET3E LO O. LOLOQ.
\y CD C O- C O- « . « . .
^^ to O3 Q_ O3 O_ LO OJ CO O CXI 1 O
U) O » I » ' *"*
E ~o CT* o^ r^- oo r*^ ** « «
. . . .' "O LO "O LO O "O
OLO oo r~- LO « t "C «c: ..c
t 1 OOr-~CVlLO LOO3 T-HO3 OLOO3
O.T3
. 3 CU
O X O «-
C O) t_ <4-
CO CD-i-
U- O U- U- U- U.
J= CU QJ
oBOtOO-oB °B oB oB
03 O to
s: QJ T3 2: s: 2: s:
***«». 1 % ""**>» *^"S1». ****>. "**^^
LO'J-COO LO O LO
CTlO-r-CLO CM CM CNJ
T3
c
cu
QJ
(- C
o3 t-
4-> 0
to _a ^
r- 00 Q U- U-
° 4-53 °
-------
TABLE 15. INCIDENCES OF LIVER LESIONS IN C3H MICE
TREATED WITH HEPTACHLOR OR HEPTACHLOR EPOXIDE
Dose group
Controls
Heptachlor (10 ppm)
Heptachlor
epoxide (10 ppm)
Hepatic
hyperplasia
38/200
108/200
65/200
Benign liver
tumors only
30/200
51/200a
85/200b
Malignant
liver tumors only
21/200
10/200
13/200
^Statistically significant (p < 0.0064),
^Statistically significant (p < 10~9).
SOURCE: Davis, 1965.
TABLE 16. INCIDENCES OF HEPATOCELLULAR CARCINOMA IN C3H MICE
TREATED WITH HEPTACHLOR OR HEPTACHLOR EPOXIDE
Dose group
Males
Females
Controls
10 ppm heptachlor
10 ppm heptachlor epoxide
22/78 (28%)
64/87 (73%)
73/79 (92%)
2/54 (4%)
57/78 (73%)
77/81 (95%)
SOURCE: Davis, 1965 (as diagnosed by Reuben, 1977b)
4-54
-------
Reuben's findings yielded a highly significant (p = 5 x 1CT8 to <1 x 1(T9)
increase in incidences of liver carcinoma in both sexes for heptachlor and
heptachlor epoxide. Four other independent pathplogists (Drs. R. Squire, R.
Sternberg, H. Stewart, and 6. Williams) reviewed a sample of 19 slides and
generally concurred in the Reuben diagnoses (U.S. EPA, 1985b).
In addition to liven tumors, Reuben also diagnosed nontumon liver lesions,
primarily hyperplasia, nodules, hepatic vein thrombosis, and cirrhosis, in the
heptachlor and heptachlor-epoxide-treated mice.
4.4.1.2.1.2. National Cancer Institute (1977b). In the NCI carcinogen-
icity bioassay conducted at Gulf South Research Institute, technical grade
heptachlor (approximately 73 percent heptachlor, 22 percent trans-chlordane,
and 5 percent noriochlor) was fed to groups of 50 B6C3F1 mice of each sex for
80 weeks, followed by an observation period of 10 weeks. The TWA high-dose
and low-dose concentrations were 6.1 and 13.8 ppm (approximately 0.87 and
1.97 mg/kg bw) for males and 9.0 and 18.0 ppm (approximately 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 compared
with matched controls, while the incidence in low-dose males was comparable to
that of the control groups. The difference in hepatocellular carcinoma inci-
dence 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 dif-
ference between the high- and low-dose groups. Other tumor types were observed
with low frequency among all groups. The incidence of hepatocellular carcinoma
in mice is presented in Table 17.
4-55
-------
TABLE 17. INCIDENCE OF HEPATOCELLULAR CARCINOMA
IN B6C3F1 MICE FOLLOWING
CHRONIC DIETARY EXPOSURE TO HEPTACHLOR/CHLORDANE MIXTURE9
Groups (ppm diet)
Incidence9
Males
0 (pooled)
0 (matched)
6.1
13.8
17/92 (18%)
5/19 (26%)
11/46 (24%)
34/47 (72%)b
Females
0 (pooled
0 (matched)
9.0
18.0
3/78 ( 4%)
2/10 (20%).
3/47 ( 6%)
30/42 (71%)c
Incidence expressed as No. of tumor-bearing mice
. No. of tissues examined
DStatistically different from matched controls (p = 0.0001); also dose-related
increase in male mice (p < 0.0001).
cStatistically different from matched controls (p < 0.005);
also dose-related increase in female mice (p < 0.0001).
SOURCE: NCI, 1977b.
4-56
-------
There were also many nodules in the mice that did not have liver carcinomas.
4.4.1.2.1.3. International Research and Development Corporation (1973b).
IRDC, under contract with Velsicol Chemical Corporation, fed a 25:75 mixture
of heptachlor:heptachlor epoxide in the diet, at concentrations of 1, 5, or
10 ppm (approximately 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 were fed an insecticide-free diet. The incidences of hepato-
mas were lower among the higher dose groups than in the 1 mg/kg diet group
and controls. However, the incidences of nodular hyperplasia (Table 18) 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 (U.S. EPA,
1985b). Five other pathologists (Drs. J. Rust, P. Newberne, R. Squire, H.,,.
Stewart, and G. Williams) examined a portion of the slides and agreed with4,
Reuber that the incidence of hepatic carcinoma was considerably underdiag-
nosed in the original analysis (U.S. EPA, 1985b). The incidences of liver""
carcinoma as diagnosed by Reuber are presented in Table 19.
4.4.1.2.2. Studies with Rats.
4.4.1.2.2.1. Cabral et al. (1972). Heptachlor (96.8 percent pure) in
corn oil was administered by gavage to Wistar rats in five doses of 10 mg/kg
bw each on alternate days starting at 10 days of age. The heptachlor-treated
group contained 95 Wistar rats, 7 of which died before weaning. The controls
consisted 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 to 110 weeks. There was no indication of a treat-
4-57
-------
TABLE 18. INCIDENCE OF NODULAR HYPERPLASIA IN CD-I MICE
EXPOSED TO HEPTACHLOR/HEPTACHLOR EPOXIDE (25:75) MIXTURE
Dose (ppm)
Males
Females
0
1
5
10
0/50 (0%)
2/53 (4%)
24/57 (42%)
53/69 (77%)
1/67 (1%)
0/63 (0%)
9/56 (16%)
28/46 (61%)
SOURCE: IRDC, 1973b.
TABLE 19. INCIDENCE OF HEPATIC CARCINOMA IN CD-I MICE
FOLLOWING CHRONIC DIETARY EXPOSURE TO HEPTACHLOR EPOXIDE (25:75)a
Groups (ppm diet)
Incidence*5
Males
0
1.0
5.0 ,
10.0
Females
0
1.0
5.0
10.0
0/62 ( 0%)
2/68 ( 3%)
18/61 (26%)
52/80 (65%)c
6/76 ( 8%)
1/70 (10%)
6/65 ( 9%)
30/57 (53%)c
aBased on Reuben's revaluation of IRDC (1973b) slides (U.S. EPA, 1985b),
"Incidence expressed as No. of tumor-bearing mice
No. of tissues examined
cSignificantly different from control value (p _< 0.001).
4-58
-------
merit-related increase in tumors.
4.4.1.2.2.2. Kettering Laboratory (Jolley et al., 1966). In this study,
a 75:25 mixture of"heptachlorzheptachlor epoxide 'was administered to groups of
. >
25 female CD rats in the diet at concentrations of 5, 7.5, 10, and 12.5 ppm
(approximately 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 fibroade-
nomas, 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. ^
4.4.1.2.2.3. National Cancer Institute (1977b). In the NCI bioassay
conducted at Gulf South Research Institute, technical grade heptachlor was fed
for 80 weeks to groups of 50 male and 50 female Osborne-Mendel rats at a time-
weighted average dietary level of 38.9 or 77.9 ppm (approximately 1.95 or 3.90
mg/kg bw) for males and 25.7 or 51.3 ppm (approximately 1.29 or 2.57 mg/kg bw)
for females. Animals were maintained for an additional 30 weeks on a hepta-
chlor-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 treatment.
However, no hepatocellular carcinomas were observed in any of the rats, with
one cholangiocarcinoma diagnosed in one low-dose male. Neoplastic 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 20. A
statistically significant exact test for a dose-related trend (p < 0.002) was
. ~ ' . ..-'< :i :" -., ;-, 2;, -
found for follicular-cell carcinomas of the thyroid of females, but not in,;
males, when they were combined with adenomas. However, this finding was dis-
4-59
-------
counted 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.
TABLE 20. INCIDENCES OF NEOPLASTIC NODULES IN OSBORNE-MENDEL RATS'
FOLLOWING CHRONIC DIETARY EXPOSURE TO HEPTACHLORa
Groups (ppm diet)
Incidence'3
Males
0 (pooled)
0 (matched
38.9
77.9
Females
0 (pooled)
0 (matched)
25.7
51.3
2/58 (3%)
1/10 (10%)
3/44 (7%)
6/49 (12%)
5/59 (8%)
1/10 (10%)
9/48 (19%)
5/46 (11%)
aNot significant (p £ 0.05) by either exact test or life-table adjustment.
^Incidence expressed as No. of nodule-bearing rats
No. of tissues examined
SOURCE: NCI, 1977a.
4.4.1.2.2.4. Kettering Laboratory (Witherup 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
(approximately 0.075, 0.15, 0.25, 0.35, and 0.5 mg/kg bw) for 110 weeks.
4-60
-------
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.
4.4.1.2.2.5. Kettering Laboratory (Hitherup 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 (approximately 0.025, 0.125, 0.25, 0.375, and 0.5
mg/kg bw) for 108 weeks. Similar groups of controls were maintained on hepta-
chlor epoxide-free diets. In the Kettering analysis, malignant and benign
tumors occurred randomly among the test groups and were not related to hepta-
chlor epoxide treatment. A reexamination of the histologic slides was conduc-
ted by two pathologists, Drs. M. 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 in males. Table 21 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 Witherup (U.S. EPA, 1985b).
4.4.1.2.3. Studies with Dogs.
4.4.1.2.3.1. 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
4-61
-------
TABLE 21. INCIDENCE OF HEPATIC CARCINOMA AND NEOPLASTIC NODULE'S IN CFN RATS
FOLLOWING CHRONIC DIETARY EXPOSURE TO HEPTACHLOR EPOXIDE3
Dose
(ppm)
Reuber
"''Williams
Males
0
0.5
2.5
5.0
7.5
10.0
Females
Carcinomas
1/24 (4%)
1/22 (5%)
0/19 (0%)
1/23 (4%)
1/25 (4%)
4/22 (18%)
Nodules
6/24 (25%)
6/22 (27%)
7/19 (37%)
9/23 (39%)
2/25 (8%)
7/22 (32%)
Carcinomas Nodules
0/24 (0%)
1/23 ('4%)
0/21 (0%)
1/22 (5%)
0/25 (0%)u
1/22.(5%)b
0/24 (0%)
0/23 (0%)
0/21 (0%)
0/22 (0%)
1/25 (4%)
4/22 (18%)b
0 -'"
0.5
2.5
5.0
7.5"
10.0
0/17 (0%)
3/22 (14%)
3/18 (17%)
7/22 (32%)c
3/21 (14%)
5/19 (26%)c
7/17 (41%)
8/22 (36%)
4/18 (22%)
10/22 (45%)c
14/21 (67%)-
8/19 (42%)c
0/17 '(0%)
0/20 (0%)
0/18 (0%)'
1/22 (5%)
0/23 (0%)
1/19 (5%)
4/17 (24%).
5/20 (25%)
2/18 (11%)"
2/22 (9%)
' 5/23 (22%)
1/19 (5%)
Incidence is expressed as No. of tumor-bearing rats
No. of rats examined
"Statistically significant (p = 0.019) for combined incidence of carcinomas and
nodules (U.S. EPA, 1985b).
Statistically significant (p = 0.05) for combined incidence of carcinomas and
nodules (U.S. EPA, 1985b).
SOURCE: Witherup et al., 1959. Data from the Reuber/Williams diagnoses pre-
sented in U.S. EPA, 1985b. - : ' '
4-62
-------
which time they were sacrificed and'autopsied. No tumors were reported. While
the liver weights of both males and females tended to increase logarithmically
in proportion to the amounts of heptachlor epoxide in 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.
4.4.2. Epidemiologic Studi es
4.4.2.1. Infante et al. (1978)Infante et al. (1978) presented data on five
case reports of neuroblastoma and six case reports of aplastic anemia and acute
leukemia. Between December 1974 and February 1976, 14 cases of neuroblastoma
were diagnosed at a single pediatric hospital. Of these, a history of exposure
to toxic agents indicated that five had previous exposure to chlordane. The
cases ranged in age from 2 years, 8 months to 6 years, 5 months. Exposure in
each case was due to the use of chlordane in the home due to pest infestation.
In two of the five cases, the first exposure occurred during the first trimester
of pregnancy in the mother. One of these included additional exposure to a
halothane-nitrous oxide anesthetic during the first month of pregnancy. In the
remaining three cases, exposure occurred after birth. Two of the five cases
had postnatal exposure to X-rays. The authors stated that of the other nine
cases admitted to the hospital during this time period, a history of exposure
to chlordane was not known.
According to the authors, three cases of aplastic anemia and three cases
of leukemia were reported "at several hospitals in the past several years."
The three aplastic anemias were in males with exposure to chlordane in the
home. In each case, the man had actually applied the chemical himself. In
two cases, exposures to other toxic agents, including other insecticides,
4-63
-------
paints, thinners, and varnishes were reported. The ages of the three men were
15, 28, and 68.
Of the three leukemia cases, only one was reported to have been exposed to
other toxic substances besides chlordane. This 23-year-old man was employed by
a lawn care company for which he sprayed lawns with Banvel D, diazinon, and
2,4-D, as well as chlordane. The second case was a 9-year-old girl who had
been exposed to chlordane annually since birth through the use of chlordane in
the home to treat termites. The third leukemia case was a 37-year-old male who
had used chlordane around his house for a period of 10 years.
The authors reported on several case studies of exposure to chlordane or
heptachlor. As no control group was used as a comparison population, these
data provide inadequate information with which to assess the association of
cancer risk and chlordane or heptachlor exposure.
4.4.2.2. Wang and MacMahon (1979a)--A retrospective cohort mortality study of
16,126 professional pesticide applicators was undertaken to study the mortality
patterns of these workers. The cohort was selected from three pest control
companies with offices in over 40 states. Personnel records for all persons
in job categories potentially involving exposure to various pesticides and
employed between January 1, 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; therefore, this date was used as the ear-
liest 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 follow-
ing 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 4,000 were examined. Eleven percent had been
excluded because they were female, 71 percent because they were employed for
4-64
-------
-------
SMRs were observed for skin cancer (SMR = 173, statistically nonsignificant)
and for bladder cancer (SMR = 277, corrected observed = 3.5, 95 percent CI
101-761). Statistically significant SMR deficits were observed for malignant
neoplasms of the digestive organs and peritoneum (SMR = 46, corrected ob-
served = 6.9, 95 percent CI 22-95), respiratory system diseases (SMR = 29,
corrected observed = 4.6, 95 percent CI 12-70), diseases of the digestive
system (SMR = 55, corrected observed = 11.6, 95 percent CI 31-98), and all
other causes (SMR = 355, corrected observed = 15, 95 percent CI 33-90).
SMRs for various specific causes of death using the true number of ob-
served deaths can be calculated and are presented here. For the total cohort
they are as follows: SMR for all causes of death is 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 is 231 (observed = 3); for
non-malignant diseases of the respiratory system, the SMR is 25 (observed = 4);
and for non-malignant diseases of the digestive system, the SMR is 48 (observed
= 10).
Analyses were also conducted for those workers classified as termite con-
trol operators, since they were thought to have had the most exposure to chlor-
dane 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 applicators = 187), and cancer of the bladder (SMR for ter-
mite control operators = 215, SMR for all other applicators = 187). None of
4-66
-------
the above-mentioned SMRs was statistically significant. The authors stated
" . .. '.- '3W
that the only significant SMRs (p < 0.05) observed for termite control opera-
tors were for cancer of the digestive organs (SMR = 20, corrected observed =
1.2) and for cerebrovascular disease (SMR = 39, corrected observed = 2.4).
fi£ '
The authors also stated that there were no significant findings in the "all
. . -(-.'"'
,».
other applicator" group, nor were there significant differences in SMRs between
the two groups. (The method used to determine significant differences between
' > , . ' '7!**-' .
the two groups was not reported.) A nonsignificant increase in lung cancer was
restricted to the "all other applicator" group (SMR =131, corrected observed
= 16.9). It is possible to calculate SMRs using the true number of observed
' it'.':
deaths for cancer of the skin and bladder. For skin cancer, the SMR for ter-
mite 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.
C' >'
Workers were classified according to intensity of exposure into three
groups: minimal, intermediate, and highest exposure. None of the causes of
': C 7
death exhibited an increase in SMR with an increase in intensity of exposure.
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
138, 120, and 87 were observed for minimal, intermediate, and highest exposure,
:. i>
respectively, and for skin cancer SMRs of 198, 187, and 138 were observed,,
respectively. The authors did not discuss the statistical significance of
- -,'' Jf*;:
these findings.
, 0'"*
A latency analysis was performed for lung cancer. However, few workers
were followed for more than 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
a::-
4-67
-------
increased.
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 different, 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 possi-
ble 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 control of con-
founding factors such as smoking and alcohol consumption. This study provides
inadequate evidence on the carcinogenicity of chlordane and heptachlor.
4.4.2.3. Wang and MacMahon (1979b)A retrospective cohort mortality study was
undertaken in 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, and for 1425 workers who had
ever worked at a heptachlor and endrin plant in Memphis, TN before the spring
of 1976. Of these, 1,403 subjects were identified who met the following cri-
teria: (1) male, (2) who had worked for more than 3 months, and (3) "adequate"
4-68
-------
identifying information was available. Of the 973 excluded, 7 percent were
excluded because they were female, 64 percent because they worked less than 3
months, and 29 percent 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 con-
duct 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 percent of those ascertained)
and were coded according to the 8th revision of the ICD by one of the authors
(Wang). Cause-specific SMRs were calculated by means of Munson's (1974) com-
puter program, using national mortality rates for white males by age and calen-
dar 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 mid-p values.
The authors attempted to correlate the intensity of exposure with mortal-
ity 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 job category;
however, no data were presented. Thirty-four percent of the study cohort had
less than 10 years duration of follow-up, 25 percent had 20 to 29 years of
follow-up, and 11 percent had more than 29 years of follow-up.
The overall SMR for all causes of death was 72 (observed = 113, 95 percent
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
4-69
-------
cancer of the digestive organs and peritoneum, the SMR was 82; for lymphatic
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 (SMR = 183; observed = 17, 95 percent CI 110-287).
OnljTone death was attributed to liver cancer: an 81-year-old man who died in
1958 and had worked for the company for 5 years beginning in 1944. A signifi-
cant deficit was observed for ischemic heart disease (SMR = 69, observed = 37,
95 percent CI 49-94).
Because the SMR for lung cancer was elevated (SMR = 134), though not sta-
tistically significant, the distribution of lung cancer deaths across other
variables was investigated. For workers less than 35 years old at entry into
occupation and less than 50 years old at observation, the authors noted that
the difference between observed and expected (5 and 1.2, respectively) was
statistically significant (p < 0.01). The relationship between duration of
employment and duration of follow-up or latency for lung cancer was studied.
The numbers were small and no overall pattern was observed. However, the
results indicate that there was a statistically significant deficiency (p-value
not reported) in lung cancer deaths (observed = 1, expected = 4) among those
exposed for 20 or more years. Conversely, among those employed for 10 to 19
years and followed for 10 to 19 years, there was an excess risk (observed = 6,
, 1(... * i- - ' '* .
expected = 2.1).
> , c f-
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
endrin exposure, a pesticide also manufactured at the Memphis plant.
the cohort included all plant employees, including those with little
4-70
-------
potential for exposure, such as office workers; and therefore, the risk of can-
cer 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 if one was present.
The authors did not individually follow up each cohort member. Therefore,
the number of deaths may have been underreported. Excluded from the cohort
were 282 individuals with missing data, representing about 29 percent 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 for
which smoking is a well-documented risk factor. The SMR 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
(SMR = 134) may have been an occupationally related increase. Finally, race
was assumed to be white for all study subjects. As no deaths among non-whites
were observed, this assumption may be a valid one. Thus, this study provides
inadequate evidence for the carcinogenicity of chlordane and heptachlor.
4.4.2.4. Ditraglia et al. (1981)A retrospective cohort mortality study of
employees at four organochlorine pesticide manufacturing plants was undertaken.
All workers (race and sex not specified) who had at least 6 months of employ-
ment in pesticide manufacture prior to December 31, 1964 were included in the
study population. Vital status was determined for each worker as of December
4-71
-------
31, 1976, through the SSA, State motor vehicle offices, U.S. Postal Mail Cor-
rective 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 dicyclopentadiene.
The cohort at plant 1 consisted of 327 individuals, representing 8,354 person-
years of observation. Three percent were lost to follow-up. Plant 2 had
manufactured heptachlor since 1951. Endrin was also produced at this plant in
addition to chlorine, chlorendic anhydride, hexachlorocyclopentadiene, and
vinyl chloride. The cohort at this plant consisted of 305 workers (5,672
person-years of observation), and 5 percent 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 Wang 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 (Ditraglia et al., 1981).
Death certificates for all known decedents were obtained and coded by a
nosologist 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 Poisson distribution. Confidence intervals were presented for
the SMR estimates, but the method of calculation was not reported by the authors.1
Statistically significant (p < 0.05) deficits were observed for all causes
of death at the chlordane plant (SMR = 68, observed = 59, 95 percent CI 52-87)
4-72
-------
and at the heptachlor plant (SMR = 66, observed = 24, 95 percent CI 42-98).
For deaths due to 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 intestinal cancer (SMR-,,5=
175), respiratory system cancer (SMR = 122), and cancer of the bladder and uri-
nary 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) less than 10 years since first employed, (2) 1.0;
to 19 years since first employed, and (3) 20 or more years since first employ-.
ed. 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 less than 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 statistically, .-,.-.
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
; - ' . i i t, -. i'
workers at these two plants. While none of the SMRs was statistically signifi-.
cant, the SMR of 303 (observed = 3) for stomach cancer in the chlordane,pi ant
may be important. However, there are limitations to this study. ,.
No information on quantitative exposures were provided. It was not pos-
4-73
-------
sible 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 carcin-
ogenic. No attempt was made to exclude or adjust for the effects of sex or
race, nor was there an effort to control for other confounding variables 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 in 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 in 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.
4.5. STRUCTURE-ACTIVITY RELATIONSHIPS
Seven chemicals were identified as important in the analysis of the struc-
ure-activity relationships of chlordane and heptachlor for carcinogenicity.
These chemicals are aldrin, chlorendic acid, dieldrin, endrin, endosulfan,
hexachlorocyclopentadiene, and isodrin; their structures are presented in
Figure 4.
4.5.1. Aldrin
4.5.1.1. Evidence for Carcinogenicity - Human Exposure (IARC. 1982)Specific
mention of aldrin in analytic epidemiologic studies is limited to reports of
the follow-up of a cohort of men employed in its manufacture at a plant where
dieldrin, and later endrin and telodrin, were also manufactured. In the most
recent report, of 166 men exposed to these compounds for more than 4 years and
for 15 or more years before the end of follow-up, two cases of cancer were
observed. No estimate was provided of the expected number of cancers, and the
4-74
-------
Cl
Dieldrin
Cl
.3=0
Endosulfan
Cl
OH
Cl Chlorendic Acid
Endrin
Cl,
Cl
ci ci
Hexachlorocyclopentadiene
Cl-
Cl Isodrin
Figure 4. Chemicals selected for structure-activity analysis.
4-75
-------
description of follow-up was limited. These data are inadequate to assess the
carcinogenicity of aldrin in humans.
4.5.1.2. Evidence for Carcinogenicity - Animal Studies (IARC, 1982)Aldrin
has been tested by the oral route in mice and rats. It was carcinogenic in
mice at 10 ppm in the diet, producing malignant liver neoplasms: three studies
in rats were negative or equivocal and one was inadequate due to decreased
survival rates. These data provide sufficient evidence for the carcinogenicity
of aldrin.
4.5.1.3. Evidence for Activity in Short-Term Tests (IARC. 1982)Aldrin did
not produce damage to Escherichia coli plasmid DNA and was not mutagenic to
Salmonella typhimurium or to yeast. It did not induce recessive lethal muta-
tions in Drosophila melanogaster. Aldrin induced DNA repair in cultured human
fibroblasts and lymphocytes, but it did not elicit unscheduled DNA synthesis
in cultured rat hepatocytes. It produced chromosomal aberrations in bone-
marrow cells of rats and mice exposed in vivo, but it did not elicit a positive
response in the mouse bone-marrow micronucleus test. No data on human cell
lines or lymphocytes were available. These data are inconclusive in assessing
the carcinogenicity of aldrin.
4.5.2. Chlorendic Acid
4.5.2.1. Evidence for Carcinogenicity - Human Exposure (NTP, 1985)Chlorendic
acid is manufactured in an essentially closed system. Although this procedure
would seem to minimize human exposure, there are no published data on the level
of occupational exposure to Chlorendic anhydride or Chlorendic acid.
4.5.2.2. Evidence for Carcinogenicity - Animal Studies (NTP, 1985)In an NTP
bioassay, there was clear evidence of carcinogenicity for Chlorendic acid in
male F344/N rats as shown by increased incidences of neoplastic nodules of the
liver and acinar cell adenomas of the pancreas. Increased incidences of alve-
4-76
-------
olar/bronchiolar adenomas and preputial gland carcinomas may also have been
related to the administration of chlorendic acid. There was evidence of car-
cinogenicity for chlorendic acid in female F344/N rats as shown by increased
incidences of neoplastic nodules and of carcinomas of the liver. There was
evidence of carcinogenicity for chlorendic acid in male B6C3F1 mice as shown
by increased incidences of hepatocellular adenomas and hepatocellular car-
,'S'"
cinomas. There was no evidence of carcinogenicity for chlorendic acid in
female B6C3F1 mice given chlorendic acid in the diet at concentrations of 620
or 1,250 ppm for 103 weeks. These data provide sufficient evidence for the
carcinogenicity of chlorendic acid.
4.5.2.3. Evidence of Activity in Short-Term Tests (NTP, 1985)--Chlorendic
acid was not mutagenic in strains TA100, TA98, TA1535, or TA1537 of SalmonelTa
typhimurium in the presence or absence of Aroclor 1254-induced male Sprague-
t
Dawley rat or male Syrian hamster liver S9 when tested according to the pre-
incubation protocol. Chlorendic acid was mutagenic in the L5178Y/TK"1"/" mouse
lymphoma cell forward mutation assay in the absence of S9 and was not tested in
the presence of S9. There was no mutagenic response in the absence of severe
toxicity. The toxicity curve was sharp, going from relative total growth of
74 percent to 5 percent at doses of 1,600 to 1,700 yg/mL. With such a sharp
toxic response, the increase in mutant count and mutant frequency was observed
only at the higher dose; this response was replicated in another experiment.
A response in which only one toxic response is positive raises the question of
whether the mutagenic response is an indirect mechanism in which the mutation
is not due to the direct interaction of the chemical with DNA. This assay, as
performed, does not answer this question. These data are inconclusive in
assessing the carcinogenicity of chlorendic acid.
4-77
-------
4.5.3. Dieldrin
4.5.3.1. Evidence for Cardnogenicity - Human Exposure (lARCjj 1982)Mean
tissue levels of dieldrin were elevated in one autopsy study of 50 cancer
patients and 42 comparison subjects. Mean serum levels were also reported to
be elevated in eight cancer patients compared with seven controls. Follow-up
of sdrte 233 workmen (166 employed for more than 4 years and followed for 15 or
more years) showed two cancer deaths at a plant where aldrin and dieldrin, and ;
later endrin and telodrin, were manufactured. These data are inadequate to
assess the carcinogenicity of dieldrin.
4.5.3.2. Evidence for Carcinogenicity - Animal Studies (IARC, 1982)--Die1drin
is carcinogenic to mice, producing benign and malignant liver neoplasms in many
strains following oral administration. Studies in rats are negative or equivo-
cal. No carcinogenic effect was observed in feeding studies with hamsters.
Feeding studies with dogs and monkeys were considered to be inadequate for a
carcinogenicity evaluation. These data provide sufficient evidence for the
carcinogenicity of dieldrin.
4.5.3.3. Evidence for Activity in Short-Term Tests (IARC, 1982)--Die1drin did
not produce DNA breaks in an Escherichia coli plasmid. It did elicit DNA re- i
pair in cultured human fibroblasts and human lymphocytes, but it did.not do so
in cultured rat hepatocytes and did not produce DNA damage in cultured Chinese"
hamster cells. It was not mutagenic to Salmonella typhimurium, to yeast, to
Drosophila melanogaster (recessive lethals) or to the wasp, Bracon hebetor.
In a limited study, it was weakly mutagenic to Chinese hamster V79 cells. It
produced chromosomal aberrations in human embryonic lung cells in vitro and ..
chromosomal damage to mouse bone-marrow cells-in vivo in one study but not in .
another, and not in Chinese hamster bone-marrow cells. It did not induce cell,
transfdrmaton in vitro (in BHK cells). It was negative in the mouse dominant^
4-78
-------
lethal test and did not induce mitotic gene conversion in yeast in a host-medi-
ated assay. No data on human cell lines or lymphocytes were available. These
data are inconclusive in assessing the carcinogenicity of dieldrin.
4.5.4. Endrin
4.5.4.1. Evidence for Carcinogenicity - Human Exposure (IARC, 1974)A study
was conducted in 1968 of 233 workers employed in a factory that had been manu-
facturing aldrin and dieldrin since 1954-1955, endrin since 1957, and telodrin
during 1958-65. Lengths of exposure ranged from 4 to 13.2 years (average, 7.6
years). One hundred and eighty-one workers were still employed by the same
firm at the time of the study, and their average age was 41 years (range, 22 to
64). Only two deaths had occurred, and one was due to stomach cancer. The
52 workers who had left the company were the subject of a subsequent report*
Average age at the time of this survey was 47.4 years (range, 29 to 72), aver-
age occupational exposure was 6.6 years (4.0 to 12.3), and average time since
end of exposure was 7.4 years (4.5 to 16). Only one death was recorded, and
this had not been caused by cancer. These data are inadequate to assess the
carcinogenicity of endrin.
4.5.4.2. Evidence for Carcinogenicity - Animal Studies (IARC. 1982)--There was
no evidence of carcinogenicity in B6C3F1 mice or Osborne-Mendel rats fed endrin,
although there' was early mortality in both species. Another rat study was .
inadequate. These data are inadequate to assess the carcinogenicity of endrin.
4.5.4.3. Evidence for Activity in Short-Term Tests (NTP, 1985)Endrin did
not cause mutations in NTP Salmonella mutagenicity tests (NTP, 1985). These
data are inadequate to assess the carcinogenicity of endrin.
4.5.5. Endosulfan
4.5.5.1. Evidence for Carcinogenicity - Human Exposure (NCI, 1978)Specific
mention of endosulfan in analytic epidemiologic studies was not found. Medical
4-79
-------
reports mention fainting, convulsions, and unconsciousness following exposure
to endosulfan; however, recovery was rapid. These data are inadequate to
assess the carcinogenicity of endosulfan.
4.5.5.2. Evidence for Carcinogenicity - Animal Studies (NCI, 1978)In an NCI
bioassay, no conclusion was drawn on the carcinogenicity of endosulfan for male
rats or male mice because of early deaths in these groups. Under the condi-
tions of the bioassay, endosulfan was not carcinogenic in female Osborne-Mendel
rats or in female B6C3F1 mice. These data are inadequate to assess the carcin-
ogenicity of endosulfan. .
4.5.5.3. Evidence for Activity in Short-Term Tests (NTP. 1985)No evidence
of the mutagenicity of endosulfan was noted in the standard Ames test and in
the mouse micronucleus test. These data are inadequate to assess the carcin-
ogenicity of endosulfan.
4.5.6. Hexachlorocyclopentadi ene
4.5.6.1. Evidence of Carcinogenicity - Human Exposure (U.S. EPA, 1984)
Although there is human experience with respect to mortality, there is only
limited information on the morbidity results in those exposed to hexachloro-
cyclopentadiene (HEX). Acute inhalation produces a high prevalence of head-
aches and severe irritation of the eyes, nose, throat, and lung. Dermal con-
tact can cause severe burns. Epidemiologic studies have generally shown no
significant differences in mortality between workers exposed to HEX in the
j workplace and the general population. Although a significant excess of deaths
from cerebrovascular disease was reported in one study, the deaths showed no
consistent pattern with respect to duration of employment or follow-up. Cur-
rent human exposure is limited to improper handling and disposal and proximity
to either manufacturing sites utilizing HEX or disposal sites. These data are
inadequate to assess the carcinogenicity of HEX.
4-80
-------
4.5.6.2. Evidence of Cardnogenicity - Animal Studies (U.S. EPA, 1984)--In
vivo bioassays have not been conducted; however, an inhalation bioassay of HEX
has been scheduled by NTP.
4.5.6.3. Evidence for Activity in Short-Term Tests (U.S. EPA. 1984)The
available evidence suggests that HEX is not a mutagen. Negative mutagenicity
results were obtained in bacteria, liver epithelial cells, Drosophila, mouse5
lymphoma cells, and in the mouse dominant lethal test. In addition, HEX didi;
not induce unscheduled DNA synthesis in rat hepatocytes. HEX was not a carcin-
ogen Jji_ vrtro in transformation assays using BALB/3T3 cells. These data are
inadequate to assess the carcinogenicity of HEX.
4.5.7. Isodrin
4.5.7.1. Evidence of Carcinogenicity - Human Exposure (HSDB, 1985)No data*of
occupational exposure to this isomer of aldrin were found.
4.5.7.2. Evidence of Carci_nqgem'c1ty - Animal Studies (HSDB, 1985)The' tox-
icity of isodrin is stated to be twice that of aldrin (species tested and
parameters examined were not specified). No other data on the carcinogenicity
of isodrin were available.
4.5.7.3. Evidence for Activity in Short-Term Tests (HSDB, 1985)Isodrin is
converted to endrin by liver microsomes, and endrin did not cause mutations in
NTP Salmonella mutagenicity tests. These data are inadequate to assess the
carcinogenicity of isodrin.
4-81
-------
-------
5. RISK ESTIMATION FROM ANIMAL DATA
5.1. SELECTION OF DATA
f
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 extrapolation. 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 is statistically significantly higher in
at least one test dose level as compared to controls and/or where the tumor
incidence rate shows a significant trend with respect to dose level. Both bio-
logical and statistical considerations have been used to select the most appro-
priate 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 aver-
aged to estimate potency for the general population. Because some subpopula-
tions 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 EPA's guidelines for carcinogen risk assessment (U.S. EPA,
1986).
5.1.1. Chlordane
As described in section 4.4, four data sets showed a significant increase
in hepatocellular carcinomas in treatment groups compared to controls in mice.
These are male and female mice in the IRDC study, and male and female mice in
the NCI study. Tables 22-25 give tumor incidence data for these studies. In
5-1
-------
TABLE 22. 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
Analytical grade chlordane
Mouse, CD-I, female
0.030 kg (assumed)
19-1/2 months
18 months
Liver, carcinoma
Oral, diet
2.98 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
5
25
50
0
0.65
3.25
6.50
0
0.052
0.260
0.520
0/45
0/61
32/50
26/37
SOURCE: IRDC, 1973a.
5-2
-------
TABLE 23. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF CHLORDANE
FROM HEPATOCELLULAR CARCINOMAS IN MALE MICE
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle: ^
Human potency (q^):
Analytical grade chlordane
Mouse, CD-I, male
0.030 kg (assumed)
19-1/2 months
18 months
Liver, carcinoma
Oral, diet
4.74 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
5
25
50
0
0.65
3.25
6.50
0
0.052
0.260
0.520
3/33
5/55
41/52
32/39
SOURCE: IRDC, 1973a.
5-3
-------
TABLE 24. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF CHLORDANE
FROM HEPATOCELLULAR CARCINOMAS IN MALE MICE
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle: ^
Human potency (q):
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
animal
dose (ppm)
Average animal
dose (mg/kg/day)
Equivalent human
dose (mg/kg/day)
Tumor incidence
No. responding/
No. examined
0
29.9
56.2
0
3.9
7.3
0
0.31
0.58
2/18
16/48
43/49
SOURCE: NCI, 1977a.
5-4
-------
TABLE 25. 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
Technical grade chlordane
Mouse, B6C3F1, female
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
0.25 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
30.1
63.8
0
3.9
8.3
0
0.31
0.66
0/19
3/47
34/49
SOURCE: NCI, 1977a.
5-5
-------
rats, only the RIASBT study from Japan showed a significant increase in tumors.
The incidence of hepatocellular carcinomas in male rats as reevaluated by
Williams was significantly increased in the high-dose group when compared to
controls. Table 26 gives the tumor incidence for this study. These five data
sets were used to quantify human risk.
5.1.2. Heptachlor/Heptachlor Epoxide
Eight data sets showed significant increases in the incidence of hepato-
cellular carcinomas in treated groups compared to controls. Tables 27-34
present the tumor incidence for these data sets. In rats, a significant in-
crease in hepatocellular carcinomas was diagnosed by Reuben. Incidence data
are shown in Table 35. These studies were used to quantify the carcinogenic
risk due to heptachlor/heptachlor epoxide exposure.
5.2. CHOICE OF EXTRAPOLATION MODELS
Mathematical models are used to estimate human-excess cancer risks associ-
ated with exposure to a given chemical. The models are fitted to tumor inci-
dence data from animal bioassays, and an extrapolation is made to low doses to
estimate human risk. The animal bioassay data used to predict human cancer
risk from exposure to chlordane and heptachlor are described in the previous
section.
Although no single mathematical model is recognized as the most appropri-
ate, some mechanisms of the process are known. If one accepts the linearity
of tumor formation and if the carcinogenic agent of interest acts in the same
manner as carcinogenic agents acting in the background, then the added effect
of the carcinogen on tumor formation at low doses is linear. On this basis, a
model that is linear at low doses is appropriate.
The linearized multistage model is used in this assessment to calculate
human risk. This model takes the following form:
5-6
-------
TABLE 26. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF CHLORDANE
FROM LIVER ADENOMAS AND CARCINOMAS IN MALE RATS
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle: ^
Human potency (q-):
Technical grade chlordane
Rat, F344, male
0.35kg
130 weeks
130 weeks
Liver, adenoma and carcinoma
Oral, diet
1.11 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
1 0.05
5 0.25
25 1.25
0
0.17
0.85
4.25
1/64 *'
1/64
4/64
9/64
SOURCE: RIASBT, 1983b.
5-7
-------
TABLE 27. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR
FROM HEPATOCELLULAR CARCINOMAS IN MALE MICE
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency
Heptachlor
Mouse, C3H, male
0.030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
12.4 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 0.00
10 1.43
0.000
0.108
22/78
64/87
SOURCE: Davis, 1965, as diagnosed by Reuber, 1977b.
5-8
-------
TABLE'28; CANCER'DATA SHEET FOR DERIVATION OF POTENCY.OF HEPTACHLOR
FROM HEPATOCELLULAR CARCINOMAS IN FEMALE MICE
Compound: . '
Species, strai n, 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!
ExpenmentaT'
" abimail- '
dose (ppnij
Average animal
dose-'(mg/kg/day)
Equivalent human
dose (mg/kg/day)
Tumor inc'ldehce
No. respondi'hg/
No. examined
10 'fl^'''
b.oo
1.43
o'.ooo
0.108
2/54
57/78
SOURCE: Davis, 1965, as diagnosed by Reuber, 1977b.
5-9
-------
TABLE 29. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR
FROM HEPATOCELLULAR CARCINOMAS IN MALE 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, male
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
2.79 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
6.1
13.8
0
0.79
1.79
0
0.063
0.14
5/19
11/46
34/47
SOURCE: NCI, 1977b.
5-10
-------
TABLE 30. 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
Technical grade heptachlor
Mouse, B6C3F1, female
0.030 kg (assumed)
90 weeks
80 weeks
Liver, carcinoma
Oral, diet
0.83 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
9.0
18.0
0
1.17
2.34
0
0.094
0.18
2/10 -
3/47
30/42
SOURCE: NCI, 1977b.
5-11
-------
TABLE 31. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF!HEPT;ACHLOR EPOXIDE
FROM HEPATOCELLULAR CARCINOMAS IN MALE'MICE :
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency
Heptachlor epoxide
Mouse, C3H, male
0.030 kg (assumed)
24 months
24 months . ,
Liver carcinoma '
Oral, diet ;; -..
27.7 per mg/kg/day
Experrj mental
animal
dose (ppm)
Average animal
dose (mg/kg/day)
Equivalent human
dose (mg/kg/day)
Tumor incidence
No. responding/
No. examined ;
0
10
0.00
1,43
0.000
0.108
22/78 ;:
73/79 ;:,
SOURCE: Davis, 1965, as diagnosed by Reuber, 1977b.
5-12
-------
TABLE 32. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR EPOXIDE
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
Mouse, C3H, female
0.030 kg (assumed)
24 months
24 months
Liver carcinoma
Oral, diet
36.2 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 0.00
10 1.43
o.ooo
0.108
2/54
77/81 -.-
SOURCE: Davis, 1965, as diagnosed by Reuber, 1977b.
5-13
-------
TABU 33. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR EPOXIDE
FROM HEPATIC CARCINOMAS IN FEMALE MICE
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency
25:75 mixture of heptachlor/heptachlor epoxide
Mouse, CD-I, female
0.030 kg (assumed)
19 months, 3 weeks
18 months
Liver, carcinoma .
Oral, diet
1.04 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
1 0.13
5 0.65
10 1.30
0
0.01
0.052
0.10
6/76 .:
1/70
6/65
30/57
SOURCE: IRDC, 1973b, as reevaluated by Reuber.
5-14
-------
TABLE 34. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR EPOXS-DE
FROM HEPATIC CARCINOMAS IN MALE MICE
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency
25:75 mixture of heptachlor/heptachlor epoxide
Mouse, CD-I, male
0.030 kg (assumed) :
19 months, 3 weeks
18 months t
Liver, carcinoma
Oral, diet
6.48 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
1 0.13
5 0.65
10 1.30
0
0.010
0.052
0.10
0/62 .>
2/68 .-
18/68
52/80
SOURCE: IRDC, 1973b, as reevaluated by Reuben.
5-15
-------
TABLE 35. CANCER DATA SHEET FOR DERIVATION OF POTENCY OF HEPTACHLOR EPOXIDE
FROM HEPATIC CARCINOMAS IN FEMALE RATS
Compound:
Species, strain, sex:
Body weight:
Length of experiment:
Length of exposure:
Tumor site and type:
Route, vehicle:
Human potency
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
No. responding/
No. examined
0
0.5
2.5
5.0
7.5
10.0
0
0.025
0.125
0.250
0.375
0.500
0
0.0043
0.021
0.043
0.064
0.085
0/17
3/22
3/18
7/22
3/21
5/19
SOURCE: Witherup et al., 1959, as reevaluated by Reuber.
5-16
-------
P(d) = l-exp[-(q0
q2d
qkdk)]
where
q-j >_ 0, i = 0, 1, ..., k dose levels and d = dose.
Cancer risks can also be estimated using models such as the probit, logit,
Wei bull, one-hit, and gamma multihit. The linearized multistage model leads to
an upper limit on risk.
5*3. INTERSPECIES DOSE CONVERSION ''':"
The low-dose extrapolation process involves the use of animal tumor inci-
dence data to predict human cancer risk. Differences exist between humans-^and
experimental test animals with respect to lifespan, body size, and pharmaco-
kinetic mechanisms. Hence, animal doses need to be converted to equivalent
.' '* f"
human doses. The conversion accounts for noncontiguous feeding, differences in
lifetime, and size differences. In the absence of information to the contrary,
this assessment uses the surface-area correction recommended by EPA's Guide-
lines for Carcinogen Risk Assessment (U.S. EPA, 1986). Therefore, a factor of
the cube root of the ratio of the animal to human body weights is applied to
animal doses to calculate equivalent human doses. This equation is
DH = DA (WA/70)1/3
where
DH = equivalent human dose (mg/kg/day)
DA = animal dose (mg/kg/day)
WA = weight of animal (kg): for mice, 0.03 kg; for rats, 0.35 kg
70 = weight of a human (kg)
5-17
-------
In the absence of information on human absorption, tissue distribution,
metabolism, and excretion, this assessment makes no adjustment for potential
differences between animals and humans.
5.4. ESTIMATION OF CARCINOGENIC POTENCY
Estimates of carcinogenic potency can be obtained by fitting the linear-
ized multistage model, described in Section 5.2, to each data set described in
Section 5.1. Table 36 summarizes 14 potency estimates obtained in this way.
5.4.1. Chlordane
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 is estimated at 4.7 per
mg/kg/day.
The most sensitive species tested is mice. There are four potency esti-
mates, 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 is consistent with the potency
estimate from rats of 1.1 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 1.3 per mg/kg/day.
These estimates are plausible upper bounds for the increased cancer risk
from chlordane, meaning that the true risk is not likely to exceed these esti-
mates and may be lower. These estimates supersede the potency of 1.61 per
mg/kg/day previously calculated by the EPA (U...S. EPA, 1980a).
The molecular potency index, which is the potency expressed in terms
of molecular weight, has been used to rank suspect carcinogens according to
potency. The index is computed by multiplying the general-population potency
by the molecular weight. The molecular potency index for chlordane is 5.2 x
102 per mmol/kg/day. This places chlordane in the upper middle quartile of
5-18
-------
TABLE 36. HUMAN POTENCY ESTIMATES BY CHEMICAL
Chemical
Chlordane
Chlordane
Chlordane
Chlordane
Chlordane
Heptachlor
Heptachlor
Heptachlor
Heptachlor
Heptachlor
epoxide
Heptachlor
epoxide
Heptachlor
epoxide
Heptachl or
epoxide
Heptachlor
epoxide
Sex,
species,
strain
Male mice
CD-I
Female mice
CD-I
Male mice
B6C3F!
Female mice
B6C3F!
Male rats
F344
Male mice
C3H
Female mice
C3H
Male mice
B6C3F!
Female mice
B6C3F-L
Male mice
C3H
Female mice
C3H
Female mice
CD-I
Male mice
CD-I
Female rats
CFN
Tumor
site, Potency
type (mg/kg/day)-1 Reference
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
adenoma and
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
Liver,
carcinoma
4.74
2.98
0.76
0.25
1.11
12.4
14.9
2.79
0.83
27.7
36.2
1.04
6.48
5.76
IRDC (1973a)
IRDC (1973a)
NCI (1977a)
NCI (1977 a)
RIASBT (1983b)
Davis (.1965)/
Reuber
Davis (1965)/
Reuber
NCI (1977b)
NCI (1977b.)
Davis (1965)/
Reuber
Davis (1965)/
Reuber
IRDC (1973b)/
Reuber
IRDC (1973b)/
Reuber
Witherup et al .
(1959) /Reuber
5-19
-------
suspect carcinogens ranked by CA6.
The unit risk in air, which is the potency expressed in terms of pg/.m3
ambient air concentration, is used to estimate risk when exposures are ex-
pressed as air concentrations. The unit risk in air is the increased cancer
o
risJs to a person who throughout life breathes air contaminated with 1 pg/m-3
of a pollutant. With a linear dose-response curve, risks at any concentration
can be computed by multiplying the unit ri.sk by the concentration. The unit
risk is computed by converting a 1 pg/m3 concentration.to a mg/kg/day dose
and then multiplying by the potency. For a 70-kg person, who, breathes 20 m3 a
day, an air concentration of 1 pg/m3 is equivalent to a dose of:,
(1 pg/m3) (10-3 mg/pg) (20 m3/day) / (70 kg) = 2.9 x 10-4 mg/kg/day.
Multiplying this dose by the potencies calculated above gives unit risks of
3.7 x ID'4 per pg/m3 for the general population and 1.3 x 1Q-3 per pg/m3 for
sensitive subpopulations.
The unit risk in water, which is the potency expressed in terms of pg/L
drinking water concentrations, is used to estimate risk .when exposures are
expressed as water concentrations. The unit risk in water is the increased
cancer risk to a person who throughout life drinks water contaminated with
1 yg/L of a pollutant. With a linear dose-response curve, risks at any con-
centration can be computed by multiplying the. unit risk by the concentration.
The unit risk is computed by converting a 1 pg/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 pg/L is equivalent to a dose of:
(1 pg/L) (10-3 mg/pg) (2 L/day) / (70 kg) = 2.9 x 10-5 mg/kg/day.
5-20
-------
Multiplying this dose by the potencies calculated above gives unit risks of
3.7 x ID-5 per yg/L for the general population and 1.3 x 10'4 per yg/L for
sensitive subpopulations.
5.4.2. Heptachlor
Four data sets involve heptachlor: male and female C3H mice, and male arid
female B6C3F1 mice. The most sensitive sex and strain tested is female C3H
mice. From these, the potency is estimated at 14.9 per mg/kg/day.
The most sensitive species tested is mice. There are four potency esti-
mates, 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 are plausible upper bounds for the increased cancer risk
from heptachlor, meaning that the true risk is not likely to exceed these es-
timates and may be lower. These estimates supersede the potency of 3.37 per
mg/kg/day previously calculated by the EPA (U.S. EPA, 1980b).
The molecular potency index for heptachlor is 1.7 x 103 per mmol/kg/day.
This places heptachlor in the upper middle quartile of suspect carcinogens
ranked by the CA6.
The unit risk in air is 1.3 x lO"3 per yg/m3 for the general population
and 4.3 x lO"3 per yg/m3 for sensitive subpopulations. The unit risk in water
is 1.3 x 10-4 per yg/L for the general population and 4.3 x 10-4 per yg/L for
sensitive subpopulations.
5.4.3. Heptachlor Epoxide
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 is estimated at 36.2 per
5-21
-------
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/day, with a geometric mean of
9.1 per mg/kg/day. This geometric mean from mice is 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 are plausible upper bounds for the increased cancer risk
from heptachlor epoxide, meaning that the true risk is not likely to exceed
these estimates and may be lower. These estimates supersede the potency of
57.86 per mg/kg/day previously calculated by the EPA (U.S. EPA, 1985a).
The molecular potency index for heptachlor epoxide is 3.5 x 103 per mmol/
kg/day. This places heptachlor epoxide in the most potent quartile of suspect
carcinogens ranked by CAG.
The unit risk in air is 2.6 x 10~3 per yg/m3 for the general population
and 1.0 x 10~2 per yg/m3 for sensitive subpopulations. The unit risk in water
is 2.6 x 10.-4 per yg/L for the general population and 1.0 x 10~3 per yg/L for
sensitive subpopulations.
5-22
-------
6. SUMMARY
6.1. QUALITATIVE DATA
6.1.1. Human Studies
6.1.1.1. Case ReportsThere were 11 case reports involving CNS effects where
, .-*. ;rdr-.-
the author looked at the toxic effects of chlordane/heptachlor, eight case
" . . i f,
studies involving blood dyscrasias, and five case studies of neuroblastomas in
children with pre-/postnatal exposure to chlordane or heptachlor. The blood
dyscrasias 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 myelomonocytic leukemia.
».-...-;
6.1.1.2. Epidemiologic StudiesThree epidemic!ogic studies of workers exposed
to chlordane and/or heptachlor have been reported. One of these studies, con-
ducted in chlordane/heptachlor applicators, was considered inadequate in sample
size and duration of follow-up since initial exposure. 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 (SMR = 183, p <_ 0.05). Of the 1,043 men
involved in the study, only one liver cancer was reported. The third study
involved 2,141 workers exposed to organochlorine 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.
6-1
-------
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 chem-
icals. Adjustments for these other chemical exposures and other confounding
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 percent of the decedents reported by the Social Security Admin-
istration.
Because of these methodological limitations and the limited data, it is
difficult to establish either a negative or positive association between chlor-
dane/heptachlor and carcinogenicity. Hence, these studies are considered in-
adequate epidemiologic evidence.
6.1.2. Animal Studies
6.1.2.1. Chlordane
6.1.2.1.1. Mice. Four chlordane carcinogenesis bioassays in mice have been
reported. The strains tested include C57B1/6N, CD-I, B6C3F1, and ICR. In
C57B1/6N mice fed 25 or 50 ppm for 18 months, hepatocellular carcinomas were
observed in 27 percent (16) of the survivors. This mouse strain rarely devel-
ops 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 approximately 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.
6-2
-------
6.1.2.1.2. Rats. Four chlordane carcinogenesis bioassays 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,, i&O,
or 300 pprtr for 2 years, hepatic toxicity was noted at 150 and 300 ppm, ,bufe,no
liver tumors 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 sig-
nificantly increased. In light of historical data for Osborne-Mendel 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 25 ppm. Hepatocellular swelling
was significant in females fed 25 ppm. The hepatocellular adenomas occurred
only in males surviving longer than 104 weeks.
6.1.2.2. :Heptach1or/Heptachlbr Epoxide
6.1.2.2.1. Mice. Three heptachlor/heptachlor epoxide carcincgenesis bioassays
in mice have been reported. The strains studied include C3H, B6C3F1, a'nd 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 gen-
erally large and frequently multiple tumors, especially in the epoxide group in
respect to the controls. For B6C3F1 mice fed technical grade (containing 22
percent chlordane) at concentrations of 6.1 or 13.8 ppm (males) or 9 or-ISrppm
(females^ respectively, for 80 weeks and held for an additional 10 weeks^
hepatocellular carcinomas were significantly (p < 0.001) increased in both::male
6-3
-------
and female mice. In CD-I mice fed a mixture of heptachlor epoxide/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.
6.1.2;.2.2. Rats. Five heptachlor/heptachlor epoxide carcinogenesis bioassays
in rats have been conducted. The strains of rats studied include Wistar,
Osborne-Mendel, CD, and CFN. In Wistar rats given 5 doses.of 10 mg/kg bw of
heptachlor and held for 106 to 110 weeks, no treatment-related tumors were ob-
served. For Osborne-Mendel rats fed technical grade heptachlor at concentra-
tions 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 neoplas-
tic nodules were found in both treated and control rats. In CD 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 nonneoplas-
tic 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 in 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.
6.1.3. Supporting Evidence
6.1.3.1. MutagenicityThe published literature on mutagenicity tests of chlor-
dane and heptachlor/heptachlor epoxide is quite similar, with most studies
reporting 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
6-4
-------
for both chemicals, the absence of direct cytogenetic tests in both germinal
and somatic cells precludes a conclusion as to their potential for causing
chromosomal aberrations.
6.1.3.2. Structural RelationshipThree compounds, structurally related to
chlordane/heptachlor/heptachlor epoxide, have induced malignant liver tumof-s in
animals. Aldrin, dieldrin, and chlorendic acid have produced liver tumors"6fn
mice and chlorendic acid has also produced liver tumors in rats.
6.2. QUANTITATIVE ANALYSIS
In the absence of information on human absorption, tissue distribution,
metabolism, and excretion, this assessment makes no adjustment for potential
differences between animals and humans.
6.2.1. Chlordane
Five data sets involve chlordane: male and female CD-I mice, male and fe-
male B6C3F1 mice, and male F344 rats. The most sensitive sex and strain tested
is male CD-I mice. From these, the potency is estimated at 4.7 per mg/kg/day.
The most sensitive species tested is mice. There are four potency esti-
mates, 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 is consistent with potency
estimate from rats of 1.1 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 1.3 per mg/kg/day.
These estimates are plausible upper bounds for the increased cancer risk
from chlordane, meaning that the true risk is not likely to exceed these esti-
mates and may be lower. These estimates supersede the potency of 1.61 per
mg/kg/day previously calculated by the EPA (U.S. EPA, 1980a).
The molecular potency index, which is the potency expressed in terms of
molecular weight, has been used to rank suspect carcinogens according to po-
6-5
-------
tency. The index is computed by multiplying the general-population potency by
the molecular weight. The molecular potency index for chlordane is 5.2,,x 10^ ;.
per mmol/kg/day. This places chlordane in the upper middle quartile ,of ,suspect
carcinogens ranked by CAG. -.*,««
6.2.2? Heptachlor
Four data sets involve heptachlor: male and female C3H mice, and;male'.and
female B6C3F1 mice. The most sensitive sex and strain tested is-female C3H
mice. From these, the potency is estimated at 14.9 per mg/kg/day. ,
The most sensitive species tested is mice. There are four potency esti-
mates, 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 are plausible upper bounds for the increased cancer risk
from heptachlor, meaning that the true risk is not likely to exceed these
estimates and may be lower. These estimates supersede the potency of 3.37 per
mg/kg/day previously calculated by EPA (U.S. EPA, 1980b).
The molecular potency index for heptachlor is 1.7 x 103 per mmol/kg/day.
This places heptachlor in the upper middle quartile of suspect carcinogens
ranked by CAG.
6.2.3. Heptachlor Epoxide
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 is 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/day, with a geometric mean of
6-6
-------
9.1 per mg/kg/day. This geometric mean from mice is 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 are plausible upper bounds for the increased cancer Ri-sk
from heptachlor epoxide, meaning that the true risk is not likely to exceed
these estimates and may be lower. These estimates supersede the potency of
57.86 per mg/kg/day previously calculated by the EPA (U.S. EPA, 1985a).
The molecular potency index for heptachlor epoxide is 3.5 x 10^ per mmol/
kg/day. This places heptachlor epoxide in the most potent quartile of suspect
carcinogens ranked by the CAG.
6-7
-------
-------
REFERENCES
Ahmed, F.E.; Hart, R.W.; Lewis, N.J. (1977a) Pesticide induced DNA damage and
its repair in cultured human cells. Mutat. Res. 42:161-174.
Ahmed, F.E.; Lewis, N.J.; Hart, R.W. (1977b) Pesticide induced ouabain resis-
tant mutants in Chinese hamster V79 cells. Chem. Biol. Interact. 19:369-
374.
Aldrich, F.D.; Holmes, J.H. (1969) Acute chlordane intoxication in a child.
Arch. Environ. Health 19:129.
Ambrose, A.M.; Christensen, H.E.; Robbins, D.J.; Rather, L.J. (1953a) lexico-
logical and pharmacological studies on chlordane. Ind. Hyg. Occup. Med.
7:197-210.
Ambrose, A.M.; Christensen, H.C.; Robbins, D.J. (1953b) Pharmacological ob-
servations on chlordane. Fed. Proc. Am. Soc. Exp. Biol. 12:298.
Anonymous. (1962) Registry on blood dyscrasias: report to council. J. Am.
Med. Assoc. 179:888-890
Arnold, D.W.; Kennedy, G.L. Jr.; Keplinger, M.L.; Calandra, J.C.; Calo, C.J.
(1977) Dominant lethal studies with technical chlordane, HCS-3260 and
heptachlor:heptachlor epoxide. J. Toxicol. Environ. Health 2:547-555.
Arthur, R.D.; Cain, J.D.; Barrentine, B.F. (1975) The effect of atmospheric
levels of pesticides on pesticide residues in rabbit adipose tissue and
blood sera. Bull. Environ. Contam. Toxicol. 14:760.
Ashwood-Smith, M.; Trevino, J.; Ring, R. (1972) Mutagenicity of dichlorvos.
Nature 240:482-520.
Balba, H.M.; Saha, J.G. (1978) Studies on the distribution, excretion and
metabolism of alpha and gamma isomers of [14C] chlordane in rabbits. J.
Environ. Sci. Health B13(3):211-233.
Barnes, R. (1967) Poisoning by the insecticide chlordane. Med. J. Aust. 54:
972.
Barnett, J.R.; Dorough, H.W. (1974) Metabolism of chlordane in rats. J.
Agric. Food Chem. 22:612-619.
Becker, F.F.; Sell, S. (1979) Alpha-fetoprotein levels in hepatic alterations
during chemical carcinogenesis in C57BL/6N mice. Cancer Res. 39:3491-3494.
Benes, V.; Sram, R. (1969) Mutagenic activity of some pesticides in Drosophila
melanogaster. Ind. Med. 38:50-52.
7-1
-------
Bernstein, U, Gold, L.S.; Ames, B.N.; Pike, M.C.; Hoel, D.6. (1985) Some
tautologous aspects of the comparison of carcinogenic potency in rats and
mice. Fundam. Appl. Toxicol. 5:79-86.
Blevins, R.D.; Sholes, I.E. (1978) Response of HeLa cells to selected pesti-
cides and hallucinogens. Growth 42(4):478-485.
Boyd, E.M.; Taylor, F.I. (1969) The acute oral toxicity of chlordane in albi-
no rats. Ind. Med. 38:42.
Brandt, W.; Flauim, W.; Bernheim, N. (1977) The value of hydroxyureas in
assessing repair synthesis of DNA in HeLa cells. Chem.-Biol. Interact. 5:
327-339.
Bresch, H.; Arendt, U. (1977) Influence of different organochlorine pesti-
cides on the development of the sea urchin embryo. Environ. Res. 13:
121-128.
Brimfield, A.A.; Street, J.C.; Futrell, J.; Chatfield, D.A. (1978) Identifi-
cation of products arising from the metabolism of cis- and transchlordane
in rat liver microsomes in vitro; outline of a possible metabolic pathway.
Pestic. Biochem. Physio!. 9(l):84-95.
Brodie, B.B. (1962) Part VI. Difficulties in extrapolating data on metabolism
of drugs from animal to man. Clin. Pharmacol. Ther. 3(3):374.
Brooks, G.T.; Lewis, S.E.; Harrison, A. (1968) Selective metabolism of cyclo-
diene insecticide enantiomers by pig liver microsomal enzymes. Nature
(London) 220:1034-1035.
Brooks, G.T.; Harrison, A.; Lewis, S.E. (1970) Cyclodiene epoxide ring hydra-
tion by microsomes from mammalian liver and houseflies. Biochem. Pharmacol.
19:255-273.
Cabral, J.R.; Testa, M.C.; Terracini, B. (1972) Lack of long-term effects of
the administration of heptachlor to suckling rats. Tumori 58:49-53.
(Ita., Eng. Abstr.)
Cavender, F.; Salasin, J. (1982) Analysis of human inhaled dose estimates
from animal oral dose data on selected chemicals. WP-82W00403. The MITRE
Corporation, McLean, VA.
Cerey, K.; Izakovrc, V.; Ruttkay-Nedecka, J. (1973) Effects of heptachlor on
dominant lethality and bone marrow in rats. Mutat. Res. 21:26.
Conney, A.H.; Coutinho, C.; Koechlin, B.; Swarm, R.; Cheripko, J.A.;
Impellizzeri, C.; Baruth, H. (1974) From animals to man: metabolic
considerations. Clin. Pharmacol. Ther. 16:176.
Curley, A; Garrettson, L.K. (1969) Acute chlordane poisoning. Arch. Environ.
Health 18: 211-215.
7-2
-------
Curley, A.; Copeland, M.F.; Kimbrough, R.K. (1969) Chlorinated hydrocarbon
insecticides in organs of stillborn and blood of newborn babies. Arch.
Environ. Health 19:628-632.
Dadey, J.L.; Kammer, A G. (1953) Chlordane intoxication. ,J. Am. Med. Assoc.
153:723. _ ' '
Davidow, B.; Radomski, J.L. (1953) Isolation of an epoxide metabolite from .
fat tissues of dogs fed heptachlor. J. Pharmacol. Exp. then. 107:259. '
Davis, H.J. (1965, July 19) Pathology report on mice fed aldrin, dieldrin?,K
heptachlor. or .heptachlor epoxide for two years. Internal FDA memorandum
to Dr. A.J. Lehman.
Deichmann, W.B.; Keplinger, M.L. (1966) Effect of combinations of pesticides
on reproduction of mice. Toxicol. Appl. Pharmacol. 8:337-338.
Derbes, V.J.; Dent, J.H.; Forrest, W.W.; Johnson, M.F. (1955) Fatal chlordane
poisoning. J. Am. Med. Assoc. 158:1367-1369.
Ditraglia, D.; Brown, D.P.; Namekata, T.; Iverson, N. (1981) Mortality "study
of workers employed at organochlorine pesticide manufacturing plants.
Scand. J. Work Environ. Health 7 (Suppl. 4):140-146.
Ermakov, V.V. (1977) Metabolism of heptachlor and dilor in animals. S-kh/
Biol. 12(3):402-411. (Rus.) CA 87:128395w'
Fishbein, W.I.; White, J.V.; Isaacs, H.J. (1964) Survey of workers exposed
to chlordane. Ind. Med. Surg. 33:726-727. ,
Forrest, I.S.; Aber, R.C. (1968) Metabolic pathways for the detoxication of
chlorpromazine in various mammalian species. Agressologie 9(2):259.
Occup. Med. 6:357.
Fouts, J.R. (1970) Some effects of insecticides on hepatic microsomal enzymes
in various animal species. Rev. Can. Biol. 29:377-389.
Furie, B.; Trubowitz, S. (1976) Insecticides and blood dyscrasias: chlordane
exposure and self-limited refractory megaloblastic anemia. J. Am. Med.,
Assoc. 235:1720-1722.
Gentile, J.M.; Gentile, G.J.; Bultman, J.; Sechriest, R.; Wagner, E.E.; Plewa,
M.J. (1982) An evaluation of the genotoxic properties of insecticides.
following plant and animal .activation. Mutat. Res. 101(l):19-29.
Gold, L.S.; Sawyer, C.B.; Magaw, R.; Backman, G.; DeVeciana, M.; Levinson, R.,;
Hooper, N.K.; Havender, W.R.; Bernstein, L.; Peto, R.; Pike, M.C.; Ames,
B.N. (1984) A carcinogenic potency database of the standardized results
of animal bioassays. Environ. Health Perspect. 58:9-322.
Goodman, D.G.; Ward, J.M.; Squire, R.A.; Paxton, M.B.; Reichardt, W.D.; Chu,
K.C.; Linhart, M.S. (1980) Neoplastic and nonneoplastic lesions in aging
Osborne-Mendel rats. Toxicol. Appl. Pharmacol. 55:433-447.
7-3
-------
Grant, W. (1978) Chromosome aberrations in plants as a monitoring system.
Environ. Health Perspect. 27:37-43.
Griffin, D.; Hill, W. (1978) In vitro breakage of plasmid DNA by mutagens and
pesticides. Mutat. Res. 52:161-169.
Halacka, K.; Dvorak, M.; Rysanek, K.; Jagos, P.; Cerny, E. (1975) Effect of
small orally administered doses of heptachlor on ultrastructure of hepa-
tocytes and their enzyme systems in experimental animals. Cesk. Hyg.
20:286-291.
Hanley, T.; Udall, V.; Weatherall, M. (1970) An industrial view of current
practice in predicting drug toxicity. Br. J. Med. 26(3):203-207.
Haseman, O.K. (1985) Issues in carcinogenicity testing: dose selection.
Fundam. Appl. Toxicol. 5:66-78.
Hazardous Substances Data Base (HSDB),
of Medicine, TOXNET.
(1985) Bethesda, MD: National Library
Huang, J.C. (1980) Fate of organic pesticides in the aquatic system. Eng.
Bull. Purdue Univ. Eng. Ext. Series, pp. 449-457.
Infante, P.F.; Epstein, S.S.; Newton, W.A., Jr. (1978) Blood dyscrasias and
childhood tumors and exposure to chlordane and heptachlor. Scand. J. Work
Environ. Health 4:137-150.
Ingle, L. (1952) Chronic oral toxicity of chlordane to rats. Arch. Ind. Hyg.
Occup. Med. 6:357.
International Agency for Research on Cancer (IARC). (1974) Evaluation of car-
cinogenic risk of chemicals to man. Some organochloride pesticides. Vol.
5. Lyon, France.
International Agency for Research on Cancer (IARC). (1982) Aldrin. In:
Chemicals, industrial processes and industries associated with cancer in
humans. IARC Monographs Supplement 4:25-27. Lyon, France: World Health
Organization, IARC.
International Research and Development Corporation (IRDC). (1973a, Dec. 14)
Eighteen-month oral carcinogenic study in mice. Unpublished report to
Velsicol Chemical Corporation.
International Research and Development Corporation (IRDC). (1973b, Sept. 16)
Eighteen-month oral carcinogenic study in mice. Unpublished report to
Velsicol Chemical Corporation.
Jolley, W.P.; Stemmer, H.L.; Pfitzer, E.A. (1966, Jan. 28) The effects of
feeding diets containing a mixture of heptachlor and heptachlor epoxide to
female rats for two years. Unpublished report from the Kettering Lab. to
Velsicol Chemical Corporation. (Cited in Epstein, 1976)
7-4
-------
Kacew, S.; Sutherland, D.J.B.; Singhal, R.L. (1973) Biochemical changes fol-
lowing chronic administration of heptachlor, heptachlor epoxide and endrin
to male rats. Environ. Physiol. Biochem. 3:221-229.
Klemmer, K.W.; Budy, A.M.; Takahasdhi , W.; Haley, T.J. (1977) Human tissue
distribution of cyclodiene pesticides Hawaii 1964-1973. Clin. Toxicol.
Kutz, F.W.; Strassman, S.C.; Sperling, J.F.; Cook, B.T.; Sunshine, I.;
Tessari , J. (1983) A fatal chlordane poisoning. J. Toxicol .-Clin.
Toxicol. 20(2): 167-174.
Lensky, P.; Evans, M. (1952) Human poisoning by chlordane. J. Am. Med.
Assoc. 149:1394.
Loge, J.P. (1965) Aplastic anemia following exposure to benzene hexachloride
(Lindane). J. Am. Med. Assoc. 193:110-114.
Markarjan, D.S. (1966) Cytogenetic effect of some chlororganic insecticides on
mouse bone-marrow cell nuclei. Genetika 2:132-137.
Marshall, T.C.; Borough, W.; Swim, H.E. (1976) Screening of pesticides for
mutagenic potential using Salmonella typhimurium mutants. J. Agric. Food
Chem. 24:560.
Maslansky, C.J.; Williams, 6.M. (1981) Evidence for an epigenetic mode of
action in organochlorine pesticide heptacocarcinogenicity: a lack of
genotoxicity in rat, mouse and hamster hepatocytes. J. Toxicol. Environ.
Health. 8(1-2):121-130.
Matsumura, F. ; Nelson, J.O. (1971) Identification of the major metabolic pro-
duct of heptachlor epoxide in rat feces. Bull. Environ. Contam. Toxicol.
5:489.
Mestitzova, M. (1967) On reproduction studies on the occurrence of cataracts
in rats after long-term feeding of the insecticide heptachlor. Experientia
23:42-43.
Mizyukova, I.G.; Kurchatov, G.V. (1970) Metabolism of heptachlor. Farmacol .
Toksikol. (Moscow) 33(4):496-499. (Rus.) CA73:86885j.
Moriya, M.; Ohta, T.; Watanabe, K.; Miyazawa, T. ; Kato, K.; Shirasu, K. (1983)
Further mutagenicity studies on pesticides in bacterial reversion assay
systems. Mutat. Res. 116(3-4):185-216.
Muirhead, E.E.; Groves, M.; Guy, R.; Haldon, E.R.; Bass, R.K. (1959) Acquired
hemolytic anemia, exposure to insecticides and positive Coombs test depen-
dent on insecticide preparations. Vox Sang. 4:277-292. (Cited in Infante
et a!., 1978)
Munson, R.R. (1974) Analysis of relative survival and proportionate mortal-
ity. Comput. Biomed. Res. 7:325-332.
7-5
-------
National Academy of Sciences (NAS). (1969) Application of metabolic data to
the evaluation of drugs. Clin. Pharmacol. Ther. 10(5):607-634.
National Cancer Institute (NCI). (1977a) Bioassay of chlordane for possible
carcinogenicity. NCI Carcinogenesis Tech. Rep. Sen. No. 8. [Also publ.
as DREW Publication No. (NIH) 77-808].
National Cancer Institute (NCI). (1977b) Bioassay of heptachlor for possible
carcinogenicity. NCI Carcinogenesis Tech. Rep. Ser. No. 9. [Also publ.
as DHEW Publication No. (NIH) 77-809].
National Cancer Institute (NCI). (1978) Bioassay of endosulfan for possible
carcinogenicity. NCI Carcinogenesis Tech. Rep. Ser. No. 62. [Also publ.
as DHEW Publication No. (NIH) 78-1312].
National Toxicology Program (NTP). (1984) Report of the Ad Hoc Panel on
Chemical Carcinogenesis Testing and Evaluation. Research Triangle Park,
NC, p. 280.
National Toxicology Program (NTP). (1985a) Report of the Ad Hoc Panel on
Chemical Carcinogenesis Testing and Evaluation. Research Triangle
Park, NC, p. 280.
National Toxicology Program (NTP). (1985b) Bioassay of chlorendic acid for
possible carcinogenicity. DHEW Publication No. (NIH) 85-2560. National
Toxicology Program Carcinogenesis Technical Report Series, No. 304.
Neal, R.A. (1983) Design of carcinogenicity studies, dose selection, route,
blood levels, transformation. Toxicol. Pathol. 11(1):48.
Nye, D.E.; Dorough, H.W. (1976) Fate of insecticides administered endo-
treacheally to rats. Bull. Environ. Contam. Toxicol. 15:291.
Oehme, F.W. (1970) Species differences: the basis for and importance of
comparative toxicology. Clin. Toxicol. 3(1):5-10.
Plewa, M. (1982) Specific-locus mutation assays in Zea Mays. A report of
QQ?O Environmental Protection Agency Gene-ToxTrogram. Mutat. Res.
y y C/JL / **o j /»
Polen, P.B.; Hester, M.; Benzinger, J. (1971) Characterization of oxychlor-
dane, animal metabolite of chlordane. Bull. Environ. Contam. Toxicol.
5:521.
Poonawalla, N.H.; Korte, F. (1971) Metabolism of trans-chlordane-^C and
isolation and identification of its metabolites from the urine of rabbits.
J. Agric. Food Chem. 19(3):467-470.
Probst, G.S.; McMahon, R.E.; Hill, L.E.; Thompson, C.Z.; Epp, J.K.; Neal, S.B.
(1981) Chemically-induced unscheduled DNA synthesis in primary rat hepa-
tocyte cultures: A comparison with bacterial mutagenicity using 218 com-
pounds. Environ. Mutagen. 3(l):ll-32.
7-6
-------
Prout, M.S.; Provan, W.M.; Green, T. (1985) Species differences in response
to trichloroethylene. 1. Pharmacokinetics in rats and mice. Toxicol.
Appl. Pharmacol. 79:389-400.
Radomski, J.L.; Davidow, B. (1953) The metabolite of heptachlor, its estima-
tion, storage and toxicity. 0. Pharmacol. Exp. Ther. 107:226.
Research Institute for Animal Science in Biochemistry and Toxicology (Japan)
(RIASBT) (1983a, Dec. 1) Chlordane chronic feeding study in mice.
Unpublished report prepared for Velsicol Chemical Corporation.
Research Institute for Animal Science in Biochemistry and Toxicology (Japan)
(RIASBT) (1983b, Dec. 1) Technical chlordane chronic feeding study in
rats. Unpublished report prepared for Velsicol Chemical Corporation.
Reuber, M.D. (1977a) Hepatic vein thrombosis in mice ingesting chlorinated
hydrocarbons. Arch. Toxicol. 38:163-168.
Reuber, M.D. (1977b) Histopathology of carcinomas of the liver in mice inges-
ting heptachlor or heptachlor epoxide. Exp. Cell Biol. 45:147-157.
Reuber, M.D. (1978) Carcinomas and other lesions of the liver in mice inges-
ting organochlorine pesticides. Clin. Toxicol. 13(2):231-256. [Also
available as Reuber, M.D. (1979) Toxicol. Annu. 3:231-256].
Savage, E.P.; Tessari, J.D.; Malberg, J.W.; Wheeler, H.W.; Bagby, J.R. (1973)
Organochlorine pesticide residues and polychlorinated biphenyls in human
milk, Colorado 1971-1972. Pestic. Monit. J. 7:1-5.
Savage, E.P.; Kiefe, T.J.; Tessari, J.D.; Wheeler, H.W.; Applehaus, F.M.;
Goes, E.A.; Ford, S.A. (1981) National study of chlorinated hydrocarbon
insecticide residues in human milk, USA. Am. J. Epidemiol. 113(14):413-
422.
Shain, S.A.; Shaeffer, J.C.; Boesel, R.W. (1977) The effect of chronic inges-
tion of selected pesticides upon rat ventral prostate homeostasis. Toxicol
Appl. Pharmacol. 40(1):115-130.
Shirasu, Y.; Moriya, M.; Kato, K.; Furuhashi, A.; Kada, T. (1976) Mutagen-
icity screening of pesticides in the microbial system. Mutat. Res.
40:19-30.
Simmon, V.; Kauhaven, K.; Mortelmans, K.; Tardiff, R. (1978) Mutagenic activ-
ity of chemicals identified in drinking water. Mutat. Res. 53(2):262-263.
Sontag, J.M.; Page, N.P.; Saffiotti, U. (1976) Guidelines for carcinogen bio-
assay in small rodents. NCI-CG-TR-1/DHEW Publication No. (NIH)76-801.
National Cancer Institute, Bethesda, MD.
Stohlman, E.F.; Thorp, W.S.; Smith, M.F. (1950) Toxic action of chlordane.
Arch. Ind. Hyg. 1:13.
Strassman, S.C.; Kutz, F.W. (1977) Insecticide residues in human milk from
Arkansas and Mississippi, 1973-1974. Pestic. Monit. J. 10:130-133.
7-7
-------
Street, J.E.; Blau, S.E. (1972) Oxychlordane: accumulation in rat adipose
tissue on feeding chlordane isomers or technical chlordane. J. Aqric.
Food Chem. 20:395-397.
Takei, G.H.; Kauahikua, S.M.; Leong, G.H. (1983) Analysis of milk samples
collected in Hawaii for residues of organochlorine pesticides and PCBs.
Bull. Environ. Contam. Toxicol. 30:606-606B.
Tashiro, S.; Matsumura, F. (1977) Metabolic routes of cis- and transchlordane
in rats. J. Agric. Food Chem. 25:872-880.
Tashiro, S.; Matsumura, F. (1978) Metabolism of trans-nonachlor and related
chlordane components in rat and man. Arch. Environ. Contam. Toxicol.
Tong, C.; Fazio, M.; Williams, G. (1981) Rat hepatocyte-mediated mutagenesis
of human cells by carcinogenic polycyclic aromatic hydrocarbons but not
organic pesticides. Proc. Soc. Exp. Biol. Med. 167:572-575.
U.S. Environmental Protection Agency. (1977, Mar. 28) Risk assessment of
chlordane and heptachlor. Prepared by the Carcinogen Assessment Group.
Office of Health and Environmental Assessment.
U.S. Environmental Protection Agency. (1980a) Ambient water quality criteria
document for chlordane. Prepared by the Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office, Cincinnati,
OH, for the Office of Water Regulations and Standards, Washington, DC.
EPA-440/4-80-027. NTIS PB 81-117384.
U.S. Environmental Protection Agency. (1980b) Ambient water quality criteria
document for heptachlor. Prepared by the Office of Health and Environmental
Assessment, Environmental Criteria and Assessment Office, Cincinnati, OH,
for the Office of Water Regulations and Standards, Washington, DC.
EPA-440/4-80-052. NTIS PB 81-117632.
U.S. Environmental Protection Agency. (1984) Health assessment document for
hexachlorocyclopentadiene. Office of Health and Environmental Assessment.
EPA-600/8-84-001F. NTIS PB 85-124915.
U.S. Environmental Protection Agency. (1985a, Mar.) Drinking water criteria
document for heptachlor, heptachlor epoxide and chlordane. Prepared by
the Office of Health and Environmental Assessment, Environmental Criteria
and Assessment Office, Cincinnati, OH, for the Office of Drinking Water,
Washington, DC. Final Draft. EPA-600/X-84-197-1.
U.S. Environmental Protection Agency. (1985b) Hearing files on chlordane,
heptachlor suspension [unpublished draft]. Available for inspection at:
U.S. Environmental Protection Agency, Washington, DC.
U.S. Environmental Protection Agency. (1986) Guidelines for carcinogen risk
assessment. Federal Register 51(185):33992-34003.
7-8
-------
Velsicol Chemical Corporation. (1983a, Dec. 1) Yonemura, T.; Takamura,.F.;
Takahashi, Y. Thirty-month chronic toxicity and tumorigenicity test in
rats by chlordane technical. (Unpublished study by Research Institute
for Animal Science in Biochemistry and Toxicology, Japan. Chicago, IL.
Accession No. 252267.) ^
Velsicol Chemical Corporation. (1983b, Dec. 1) Inui, S.; Yamazaki, K.; .
Yonemura, T; et al. Twenty-four month chronic toxicity and tumorigeni-
city test in mice by chlordane technical. (Unpublished study by Research
Institute for Animal Science in Biochemistry and Toxicology, Japan.
Chicago, IL. Accession Nos. 254665 and 251815.-) "
Velsicol Chemical Corporation. (1984, June 16) Hardy, C.J.; et al. Chlordane:
a 90-day inhalation toxicity study in the rat and monkey. Unpublished
study by Huntingdon Research Centre. ,,
Vettorazzi , A.G. (1975) Toxicological decisions and recommendations result-
ing from the safety assessment of pesticide residues ,in food. Crit. Rev.
Toxicol. 4:125-183. ;/.;
Wang, H.H.; MacMahon, B. (1979a) Mortality of workers employed in the manu-
facture of chlordane and heptachlor. J. Occup. Med. 21:745-748.
Wang, H.H.; MacMahon, B. (1979b) Mortality of pesticide workers. J. Occup.
Med. 21:741-744. , ''
Wasserman, M.; Nogueira, D.P.; Tomatis, L. et al. (1972) Storage of organo-
chlorine insecticides in people of Sao Paulo, Brazil. Ind. Med. 41:22.
Wasserman, M.; Tomatis, L.; Wasserman, D. et al. (1974) Epidemiology of
organochlorine insecticides in the adipose tissue of Israelis. Pestic.
Monit. J. 8:1.
Wazeter, F.X. (1967) Unpublished report. (Cited in Vettorazzi, 1975)
Welch, R.M.; Levin, W.; Kuntzman, R.; Jacobson, M.; Conney, A.H. (1971) Ef-
fect of halogenated hydrocarbon insecticides on the metabolism and utero-
tropic action of estrogen in rats and mice. Toxicol. Appl. Pharmacol. 19:
234-246.
Witherup, S.; Cleveland, P.P.; Shaffer, F.G.; Schlecht, H.; Musen, L. (1955,
Aug. 17) The physiological effects of the introduction of heptachlor
into the diet of experimental animals in varying levels of concentration.
Unpublished report from the Kettering Laboratories to Velsicol Chemical
Corporation.
Witherup, S.; Cleveland, F.P.; Stemmer, K. (1959, Nov. 10) The physiological
effects of the introduction of heptachlor epoxide in the varying levels of
concentration into the diet of CFN rats. Unpublished report from the
Kettering Lab. to Velsicol Chemical Corporation.
Yamaguchi, I.; Matsumura, F.; Kadous,
ATPases by heptachlor epoxide in
ll(l-3):285-293.
A.A. (1979) Inhibition of synaptic
rat brain. Pestic. Biochem. Physio!.
7-9
-------
r
Zapp, J.A., Jr. (1977) Extrapolation of animal Studies to the human situation.
J. Toxicol. Environ. Health 2:1425-1433.
Zavon, M.R.; Tye, R.; Latorfe, L. (1969) Chlorinated hydrocarbon insecticide
content of the heonate. Ann. NY Acad. Sci. 160:196-200.
7-10
-------
APPENDIX A
!' I UNITED STATES ENVIRONMENTAL PROTECTION AGENCV
r WASHINGTON, D.C. 20460
OFFICE OF
PESTICIDES AND TOXIC SUBSTANCES
MEMORANDUM
SUBJECT
FROM:
TO:
THRU
The Mutagenicity Assessment (GENE-TOX PROFILE) of
Chlordane - Addendum to the Registration Standard
TB Projects: OOOf/1244
No
174
Irving Mauer, Ph.D.
Toxicology Branch ^
Hazard Evaluation Division (TS-769C)
Henry Spencer, Ph.D.
Toxicology Branch
Hazard Evaluation Division (TS-769C)
and
Amy S. Rispih, Ph.D.
Science Integration Staff
Hazard Evaluation Division (TS-769C)
Judy Hauswirth, Acting Head
Section VI, Toxicology Branch
Hazard Evaluation Division (TS-769C)
The assessment of the mutagenicity of chlordane developed
here was generated from our review of the available published
literature located by Dynamac Corporation (TB Project 1244),
as well as those articles in the files of EMIC (Environmental
Mutagen Information Center at Oak Ridge National Laboratory)
which were employed for evaluation in Phase I of the Agency's
GENE-TOX PROGRAM (Dr. Michael D. Waters, HERL/RTP). A cursory
appraisal ("Chlordane/Heptachlor Mutagenicity," memorandum:
Mauer to Spencer December 16, 1985attached as APPENDIX I)
-------
of some of these assays as summarized by the CAG
("Carcinogenicity Risk Assessment of Chlordane and Heptachlor/
Heptachlor Epoxide," DRAFT, December 1985) is also incorporated
here, since the conclusions in this more comprehensive assessment
corroborate those in that preliminary discussion. There
appears to have been no primary gene-tox studies submitted as
CBI by registrants.
Technical chlordane (CAS Registry No. 12789-03-06) is a
complex mixture of related, chlorinated methanoindane compounds.*
Approximately one-third of the published studies available
for review could not be interpreted because they did not
clearly state the nature, purity, source, and composition of
the material tested but used unspecified "chlordane/commercial
grades" of the pesticide. Collectively, however, results
from over thirty assays reviewed here permit at least a
preliminary assessment of the mutagenicity potential of the
technical/its principal isomers.
This assessment is organized as follows: A survey and
discussion (with summary tabulation), as well as evaluation
(for regulatory purposes) of all studies located by both
sources (DYNAMAC, EMIC/GENE-TOX PROGRAM) listed according to
FIFRA categories of genetic effect assayed (gene mutation,
chromosomal effects, primary DNA damage/repair, and other
mechanisms); followed by conclusions as well as recommendations
both to satisfy regulatory requirements ("data gaps") and to
indicate further testing the results from which would be
useful for risk assessment.
* Approximate composition as follows:
- cis (alpha)-chlordane (5103-71-9) 19%
- trans (gamma)-chlordane (57-74-9) 24%
- chlordene isomers 21%
- heptachlor (76-44-8) 10%
- nonachlor (3734-49-4) 7%
- Diels-Adler adduct (68441-65-6) of cyclopentadiene
(542-92-7) and pentachlorocyclopentadiene
(25329-35-5) 2%
- hexachlorocyclopentadiene (77-47-4) .......... 1%
- octochlorocyclopentadane (287-92-3) 1%
- (miscellaneous constituents) 15%
[AG-chlordane (HCS-3260, Velsicol) is an experimental 3:1
mixture (95%) of cis (a) and trans (Y) chlordane (95%
purity) employed is some toxicological assays (e.g., Arnold
et al., 1977).]
A-2
-------
Although a definitive genetic toxicology profile cannot
be established from the published literature reviewed here,
the following preliminary conclusions on the mutagenicity of
chlordane are warranted: :^
1. Neither technical chlordane nor any of its constituents
appear to be mutagenic in bacterial cells in adequately
controlled assays.
2. Although negative in acceptable tests with less well
established mammalian cell lines, technical chlordanel
and/or one of its isomers have induced positive
results in assays without activation (not tested
with S9) in mammalian cell systems with established
data bases. Hence, its potential to induce gene
mutation in adequately controlled assays (i.e., with
and without metabolic activation in the standard in
vitro mammalian assays) still needs to be determine~d.
3. Similarly, adequate germinal assays indicate this
organochlorine does not cause gross chromosome
damage, but its potential for somatic cytogenetic ?
activity has not been evaluated.
4. The combined results of DNA repair assays indicate
that chlordane (the technical and/or one or more of
its constituents) is genotoxic in yeast and fish, as
well as in some types of mammalian cell systems.
-.= *.j> -.
5. There is also evidence suggesting that chlordane may
act by epigenetic (e.g., promoter) mechanisms.
A-3
-------
MUTAGENICITY OF CHLORDANE
"y- -.'
THE DATA BASE ; . ]_ ,.,',,,,
A total of' 25' published articles were available, for review> :
containing 34 assays testing technical' chlbrdane/itscoristitutent'
isomers as well as cpmmercial preparations (TABLE 1). One-third
were judged inadequate '(UNACCEPTABLE) to support- the "negative .
results reported, because of major def icienei'es, 'and -another- y.
one-third reporting positive results were declared "liSTCOMCL'UStl.VE^
because of a combination of procedural/accouhting' inadelqua-cies.!--!
The remaining nine studies satisfy the minimal 'data- requirements
foirlall geaetic1 ^end-points.necessary for regulatory purposes ^
(geriV mutation, '-chromosomal 'damage, primary-DNA repair)', but -do
not provide a 'comprehensive'assessment of geriotoxTcpotential for
this, pesticide. * Further testing is recommended/- in order to; ".-
confl'rm some 'suspect positive results, as well as to provide ';; ;
support for assessments in-other areas -(-e.g. , evaluation -as-, a'--
suspect carcinogen).
DISCUSSION AND EVALUATION OF ASSAY RESULTS ' '\ "' "^ ' ; ^
-..-?. A- Survey-Studies ".'' ' ".,,';, .^.''^' : ' '''.''.'''"*
The published results 'of studies conducted with '
chlordane that contained little or no primary data, ;although^y ;;-
individually considered technically inadequate"and unacceptable
in'meeting regulatory requirements, ,can be used '; to support the ^' ;
overall conclusions drawn from the acceptable studies discussed- ^
below. -.,.-.... - , -. :_^^,- ;
As shown.in Table 1, chlordane was evaluated in three
modified Salmonella tvphi'murium mammalian microsome plate incorpo-
ration Ames assays "(Wildeman and Nazar 1982; Probst et dl.'1981;
Gentile et ai. 1982). Wildeman and Nazar (1982) reported that a
commercial preparation providing 50 ug/plate active ingredient.
did not induce reversion to histidine prototrophy of S. typhimurium
TA98 and TA100 either in the absence or the presence of the
following activation"systems: rat S9, S14 microsome fractions' -,
derived from corn or wheat, or combine-d ;rat S9 and plant S14
fractions." , _ ... -..n-i. ::..-.' ' .< "- ;;-.. ..- ,/ .,.-;.',!, < ^.
Qualitative negative results^ were also'-'feport'e'd by
both Probst and associates (1981) over a,.!- to 10,000-fold non-
activated and S9-activated concentration ,range, of .chlptdane in
the Concentration agar gradient test with eight S., typhimurium
and'Vwo Escherichia cbli strains', ahd by gentile et -al, (1982)
who, .reported both co^rteTcial and technical grade chlordane -(tested
at unreported concentrations) to be negative in the"conventional
Ames test using rat S:9 and in an Ames assay using corn microsome
IS fractions for activation.
A-4
-------
Both Ashwood-Smith and colleagues (1972) and Ficsor
(1972) reported negative results in E. coli reverse mutation
nonactivated spot tests with chlordane, the former assayed a
1 mg/disc sample of 10% qhlordane and the latter tested sn
unreported dose of 44.4% chlordane,.
A reverse mutation assay using Zea mays at the waxy
(wx) locus as the target was also conducted by Gentile and
associates .(1982), but provided only qualitative evidence of a
positive response induced by chlordane in a survey gene mutation
study. For this experiment/ 2.24 kg/hectare commercial grade
chlordane was sprayed on mutant seeds in situ prior to emergence
of corn seedlings. Plants were allowed to grow 12 to 14 weeks
and pollen grains were harvested, dehydrated* and stained. The
authors reported a significiant increase (p < 0.05) in back-
mutations of wx~ to wx+. Because primary data were not reported,
chlordane is classified inconclusive but a presumptive positive
mutagen in this assay. ;
These authors also included the results of a
Saccharomyces cerevisiae D4 mitotic gene conversion assay. Log-
phase S_, cerevisiae cells were exposed for 30 minutes to four
doses of commercial and technical grade chlordane over a threefold
to hundredfold dilution range in the absence and presence of rat
S9 and corn IS. Commercial grade chlordane was reported positive
for unspecified doses in the presence of both rat (S9) and corn
(IS) microsome fractions, Technical grade chlordane was active
only in the presence of rat S9 activation. Based on this quali-
tative evidence, chlordane is considered presumptively genotoxic
in yeast. L
The ability of 0.1 mg/mL chlordane of unreported purity
to interact with and damage DNA was also evaluated in purified
CoL El DNA from J3. coli K12 by Griffin and Hill (1978), who
reported the assay as negative.
The findings presented in these survey articles did not
provide sufficient data to draw unequivocal conclusions. However,
the data collectively provide qualitative evidence that chlordane
is probably not mutagenic in bacteria, but may be presumptively
mutagenic in plants. The data also provide presumptive evidence
that chlordane causes mitotic gene conversion in yeast.
B. Primary Studies
The following published results of genetic toxicology
assays performed with chlordane contained sufficient primary
data for review and evaluation according to FIFRA genetic points.
A--5
-------
1. Gene Mutation
a. Bacteria
Chlordane technical as well as trans (gamma)
chlordane were selected by the National Toxicology Program for
evaluation in the preincubation modification to the Ames test
using nonactivation and 10% rat or hamster S9 activation (Haworth
et al. 1986; Mortelmans et al. 1986). For both preparations,
five nonactivated doses (0.1 to 10 u_g/plate) did not induce
histidine prototrophy in S_. typhimurium TA1535, TA1537, TA98, or
TA100; a slight cytotoxic effect was reported for strain TA1537
at 10 ug/plate. In the presence of either hamster or rat S9
activation/ compound precipitation was reported at 3.3 and 10
mg/plate. Similarly, neither preparation induced mutants in any
strain over the assayed dose range (10 to 1000 ug/plate with
strains TA1535 and TA1537 and 100 to 10,000 ug/plate with -strains
TA100 and TA98) in the presence of either of the two rodent
species' S9 microsomal fractions. Based on the findings of this
study, both the technical as well as the trans-isomer were con-
sidered to be nonmutagenic. The test material was adequately
investigated in these properly controlled assays, and these
studies are judged acceptable.
b. Mammalian Cells
Three assays evaluated the potential of chlordane
to induce gene mutation in mammalian cells (Ahmed et al. 1977a;
Telang et al. 1982; Tong et al. 1981). In the preliminary dose
selection test Ahmed and associates (1977a) found that nonactivated
>_ 100 uM chlordane was cytotoxic to Chinese hamster lung V79 cells.
Based on these results, nonactivated 10 uM chlordane was evaluated
in the mutagenicity assay. The reported results showed that at
cell survival of 44.4%, the selected dose (30 replicate cultures)
induced a fifteenfold increase in ouabain-resistant mutants.
However, the study was incomplete because
exogenous metabolic activation was not used. These data are
needed to establish whether metabolism has an effect on the test
material's mutagenic potential. Additionally, the study,results
are considered inconclusive because:
o Only a single dose of an unspecified
preparation of chlordane was evaluated for
mutagenicity.
o The presumptive mutagenic response was not
confirmed at the concentration range expected
to give dose-response data.
A-6
-------
o Carbaryl and 2,4-D fluid, which have
previously been shown to be either weak
mutagens or nonmutagens, were reported to
give strong positive responses.
o No positive control was included to ensure
an appropriate level of sensitivity.
Although assessed as inconclusive evidence for a positive response,
nonactivated chlordane may be classified as an unconfirmed
presumptive mutagen in Chinese hamster lung cells.
Telang and colleagues (1982) exposed 4 x 105
primary adult rat liver epithelial cells (ARL) to six concentra-
tions of technical chlordane (10~3 to 10~6 M) in two independent
experiments. Chlordane was cytotoxic at 10~3 M, but 6-thioguanine-
resistant (6-TGr) mutants were not induced at any dose. Under
identical conditions the positive controls, benzo[a]pyrene (B[a]P)
or 7,12-dimethylbenz[a]anthracene (DMBA), induced definitive
mutagenic responses.
Negative results were also reported by the same
laboratory (Tong et al. 1981) in human foreskin fibroblasts
(D-550) exposed to nonactivated chlordane at the maximum non-
cytotoxic dose (10~4 M). When D-550 fibroblasts were also
cocultivated with hepatocyte primary cells (HPC), chlordane did
not increase the frequency of 6-TGr mutants in three separate
experiments. By contrast, the concurrent positive control (DMBA)
induced a significant (p < 0.05) mutagenic response in the
presence of HPC. Since chlordane was properly evaluated up to
cytotoxic levels, the study is acceptable.
The National Toxicology Program (1985) reported
forward mutation at the TK locus of L5178Y mouse cells, but only
in the absence of metabolic activation.
c. Conclusions for Gene Mutation
Based on the ir\ vitro results of two inconclusive
mammalian gene mutation assays, chlordane is presumed to be a
direct-acting mutagen for Chinese hamster lung cells and mouse
lymphoma cells. However, the two acceptable studies submitted in
the _in_ vitro mammalian cell gene mutation category provide evidence
that chlordane was not mutagenic in liver epithelial cells or
human foreskin fibroblasts cocultivated with or without HPC.
Hence, the relevance'of the direct positive responses are unclear
until tests are conducted to assay the possibility that chlordane
mutagenicity can be either enhanced or detoxified in the presence
of exogenous metabolic activity.
A-7
-------
2. Chromosomal Aberrations
a. Mammalian Studies
Technical chlordane and its major constituent
isomers have been evaluated in dominant lethal assays by two
laboratories (Arnold et al. 1977; Epstein et al. 1972).
Arnold et al. showed that single gavage or
ip administration of 50 and 100 mg/kg of the technical did not
elicit a dominant lethal effect in the offspring of male mice
(eight/dose) sequentially mated (three females/mating) for
6 weeks. The negative control groups consisted of eight males/
group/route of administration. Measured parameters included
pregnancy rates, total implants, number of early deaths, and the
embryonic index. The higher dose evaluated (100 mg/kg) was
selected based on the results of an acute toxicity study which
indicated that 300 and 1000 mg/kg produced mild to severe toxic
effects. The same investigators performed additional dominant
lethal assays in mice given 50 and 100 mg/kg AG-chlordane (3:1
mixture of alpha and gamma chlordane) orally (gavage) and ip.
Dose selection was based on the evidence of mortality/other toxic
signs at 300 and 1000 mg/kg by both routes of administration.
The dominant lethal assay was performed as described for technical
chlordane; results indicated that the alpha and gamma chlordane
mixtures were negative.
Although the Arnold et al. data are discussed
here, they have not been considered for regulatory purposes
because these studies were conducted at IBT, and never validated.
Epstein and associates (1972) also showed that
neither technical chlordane nor alpha or gamma chlordane induced
dominant lethal effects. In one series of experiments, seven and
nine male mice received 48 and 240 mg/kg chlordane, respectively,
as single ip injections, and in a second experiment 10 males were
administered 50 mg/kg/day by gavage for 5 consecutive days. Four
males receiving 240 mg/kg and two animals in the 50 mg/kg gavage
group died. All surviving males were sequentially mated (three
females/mating) for 8 weeks. Females were sacrificed 13 days
after mating and scored for pregnancy, total implants, and early
fetal deaths. At the seventh mating interval, early fetal deaths
were recorded for 55 percent of the females mated to males in the
50 mg/kg gavage group. An analysis of variance revealed no
significant effect (p < 0.05). Based on the detailed criteria
developed by the authors to evaluate assay validity and positive
responses, chlordane was classified as negative. We assess that
the study is acceptable.
Epstein et al. also tested for dominant lethal
assays with the alpha isomer of technical chlordane ([1] 58 and
A-8
-------
290 mg/kg, ip, [2] 42 and 210 mg/kg ip, and [3] 75 mg/kg/day by
gavage for 5 days) and gamma chlordane (50 mg/kg/five daily
gavage administrations). These additional studies were also
reported as negative, an assessment with which we concur that
neither alpha or gamma chlordane induced a dominant lethal effect-
the studies are acceptable. '
In a single in vitro assay, trans-chlordane was
reported negative for chromosome aberrations in Chinese hamster
ovary (CHO) cells tested up to levels of cytotoxicity (NTP 1985).
t> Conclusions for Chromosomal Aberrations "+ ^ ,
The results of dominant lethal assays conducted
with chlordane, chlordane isomers, and mixtures of chlordane
isomers demonstrated that the test material is not clastogenic
in male mouse germinal cells. Only one study investigating the
potential clastogenic effects of chlordane on somatic cells vwas
located. The reported negative result in vitro reguires confirma-
tion in an adequately conducted in vivo assay. r".' ,
3. DNA Repair ''
The results of a variety of DNA repair assays ,
conducted with chlordane preparations are variable, but appear
to be generally consistent for the specific type of assay. .Thus,
although considered inconclusive, -both assays for gene conversion
in Saccharomyces cerevisiae strain D4 recorded presumptively
positive results (Gentile et al. 1982; Chambers and Dutta 1976)..,
Maslansky and Williams (1981) assayed 10"1 to Tt)~6 M
chlordane for unscheduled DNA synthesis (UDS) in rat, mouse, and
hamster primary hepatocytes. Three separate experiments wer§ .
conducted with each,hepatocyte species and 30 morphologically;
normal cells/experiment/species were counted for nuclear grains.
at the highest noncytotoxic dose only. Chlordane at _> 10-4"'M:was
cytotoxic for all cell systems; no significant increase in net
nuclear grain counts was seen at 10~5 M. The positive control,
DMBA, gave an appropriate response thereby demonstrating the':
sensitivity of the test system to detect UDS in rat, mouse, /ind
hamster hepatocytes. The study is considered acceptable and."..
showed that chlordane assayed up to a cytotoxic level was notf"
genotoxic in hepatocytes of three rodent species.
Probst and associates (1981) also reported a negative
UDS response in rat hepatocytes. No significant increase in"net
nuclear grain counts was seen in 20 morphologically normal""
hepatocytes per dose group exposed for 5 and 18 to 20 hoursto
the maximum noncytotoxic dose of chlordane (100 nmol/mL). T'he
study appears as have been properly controlled and is acceptable.
A-9
-------
Brandt et al. (1972) reported that treatment of
nonirradiated and UV-irradiated HeLa cells with 0.04 mM alP^-
Shlirdane did not cause UDS or inhibition of DNA repair. Although
reported as negative, the study provided no meaningful data. The
sensitivity of this assay is questionable because the positive
controls (methyl methanesulfonate and ethyl methanesulfonate)
were only marginally active in inducing UDS, and showed no
Interference with repair replication of the UV-challenged cells.
The study is therefore unacceptable.
By contrast to the negative finding in primary rat
mouse, and hamster hepatocytes, the UDS assay conduct^dq^h^V"^h
transformed human fibroblasts (VA-4) by Ahmed et al. 1977b) with
nonactivated and rat S9-activated doses of chlordane (1, 10, luu,
and 1000 uM) reported a significant increase in UDS over the
control (? < o!o5) at all doses. It should be noted that^hydroxy-
Srw treatment was used to block cell entry into scheduled
" DNA synthesis. Although quantitative data were not
s: s^s-s^s <°v :V ' HE
-'°
were seen.
To further define the type of repair mechanism
elicited by chlordane, these investigators performed a BUdR
ly long (UV-type) excision repair mechanisms. The
regions are saturated at relatively low
However, the lack of primary data for the majority
suspect for the following reasons:
n SV-40, VA-4 is a virus transformed cell line;
and it has been recommended by a panel of expertSl
1 Mitchell, A>U.; Casciano^D.A.^ltz^M.L.; Robinson, ^^^
San, R'^"g[sW^gts?Sa report of the U.S. Environmental Protection
Agenc^Gene-Tox Program. Mutat Res 123:363-410.
A-10
-------
that only nontransformed diploid human cells
should be used in this assay.
o The cells were not grown under reduced serum or
arginine deprivation conditions in conjunction
with hydroxyurea treatment to completely preclude
entry of the cells into (S-phase) DNA synthesis,
thus increasing the possibility of a false
positive result.
o The results were not confirmed.
o The publication did not indicate that the slides
were coded to eliminate bias.
o Several agents which have been repeatedly shown
to be nongenotoxic or weakly genotoxic (i.e.,
diquat, 2-4-D fluid, and carbaryl) induced
strong UDS responses. .
Based on the above rationale we consider this study
as inconclusive but classify chlordane as a presumptive but uncon-
firmed genotoxin for SV-40 transformed human cells. We recommend
that these findings be confirmed in a rigorously controlled
experiment that conforms to the currently accepted protocols for
UDS assays with human cell lines.
Sobti et al. (1983) exposed human lymphoid LAZ-007
cells to nonactivated and rat S9-activated 10~4 to 10~6 M chlordane
for 48: hours and 1 hour, respectively. Although statistically
significant increases (p < 0.01) in the incidence of sister
chromatid exchanges (SCE) were reported at 10~5 -S9 and at 10~5
and 10-o M +S9f tne elevated SCE frequencies were neither dose-
related nor twofold at any level. It should be mentioned that
experts recommend2 that for a compound to be considered positive
in the SCE assay it should either cause at least a twofold increase
over baseline SCE frequencies or demonstrate a three-point, dose-
response curve showing a progressive increase over baseline SCE
frequencies with at least one SCE value at p < 0.001 level.
Although the authors concluded that chlordane was genotoxic, the
study is considered inconclusive and should be repeated.
Vigfusson and associates (1983) described the
111 vivo exposure of 15 central mudminows (Umbra limi)/dose in
Latt, S.A.; Allen, J.; Bloom, S.E.; Carrano, A.; Falke, E;
Kram, D.; Schneider, E.; Schreck, R.; Tice, R.; Whitfield,
B; Wolff, S. (1981) Sister-Chromatid Exchanges: A Report
of the Gene-Tox Program, Mutat. Res. 87:17-62.
A-ll
-------
5 gallon well aerated aquaria (5 fish/aquaria) to four doses of,
chlordane (5.4 x 10~9 to 5.4 x 1Q-2 M) for 11 days. The positive
lindinSS of this in vivo study provided evidence of, a dose-related
and highly signiflc-anTTp < 0.01) increase in the frequency of
SCE in metaphases harvested from intestinal tissue. At the
highest doll , four fish survived treatment and the SCE frequency/
cell (19.3 + 0.51) was approximately 4.3-fold higher than the
control. Similarly, 3.8- and 1.7-fold increases were reported ..
at 5.4 x 10-10 and 5.4 x lO"11 M, respectively; the lowest
concentration assayed was negative. The sensitivity of^he
assay was demonstrated by the significant (p < 0.01) increase in
SCE frequency/cell induced by 5.4 x 10~9 M Mitomycin C. We ;
assessed, therefore, that chlordane is genotoxic in Umbra limi >. ..
and the study is acceptable.
Finally, the NTP (1985) reported trans-chlordahe
induced sister chromatid exchanges in CHO cells cultured in
vitro, but only in the absence of metabolic activation. This, is
fUFther supporting evidence for a direct DNA-damaging effect of
the trlns-ilomer in mammalian cells, and indicates the necessity
to evaluate this potential in adequately controlled mammalian
test systems which include mammalian activation. ,s
a. Conclusions for DNA Repair
The results of UDS assays appear to indicate that
chlordane is not genotoxic in primary mouse, rat, or hamster .
liver cells. However, nonactivated chlordane was reported to
induce a positive genotoxic response in SV-40 transformed human
fibroblasts but ^was negative in the presence of S9 activation.
The authors indicated that direct-acting chlordane appeared to
adequate controls.
Elevations in SCE frequency were observed in human
lymphoid cells .following exposure to both ^-activated and non-
activated doses, however, the response did not satisfy
acceded criteria for a positive effect in this system.
The single in vivo DNA repair study reported' that
chlordane induced SCE in IHteTtinal cells of fish and that the
response was dose related.
Based on the results of DNA repair assays we
conclude that chlordane is not genotoxic in rodent liver cells
A-12
-------
or human iymphoid cells, but is genotoxic in fish. The presumptive
direct-acting genotoxic effect on transformed human f ibrojiblasts
is unconfirmed. ;
4. Epigenetic Effects
Telang et al. (1982) evaluated six doses of technical
chlordane (10"4 to 10"7 M) for the potential to inhibit inter-
cellular communication between TGr mutants of ARL (HGPRT") and
ARL (HGPRT)+ cells. Chlordane was cytotoxic at 10~4 M for ARL
(HGPRT-) cells. In the presence of HGPRT+ cells, 5 x 1Q^6 through
1 x. 10"' M, chlordane markedly inhibited the metabolic cooperation
between the HGPRT- -deficient and -competent cells. At 5 x 10~6 M
an almost complete blockage of phosphoribosylated TQ. transfer
between the cells was observed as indicated by the.* > 100 and 86
percent recovery of TGr mutants cocultivated with 0?75 and
1.25 x 106 HGPRT+ cells. Increased survival was alsp reported at
10~5 and 10~6 M chlordane. The specificity of the assay to detect
only agents which interfere with cell-to-cell communication
(epigenetic) in contrast to chemicals which induce genQtoxicity
was 'demonstrated with the lyphophilic procarcinogen/promutagen,
B[a]P. Four doses of B[a]P, up to a cytotoxic level,, did hot
increase the recovery of TGr colonies.
To support the conclusion that chlordane interferes
with intracellular communication, the investigators evaluated
the effects of chlordane on guanine metabolism of treated ARL
cells. At/the cytotoxic level and at those concentrations that
profoundly affected cell-to-cell communication in the primary
assay.(10"4 to 10~6 M), chlordane did not cause ah appreciable
reduction of [14C] guanine conversion to its principal metabolites.
Although a positive control which is known to inhibit cell-to^cell
communication, such as DDT, was not included in this experiment,
we assess that the findings provide evidence that chlordane
interferes with metabolic cooperation suggesting^ that chlordane
may act as a promoter. ;
Further suggestive evidence for a promoter effect is
provided by the study of Brubaker and colleagues (1970), who
assessed that trans-chlordane was positive for cell division
inhibition but negative for inhibition of DNA synthesis in mouse
iymphoma (L5178Y) cells. ; /'
OVERALL INTERPRETATION OF STUDY RESULTS '.;
The individual published studies were insufficient to
establish a complete genetic toxicology profile for chlordane.
When considered collectively, however, some preliminary assess-
ments can be made.
A-13
-------
The single bacterial gene mutation assay with primary data
and the survey studies with procaryotes show that chlordane is
not mutagenic in bacteria. The positive reverse mutation assay
using Zea mays- provided qualitative evidence of a mutagenic -
response induced by chlordane in a plant system. Although the
study reported no primary data, chlordane is classified as
presumptively mutagenic.
Mammalian cell gene mutation assays with chlordane showed">
that the test material was not genotoxic in ARL cells or human
fibroblasts. Although nonactivated chlordane induced an incon-
clusive, mutagenic response in Chinese hamster V79 cells, the
validity of this assay was seriously compromised because of
technical deficiencies, the reporting of positive responses with
compounds that are either weakly mutagenic or not mutagenic, and!
the lack of a positive control. Although we classified chlordane
as an unconfirmed presumptive positive mutagen for Chinese hamster
V79 cells, we have serious reservations about the validity of the
study, and recommend that these results be verified in a complete ;
assay (i.e., three or more noncytoxic doses, with and without
metabolic activation and with known mutagenic positive controls).
Although chlordane proved to be nonclastogenic in dominant
lethal assays in male mouse germinal cells, confirmation of its
potential clastogenicity in somatic cells is needed.
The results of survey studies indicate that chlordane in the
presence of rat and plant microsomes is presumptively recombinogenic
in yeast. Data from studies with sufficient primary data to draw
meaningful conclusions, however., indicate that chlordane is not
genotoxic in primary mouse, rat, or hamster hepatocytes. Although
elevations in the SCE frequency were seen in human lymphoid
cells, the increases were not considered indicative of a genotoxic
response. On the other hand, chlordane was reported to induce
SCE in central mudminnows, and in CHO cells without activation.
There was a statistically significant increase in UDS reported
in SV-40 transformed human fibroblasts exposed to nonactivated
doses of chlordane; however, the effect was eliminated in the
presence of rat S9 activation. The validity of this assay was
seriously compromised by numerous technical deficiencies, strong
positive responses by weakly genotoxic or nongenotoxic substances,
the lack of positive controls, and the use of a transformed cell
line. The UDS assay should be repeated with a nontransformed
cell line.
In preliminary results with a novel assay, Telang et al.
(1982) reported that chlordane interferes with cellular communi-
cation. As enunciated by these authors, the mechanism by
which chlordane inhibits such metabolic cooperation may involve
intercellular communication mediated by gap junctions and thus,
interference with this process could be produced by a variety of
A-14
-------
effects on the cell membrane. Since organochlorine pestic-ides
are lipophilic in nature, they consequently tend- to accumulate in
the lipid layer of the cell membrane, thereby altering membrane
functions. Therefore, these authors suggest that accumulation of
organochlorine pesticides in the cell membrane could directly
interfere with the function of gap junctions.
The combined results of gene mutation assays indicate that
chloirdane is not mutagenic in bacteria. On the other hand,
presumptive (unconfirmed) positive results have^been reported
in V79 and L5178Y cells with and without activation^ as well as
in plant assays. DNA repair assays indicate chlordane appears
to be not genotoxic in bacteria, rodent hepatocytes and human
lymphoid cells, but is an unconfirmed presumptive genotoxin in
yeast and transformed human fibroblasts.
Based upon the lack of a definitive genotoxic effect, the
positive results in the metabolic cooperation assay, and the
evidence that chlordane has properties similar to known promoters,
chlordane may act by promotional (epigenetic) mechanisms. We
recommend, however, that this indication of.promoter activity be
corroborated in additional studies.
REGULATORY REQUIREMENTS
The following assays are needed to complete the genetic
toxicology requirements for chlordane:
, o Gene mutation assays in mammalian cell systems with and
.without metabolic activation in accordance with acceptable
. procedures.
o In vivo mammalian assays for chromosome aberrations in
somatic cells (e.g., rodent bone marrow).
o in vitro assays for SCE (with and without activation) in
mammalian cell systems.
RECOMMENDATIONS
The following tests are recommended to investigate the
potential promptional activity of the test material, according to
recognized procedures :*
0 lH vitro transformation with promotion assay.
o In vivo promoting activity assay.
Weisburger, J.H.; Williams, G.M. (1984) Bioassay of carcinogens
In vitro and in vivo in Chemical Carcinogens, Vol. 2, Univ. o£
'iTrmingham, UK, Am. Chem. Soc. Monograph 182, pp. 1323-1373.
A-15
-------
lu E C >,
O B O rH
4J O
O
§
(8 C «-
CU-H <
0) (0 E-
»J U
Q, JJ .
>> a,'*
rH (A IB O
to co
O 4J
O (A
8,
CO O
w
IS rH
B XI
S.-S
O rH
o .o
O
O 9
a CQ
« K
> IS iH
0) C O
JE SO
S1
9 o
rJ
(A X
rH C O fl)
0) r-l O -U rH
> 1C -r> O J3
9 O
*> 4J 9 M
> «
C O 4>
O JJ 'O
wo e
O HI
U) >
c o o e 41
Z 9 O
en
QJ
4J 4J.9 W
o> *o o; o 01
Q C CO >
C O O C 0)
9 O 4J 9 rH
e -^
0> CM
O CO
JE 4>
i ^- o ra
-
at o
W C Cfl
O O 9
Q O
O
i W
O nj
4J 43
S
CTl
08
« '
O O
«- r- 4J >J
re ai
O O M 4J
O'W
O «- +
m
V
^ 4J..
T- O
$
4J o en e
o w «
f r- + iC.
P. 4J
U> 9
*> 4 W*
4J C0| I
(B C <
CD -H
01 0)
C -P
A-16
-------
0
o
est Materials:
f
o
rs
0 9
^
H
fM
0> 01
JJ JJ
iJ i-l
a. S
01
4) 01
«. a
c
ic
j;
C
ose (mg/kg) or
oncentration
ug, ppm) Range)
QCJ
0> 0
&tl
E" 5
O 4J
38
W E-«
u £
3 &
0) 4
C 4
$8
1
8
o
b
o,
Jj
c
I-l
*
^1
X
^f
a>
r-l
1
0 hi
P 0>
3 &
,4 . >O
5 o.S
U 4) JJ lu
* TJ 4) -i
iw U
U-i r-4 » ,4>
9 IB >, 04 C
01 |J JJ 01 O
C 3 I C -<
H TJ U 3 JJ
1
"j
O
c
o
o
H
m
^
Positi
r-4
1C.
JJ
0)
. c
o
1-1
CO
,
specified] : 20
*
.2
'01
u
f-4
IS
.2
^
CO
01
s
4)
01
o
O
^
t^i
C
I-l
I-l
§
p
to
o
o
E
3
H
l-l
i-l
51
(0
rr*
fl
0>
c
jj
a.1
H
"O
0)
H
H
U
a .
S'S
3 -M
0 JJ
-) C rs
u-i rs is
UJ IS
9 x a
01 JJ 4)
C .rf S
n s a
i
g
r-t
o
c
0
u
M
c
41
>
Positi
IS
IS
c
is
EI
c
H
to
specified] :
s
*»
JJ
0) "O
4) -
t< JJ
O>
01
00
J <
3 E-<
i' C,
.^1
O f-»
2.g
r>
^.
*^*
O
8
0 P*
§-
in 0
So
O
P P
T- IN
01
S..J
UJ .
V
d
jj
1
Insuff ic
9
g
8
M
C
H
O>
JJ
1
ITS*
O
00
en
6
IS
1
E.
s
in
1
14-1
H
O
£
U)
£
,4
u
0>
&4 '
in c
e o
with an
preparat
1
'01
i
IS
tjl
4> 00
C C^
«f
- E-
0
o o>
5
's
-4 O
o 2
o
P 00
2?
mo
tol 4) O
c
l-l
o
jj
3
9
E
E ^-"
^
7
4J
§
U
0)
2
u
01 >
to ,-'
1
JJ.
&4
1 v .'»
IS rH
C £
D IB
4)'
. >
H
JJ
IS
.. 4>
z-
i >
' "8
JJ
0> '
£> 00
O CA
0. ~
B
specified]; 1
000 (gradient),
rat S9
E2*>
u
s
o
£
I-l
a
c
01
01
Vl
u
Survey s
:'. <
a
8
o
IB
C
D
0>
>
. JJ
IS
0)
-«
jr
jj
. *i-4
W
1
1
' 01
0"
^
hnical: 1000/d
8
E-
.2
, 01
I
r-4 r-4
IS O
t v
4) «
JJ ' W 1
o
10 C
a'
01
c
c
0
activati
tests.
o>
IS
CM
e>>
v
^'
IS
JJ
*
?
JJ
9
,- O1
§
"a1
, c-
0>
- 4)
' 14
O
Survey. s
i
jj
Ct4
a>
. o
u
IS
e
D
JJ
a
01 ;
.5. '.
o
activati
tests.
1 '
IS
jj
IS
;|. .
CM
u
T
^^
O
8
i-t
fa
o
r-4.
imercial (Ortho-
B
o
IS
U4 O
o to
« Qi
*r 4)
«- 14
04
i3
2 2
Ci 4)
CO E
^r o
r- o
JZ
JJ
's
01
3
01
o
Summary
i
3
r-4
O
c
o
u
H
O
U^
j.1
>
Positi
s
r-4
IS
JJ
41
0>
H
JJ
C
4)
O
^
S
a1
IN
CN
FH
IS
H
U
VJ
O
O
C
l-l
o
f4
JJ
IS
JJ
E
C
iH
Cu
1
JJ
kj
a
4)
V
CU
r-l
IB
O
a commer
1 tion.
1
0)
0!
in
c
r4
IS
kl
o>
4'
rH
O
Oi
CM
03
Cfl
^
*"*
3
H
01
.51
o
41
Oi
01
IB
X
^
i
rs
4)
N
C
0
u
X
*
D^
C
H
c
contai
A-17
-------
a c
o c
O (8
01 -P
C O
o a;
O XI
O 0)
a
s.«
M i
s,
o»
c:
IB C (U
> -c
rl C!
X) i-! O
.-< O* ->
01 C X
O -) O
fb 0) 4J
« O Q)
4J -O
U >. W
(SOU)
O> M
0) O O
2 -P -O
4J O
H x:
O Cn «J
H X Oi ^
-p o
a) o o o i c
Z C -O T- ID -H
E cn
rfl oo
en oo
-O
r-l !-
O P-
O (0
O OS
(D T-
6 *
S q
a
O 0)
_ "O
o a, n
T- -,J O
04
O r-l
O 18
-
o
o C
0) 0)
o -' >
O CO
«
<
3 ~
t-t -r1 IT)
O 03
o c
o clo
ro o c
S
XI
U < -P O
A-18
-------
I
u
O
Z
H
EH
CO
13
U
| Test Materials:
4-1
C
(
E
E
C
C
C
1
10
m 3
EH rt
10
w
D
a) to
P 4-1
>- l-H
O 3
a co
01 (1
K fH
§
r
4-
It
t
c.
Dose (mg/kg) or
Concentration
I (ug, ppm) Range)
0) .(1)
^ w
0* 4->
3 CO
4J gi
tO EH
^
O r<
r-l O
4J tP
(!) 0)
C 4J
4> IB
O U
JJ
a
a.
o
0
**
a
3
(!)
,,j
4J
(B
o-
0)
2
*
r J
J lJ CO C
0 10 --
O u-' Qi C 4J
.,-1 Q) «-! 10
X 0! U E
O i-1 Qi 10
4-> C! C X
> - o a>
o a> r-t -H a)
4J i-H IB 4-> g:
cs
r^
r
O 0) O O (1)
^* tr* c^ * Q) CP
CM IB CM (N Cn 10
> m .. >
OlB (T3»O> r4lB
Ct7» MCCld 0)C7»
IB 0) IB IB CT> E
E o »
COO OCOfN COO
^J1 in to in ^ m -H in
H t^ 1
XX 1 X X X
Qi Oi Qi X 10
«- i-i m IB T- -H T- -H E in
J3 in to E
& 2 IB
* CM M»<« (N ro OSCD*
a
-U
c
o
a
IB
(1)
r-l
a
1 4-^
10 ^^ 01 C
IJ to CTi O
M C 10 U
m O E
XI -H IB
< 4J Q
4J
Q.
a) a)
U r-J
0 XI
IX
EH
z.
Trans-Ch lordane :
(?) +_S-9
*~*
c in u
rJ r-l ~\
r-< 0
to C) EC
O O U
>-J
4-> C
(!) IB O
C 'f-J iM
0) r-l 4->
C7> IB *H
U E -r-l
-
10 C
4J 3
rJ
r-l 4->
10 10
3
B" to «
4-' <1)
UJ r-- tO
030
to T3
>i (1)
iW >-: -C
IB (!)
3 --J O
CO 4J Oj
^
O
C
O 0)
u >
C -rJ
r-i K
£
4-)
S C
> 4-i
4J >
01 JJ r-l
O O C
QJ 10 O
ri
10
4-1
Q)
0)
r-1 *
H -H
C >
O 0)
O V4
HI
0) o
C
0)
tr> co|
4-1
ra c
IB O
!" to Q
0)
Ui
e
10 Lt
D -H
< Qi
2 ai
Q a;
r-l
C
O i-J
IB
)-; 3
IB "O
E .4!
E U
3 O
CO ^
Oi
4J
O 4-i cr
10 3 r-J
ij O -H
4J X! IB
CO 4J 4J
XI -H 01
rt IS t3
3
r-l
O
c
o a>
0 >
C -r-l
M 01
C'
*^
4J
H
CO
O
ft
10
4J
4J
3
O
CO
0)
E t-
10 Cn
x; T-
O "
[Unspecified] :
"0.7.5%"
tu
o
IB >i
0)
C >
rH VJ (!)
3 >-i
e co 3
CO TD
o tn a>
T3 C O
H O
(!) C ij
Cn 0)
C >j 3
rJ O 01
co co c
^
a
a'
o
O 0)
10 r-l
C X!
O 10
0)
^
4J
10
Cn
01
2
r-l
r-l
V-
C
0) -~
X -H t/1
10 E 0
(1) to X!
JJ (8
XI iH (N
? *i
IB W
Vi -H
4J r-l rH
M3 8
IB
Q -rH W
.
A-19
-------
XI
c
ai
o
u
§
TB
Evaluati
XI X
U i-
O4 V
K Q
«»
Test Materials
e
c
r
P
(t
4.
c
^^
M 0)
Dose (mg/kg) o
Concentration
(uq. ppm) Rang
o
Oi
!'
V)
u
r4
XI
0)
c
0)
u
1
XI
cv
O r-<
U X)
< 10
Negative up
to cytotoxic
doses
O CO
m
jt
in
c
R)
to
s
[Unspecified]:
Unscheduled DNA
en
-*
(0
§
H
r-4
S
XI
m
M
S3 XI
° i
s*
O
- in
l!
synthesis (UDS) in
primary hepatocytes
i n vi 1-ro f hena to-
1
XI
Oj
a> a)
O -!
0 XI
< id
Negative up
to cytotoxLc
dosos
,-j
XI
CO
XI
to
1
£
[Unspecified] :
cytes from rat,
mouse, hamster)
^^
CO
^
tJ
1
c
o
O >i
r- I-l
c
s °
XI
in to
o »»
o
a>
to
a> r
,u
>t
-j
T< jrt
a> c to
tn o tn
O -H Id
t3 J
a
tn a>
ai a> >
§ g-i
>* id to
O
i
XI
Qj
Unaccel
C CO
r4 10
%
a
id
Negative
i-t
id
XI
0)
XI
O
C
(d
^j
ffi
0)
c
id
O
W
id
|
id
tn
0
Inhibition of re-
,^
e'-
en
'
o
o
CO
*-^
E e
^
* in
O O">
o a)
pair (CsCl density
gradient assay) in
mammalian cells
(HeLa)
3
i-t
Inconc
Positive only
,-4
id
x>
1
^
O
"
^~
[Unspecified] :
UDS in transformed
p4
to
without acti-
vation (+S9,
no effect)
for long-
patch repair
J3
tn
*""'
^
o
0
o
*~ co
T3
C XI
id id
14
0 1
0 \
(SV-40) human
fibroblasts (VA-4)
0)
x>
M
id ,
id rd C
iH O
a> e -!
u
-1 *-" *
1 Ui U/
a> tu a> c
to tn > -i-i
o c c .-i
13 O O
Oi U >-l
c
J? u 1
o a> o a>
"Z, M C O
1
3
» 1
Inconc
a)
>
r4
to
Positive at
noncyto toxic
doses ( 1 0~5 ,
10~6 M)
H3
4J
CO
x>
o
co
Trans -Chlordane :
Sister-chromatid
-
CO
en
s'l
.D
o cn
>- co
S2
U
exchange (SCE)
in vitro (human
lymphoid cells,
LAZ-007 )
i
4J
Qi
CO
0
s
CO
-1
Q
id
Positive,
dose-related
increase
1-1
4J
a>
c
o
tn
to
^
***
iw co
en en
H t-
>
I
o
^~
kj
Technical: 5.4
i
to
c
H
1
C
bi
M
o
r4 .
S IJ
N 3
7 id
o
.- c
X '
tn
* .u
in 73 a)
4-1
o - id
4-1 r- S
tinal cells of ex-
posed central mud-
minnows , Umbra
H
H
H
o> ^ '
«
a
rt
z
a
ns
14 XI
(0 O
D, c;
B
A-20
-------
CJ
HI
EH
CO
O
H
en
.H
(8
rl
M
IV
18
4J
tn
(V
EH
j_>
C
IV
E
I
0
CJ
e
o
rl
4->
18
CD 3
EH i-H.
18
W
C
rl
O
0 6
c c
^-« , o TC
a\.ri ctf
A; 4J
«. *
0-> jj E
Sec
IV O «
01 C Oi
O O 3
Q CJ
^ e
IV (V
O< J-i
>t 01
H3 4J
3 tn
4-> 0)
W >EH
>
CJ IH
H O
4-> 01
4-)
O O
ES 18
1
3
i (
U
C
O 0)
o t>
C -rl
H CO
CT*
W
t> 4J
3 S
rH .C
tn 4J
o -r<
Cu 3
. .
tn
CO
Cn
s--
ft.
55
.»
(U
C
18
a
o
rH
X
. u cn
1 CO
01 1
c
(8 ^"*
EH
O
CJ
o
4->
.pj
c
CJ
CO
(V
tr* *
, 18 -O
E
Q -H C
18 O
rtl Qi CJ
53 IV ^-*
Q C£
4J
a.
u
o
<
LI
o
a.1
jj
rH
tn
O
*
*- 1
J-l
O
Cn
e
18
i-H
IV
EH
rH
(8
O
H
e
_r*
u
01
EH
1
18
0)
Oi
0
O
O
O
i-J
O
Xi
0
E
!_
01
,^3
o
O rJ 1C
-rJ I-" O
4J (P -rH
rJ CJ C
X! I-' 3
«-i a; E
ceo
-J -rJ CJ
,-*
OJ
CO
cn
*_"
o
4J
S W
CU
** i>j
t 3
0 -P
r- rH
3
o o
4_)
'O
* CU
I-H It C3
a) o o x
0 «H V4 ^
c i 5 fe 3
O E CU rH
rl O C C3 41
4-> O -rl B3 O
tn
E
tn
i*j
c
18
O
U
a
4J
tn
a)
4J
(V
K
o
0
(V
c
o
^
i-H
e
o
rt
z
Iu
O
tv
rJ
4->
rH
tn
S
i
IV
.U'
0)
(0
pQ
2
CQ
IV
e
ns
*s
o
i-H
_<-;
CJ
1
tn
e
18
EH
U--
O
C
o
iH
4-1
rj
XI
rH
S
M
e
0
4J
18
r-l
4J
O
(S
4J
3
O
X
4->
rl
s :
u-i c " cn ' . . . ,
O O >J 1-1
r4 ^ O W .
C 01 4J U-I (V
O -rl 0) X
^ > CD a1 4J
4J -r- b t> C
rJ T3 >J -H1 tK,
XI 18 J-> in -
iH rH (8 ''.'
X i*H C*J O^' t£
C IV C3 CU Z
rH O 13 O
r^ ^
o
r*.
m . . . .
*^ * ! ?
G\ ' ' .''
co ; -'"'.
>.
j '. . .
E
Oi,
3 1
* . . .
^^.
tn co
C rJ r-
rJ l-H ^«
IV I/)
C CJ J . .
o »-
H e .
JJ 18 O
18 -H VJ
O rH *J
rl 18 -rl . .
rH E > '?'
& 18 C
>J E -rl
A-21
-------
CHLORDANE REFERENCES
Ahmed, F.E.; Lewis, N.J.; Hart, R.W. (1977a) Pesticide induced
ouabain resistant mutants in Chinese hamster V79 cells.
Chem.-Biol. Interactions 19:369-374.
Ahmed, F.E.; Hart, R.W.; Lewis, N.J. (1977b) Pesticide induced
DNA damage and its repair in cultured human cells. Mutation
Res. 42:161-174.
Arnold, D.W.; Kennedy, G.L.; Keplinger, M.L.; Calandra, J.C.;
Calo, C.J. (1977) Dominant lethal studies with technical
chlordane, HCS-3260, and heptachlor: heptachlor epoxide.
J. Toxicol. Environ. Hlth. 2:547-555.
Ashwood-Smith, M.J.; Trevino, J.; Ring, R. (1972) Mutagenicity
of dichlorvos. Nature 240:418-420.
Brandt, W.N.; Flamm, W.G.; Bernheim, N.J. (1972) The value of
hydroxyurea in assessing repair synthesis of DNA in HeLa
cells. Chem.-Biol Interactions 5:327-339.
Brubaker, P.E.; Flamm, W.G.; Bernheim, N.J. (1970) Effect of V
(gamma)-chlordane on synchronized lymphoma cells and inhibition
of cell division. Nature 226:548-549.
Chambers, C.; Dutta, S.K. (1976) Mutagenic tests of chlordane
on different microbial tester strains. (Abstract). Genetics
83:sl3.
Epstein, S.S.; Arnold, E.; Andrea, J.; Bass, W. ; Bishop, Y.
(1972) Detection of chemical mutagens by the dominant lethal
assay in the mouse. Toxicol. Appl. Pharmacol. 23:288-325.
Ficsor, G.; Nu LoPiccolo, G.M. (1972) Survey of pesticides for
mutagenicity by the bacterial-plate assay method. Environ.
Mutagen Soc. Newsl. 6:6-8.
Gentile, J.M.; Gentile, G.J.; Bultman, J.; Sechriest, R.; Wagner,
E.D.; Plewa, M.J. (1982) An evaluation of the genotoxic
properties of insecticides following plant and animal
activation. Mutation Res. 101:19-29.
Griffin Til, D.E.; Hill, W.E. (1978) In vitro breakage of
plasmid DNA by mutagens and pesticides. Mutation Res.
52:161-169.
Haworth, S.; Lawler, T.; Mortelmans, K.; Speck, W.; Zeiger, E.
(1983) Salmonella test results for 250 chemicals. Environ.
Mutagen. 5:3-142.
A-22
-------
Maslansky, C.J.; Williams, G.M. (1981) Evidence for an epigenetic
mode of action in organochlorine pesticide hepatocarcinogenicity
A lack of genotoxicity in rat, mouse, and hamster hepatocytes.
J. Toxicol. Environ. Hlth. 8:121-130.
Maruyama, I. (1980), cited in Simmon, ej: a_l. (1977).
Mortelmans, K. ; Haworth, S.; Lawler, T.; Speck, W.; Tainer, B.;
Zeiger, E. (1986) Salmonella mutagenicity tests. II.
Rsults from the testing of 270 chemicals. Environ. Mutagen. 8
(Suppl. 7):1-119.
National Toxicology Program (1983) NTP mutagenesis (Salmonella
typhimurium) test results: Chlordane (Chemical No. 18). NTP
Bulletin No. 9 (April 1983), p, 6 (Table 3).
Probst, G.S.; McMahon, R.E.; Hill, L.E.; Thompson, C.Z.; Epp,
J.K.; Neal, S.B. (1981) Chemically induced unscheduled DNA
synthesis in primary rat hepatocyte cultures: A comparison
with bacterial mutagenicity using 218 compounds. Environ.
Mutagen. 3:11-32.
Simmon, Y.F.; Kauhanen, K.; Tardiff, R.G. (1977) Mutagenic
activity of chemical identified in drinking water. Dev.
Toxicol. Environ. Sci. 2:249-258.
Simmon, V.F.; Tanaka, G. (1977), cited in Simmon, e_t a_l. (1977).
Sobti, R.C.; Krishnan, A.; Davies, J. (1983) Cytokinetic and
cytogenetic effect of agricultural chemicals on human lymphoid
cells in vitro. Arch. Toxicol. 52:221-231.
Telang, S. ; Tong, C.; Williams, G.M. (1982), Epigenetic membrane
effects of a possible tumor-promoting type on cultured liver
cells by the nongenotoxic organochlorine pesticides chlordane
and heptachlor. Carcinogenesis 3:1175-1178.
Tong, C.; Fazio, M.; Williams, G.M. (1981) Rat hepatocyte-
mediated mutagenesis of human cells by carcinogenic polycyclic
aromatic hydrocarbons but not organochlorine pesticides.
Proc. Soc, Exptl. Biol. Med. 167:572-575.
Vigfusson, N.V.; Vyse, E.R.; Pernsteiner, C.A.; Dawson, R.J.
(1'333) In vivo induction of sister-chromatid exchange in
Umbra limT by the insecticides endrin, chlordane, diazinon,
and guthion. Mutation Res. 118:61-68.
Wildeman, A.G.; Nazar, R.N. (1982) Significance of plant
metabolism in the mutagenicity and toxicity of pesticides.
Canad. J. Genet, Cytol. 24:437-449.
. A-23
-------
-------
APPENDIX I
A-25
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. Z0460
OFFICB OF
PBSTICIDKS AND TOXIC SUBSTANCES
MEMORANDUM
SUBJECT;
PROM:
TO:
THRU!
Chlordane/Heptachlor Mutagenicity
Irving Mauer, Ph.D., Geneticist
Toxicology Branch
Hazard Evaluation Division (TS-769C)
Henry Spencer, Ph.D., Pharmacologist
Section 7, Toxicology Branch
Hazard Evaluation Division (TS-769C)
Jane E. Harris, Ph.D.
Section Head, Toxicology Branch
Hazard Evaluation Division (TS-769C)
This is in response to your request for an expeditious
appraisal of the positive mutagenicity studies for chlordane
and heptachlor/heptachlor epoxide, as reported in the CAG
document ("Carcinogenicity Risk Assessment for Chlordane and
Heptachlor/Heptachlor Epoxide"). The negative reports listed
in that document were also scanned.
Based upon recent Velsicol submissions (Accession Nos-.
254320 and 254324) and other information available to me (NTP,
EMIC), two general conclusions are warranted from this preliminary
assessment (see below for a summary of available data):
1. Although the adequacy of the data base as reported
could not be undertaken (e.g., DER's) because of the
timely response requested, it appears that all the
genetic end-points we require to be assayed (gene
mutation, chromosomal aberrations, DMA damage/repair)
have been addressed.
A-26
-------
exposed central mudminnows (Umbra limi), as well as
in CHO cells in culture and in a human lymphoid cell
line (one report each). Whereas the positive SCE in
fish was recorded at nonlethal concentrations (added
to aquarium water), both in vitro studies were only
marginally positive (less than twofold above controls)
at toxic concentrations. Only one of the four studies
assaying for unscheduled DNA synthesis (UDS) ir\ vitro
was positive (in VA-4 cells, an SV-transformed~human
cell line), but only in the absence of metabolic
activation (negative with MA). Technical chlordane
was negative for UDS in primary hepatocyte cultures
from rats, mice, and hamsters (two reports) as well
as in a human fibroblast cell line (D-550). Finally,
gene conversion was reported in yeast cells
(S. cerevisiae D4) exposed to an activation system
(negative without), but too few data are included in
the article to properly interpret the results.
2- Heptachlor/Heptachlor Epoxide. In contrast to the
positive results for (gene) mutagenicity reported for
chlordane (both technical and reagent grades), the
available data for heptachlor and its epoxide is
consistently negative (three Ames and/or E_. coli, one
! subtilis rec assay, 1 ARL-HGPRT, two Drosophila
SLRL, 1 HPC/UDS). Reversions to histidine prototrophy
in Salmonella TA-1535 and 100 under activation conditions
to an unstated dose range, as well as to the wild-type
(nonwaxy) phenotype in corn pollen grains were reported
by the same investigator who found "commercial" chlordane
positive.
Adequate reports on dominant-lethal assays in mice
were negative, but an abstract from a meeting reported
positive results, for both germinal (DLT) and somatic
(bone marrow) chromosome damage in rats fed 1 and 5
ppm of an unstated formulation of heptachlor for
three generations. A Russian study also reported
positive chromosome damage in bone marrow cells from
"white male mice" treated i.p. with "heptachlor"
(also of unstated source and purity) at a single dose
level stated to be "4% of the LD50." Too few procedural
details are included in these "positive" studies to
interpret the results reported.
As with chlordane, negative UDS results have been
reported for heptachlor technical in primary rodent
hepatocytes (two reports), but a positive recorded in
VA-4 cells (virus-transformed human fibroblasts) for
both the technical and epoxide, but only with metabolic
A-27
-------
2. Consistent with the results of mutagenicity testing
previously reported for other agents of this chemical
class (organochlorines such as Lindane, chloroform,
Inter alia), the mutagenic potential of these chemicals
are low to unsubstantial. Hence, from the available
data it would appear their oncogenic potential derives
from mechanisms other than "genetic initiation" (direct
interaction with DNA). [There is suggestive evidence
in mammalian systems (the few promotion assays available)
of an "epigenetic" mode of action for these chemicals.]
Survey of Available Literature
1. Chlordane-technical (but not reagent-grade alpha-
chlordane, gamma-chlordane, and gamma-chlordene) was
positive in adequate bacterial (Ames) assays (three
positive, one negative), but consistently so only in
the most sensitive strain (TA-100), and only at very
high concentrations (5000 ucj/plate and above). Testing
for mutagenicity in mammalian cells in culture (four
reports) revealed inconsistent results (two positive,
two negative), due in part to the different test systems
employed. The. two negatives were reported for HGPRT
in ARL (adult rat liver) and for both thioguanine and
diphtheria-toxin resistance in V79 (Chinese hamster
lung) cells. One of the positives was for ouabain
resistance in V79 cells, but reported for only a single
concentration of an unstated formulation, at less than
50 percent cell survival; the second was found in non-
activated mouse lymphoma cells (L5178Y-TK), but the
study is incomplete since metabolic activation was not
employed.
The plant systems may activate chlordane to mutagenic
derivatives is suggested by a single article reporting
both gene conversion in Saccharomyces cerevisiae D4
cultures exposed to a reagent grade (presumably alpha-
chlordane according to the CAS Number stated), and
reversion to wild type in pollen grains from the
homozygous "waxy" strain W22 of Zea mays (corn) exposed
to a '"commercial-grade formulation." However, too few
procedural details were reported to adequately interpret
these results.
Although reportedly negative for gross chromosomal
damage in vivo (two mouse DLT's) and in vitro (CHO
cells), technical chlordane or one of its principal
components (alpha-chlordane) apparently has DNA-
damaging activity, as revealed in a single study
reporting increased dose-dependent sister-chromatid
exchanges (SCE) in intestinal cells sampled from
A-28
-------
activation (in contrast to chlordane, positive only
in the absence of activation). [The mutagenicity
data bas'e for heptachlor epoxide is less than adequate
to. satisfy FIFRA guidelines.]
Discussion
Recent studies have suggested that organochlorines
(Lindane, chlordane, heptachlor, inter alia) do not interact
directly with DNA (i.e., are not " ge no toxic""), but rather act
epigenetically" by mechanisms affecting cell membrane
permeability and/or following an irreversible "initiating
event" (i.e., are "promoters" ensuring survival of preexisting
transformed cells). The following citations employing chlordane
heptachlor available for this "quick-and-dirty" review (and
listed in the CAG document) are consistent with this suggestion:
Inhibition of metabolic cooperation in mixed cultures
consisting of thioguanine-resistant and TG-sensitive
cells (several reports from both Williams1 and Trosko's
labs).
The putative positive UDS results in cells already
"initiated" (e.g., the SV-40 transformed cell line,
VA-4).
Inhibition of DNA synthesis and/or cell cycle mechanisms
by severely toxic concentrations leading to perturbation
of repair (increased UDS, SCE).
1.
cc: Dr. Amy Rispin
Science Intergration Staff
Hazard Evaluation Division (TS-769C)
A-29
-------
PRELIMINARY EVALUATION OF REPORTED POSITIVE STUDIES
Can pound
CHLOROANE
Test | Test
Hater lal 1 Assay 1 System
|
Technical (Gene Mutation Ames (all)
|
I
Technical JGene Mutation Ames (all)
1
Technical JGene Mutation) Ames (TA 98,
1
1
Reagent |Gene Mutation
(alpha) |
"Commercial n|Gene Mutation
1
(Unstated) |0uabaln
1 resist.
1
(Unstated) (Gene Mutation
1
1
Technical | In vivo SCE
1
1
Reaaant I In vitro SCE
(alpha) |
1
(Unstated) I In vitro UPS
1
Reagent |Cel 1 cycle
(gamma) | Inhlb.
Reagent |ln vitro CA/
(alpha) | SCE
I
Reagent I Gene Mutation
100)
Sacch. - 04
Z. mays
Po 1 1 en
V79 cells
ARL/HGPRT
Mudmlnnow
LAZ-007 cells
VA-4 cells
L5178Y eel 1
CHO cells
L5176Y/TK
Dose/Cone. | | TB |
Range I Reference | Evaluation)
1 1
1
Comments
20-100 (Simmon 5t_£l_. (ACCEPT. (POS. only In TA 100
| (77)
1 1
1
1
) 10-5000 (Simmon and (ACCEPT. (a) EOUIV In TA 98
) 1000-50, 000 | Tanaka (77)
I
|b> POS. In TA 100
1
5-10,000 (Maruyama (80) (ACCEPT. (POS. In TA 100; NEG.
1
I
1
(Unstated) (JBntlle et al.
| (82)
1
(Unstated) (Ibid
1
|
I
(Only 1 dose (Ahmed et al.
reported) | (77b)
1
3 -6 '
10-10 M |Telang et al .
| (82)
5.4/10~12-10~9|vigfusson et al.
j (83)
I
fi 3 '
10-10 M (Sobtl et aj .
I (83)
1
1,10, 100, 1000|Ahmed et al.
uM | (77a)
~~ I
1
4_ug/ml I Brubaker et al.
(70)
(Unstated) (NTP (85)
1
(Multiple) NTP (85)
[
I
In TA 98
1
INCONCL. |POS. only * S9; too
1
few data
I
INCONCL. (Tech not tested; too
1
few data
1
INCONCL. "Weak" at < 50<
| survival; few data
ACCEPT. (NEC. for HGPRT, but
POS. for promotion
(ACCEPT. (Oose-dependent POS.
1
j INCONCL.
1
< 2-fold Increase;
no dose response
(ACCEPT. POS. only without S9
1
| INCONCL.
INCONCL.
1
1 NCONCL
NEG. for ONA, POS. for
G2 arrest; only 1 dost
NEG. for CA; POS. for
1 SCE
1
|Not tested with S9
A-30
-------
PRELIMINARY EVALUATION OF REPORTED POSITIVE STUDIES (cont'd)
1 Test |
Compound | Material | Assay
HEPTACHLOR
1
| Technical JGene Mutation
t
1
1
1
(Unstated) |Gene Mutation
.
1
(Unstated) .,
Epoxide
(Unstated)
In vl-tro UOS
Test
System
Ames (all)
ARL/HGPRT
VA-4 eel Is
In vitro UDS
VA-4 eel Is
1
In vivo CA
Dose/Cone. |
Range | Reference
1
((Unstated) |Gentl le et al .
j (82)
TB
Evaluation
INCONCL.
10" to 10" M|Telang et^al. ACCEPT.
100,1000 uM
(82)
Ahmed et a 1 .
(77a)
10,100,1000 uMl Ibid.
Mouse BM |5 mg/kg |Markarjan (66)
| (Unstated) (OLT/BM-CA Rat repro.
1
1
| Technical
I
study
In vitro CA/
SCE
CHO eel Is j
- I 1 | |
1 , 5 ppm jCerey (74)
(Multip.le) |NTP (85)
INCONCL.
INCONCL.
INCONCL.
1 NCONCL .
1
1
|POS. In TA 1535/100,
I Out on 1 / v I tn S9
1
INEG. for HGPRT-; POS.
I for promotion
1
|POS. only with S9 , Out
| no va 1 ues for UOS
1
| no values for UOS
I
|Too few procedural
I deta its; 1 dose
1
1 1 Abstract!
|
1
I
1
|POS. for both CA and
I SCE, Out too few
deta I Is aval laOle
A-31
-------
-------
APPENDIX B
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
OFFICE OF
PESTICIDES AND TOXIC SUBSTANCES
MEMORANDUM
SUBJECT;
FROM:
TO:
THRU-:
The Mutagenicity Assessment (Gene-Tox Profile) of
Heptachlor/Heptachlor Epoxide: Addendum to the
Registration Standard
Caswell Nos. 474
474F
TB Projects: 0007
* ^-» «*rf VW V,. J. _!. JL^ \^/ ^ «
^^ f) /^
Irving Mauer, Ph.D. ^^^^^^^^^i
Toxicology Branch /^^^ /^/-^/^-
Hazard Evaluation Division (TS-769C)
Henry Spencer, Ph.D.
Toxicology Branch
Hazard Evaluation Division (TS-769C)
and
Amy S. Rispin, Ph.D.
Science Integration Staff
Hazard Evaluation Division (TS-769C)
Judy Hauswirth, Ph.D., Acting Head .
Section VI, Toxicology Branch ':
Hazard Evaluation Division (TS-769C)
td
The attached assessment of the mutagenicity of .heptachlor*
was developed from reviews of the available published literature
located by Dynamac Corporation, as well as articles filed with
Technical heptachlor is a ternary mixture of related chlorinated
methanoindene compounds, typically containing 72 to 73 percent
l,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanol-lH-
indene (CAS Registry No. 76-44-8) as principal active ingredient,
18 to 20 percent trans (gamma) and 2 to 3 percent cis (alpha)-
chlordane (CAS No. 57-74-9), and 5 percent nonachlor (CAS No.
3734-49-4), plus about 1 to 2 percent other manufacturing
impurities. Heptachlor is readily metabolized in mammals to
the epoxide (2,3,4,5,6,7,7-heptachloro-la,Ib,5,5a,6,6a-hexahy-
dro-2,5-methano-2H-ideno(1,2-b) oxirene CAS No. 1024-57-3).
B-l
-------
the Environmental Mutagen Information Center (EMIC, Oak Ridge
National Laboratory) for evaluation in Phase I of the Agency s
GENE-TOX PROGRAM. A preliminary appraisal of some of these
studies** as summarized by the Carcinogen Assessment Group
(GAG)*** is also incorporated, since the conclusions in that
nreliminary discussion are consistent with those derived from
Ihfs morJ comprehensive evaluation There appears to have.been
no submissions of .primary gene-tox (mutagenicity ) studies by
registrants (Caswell No. 474).
As provided previously for the Registration Standards of
other organochlorine insecticides (aldrin/dieldrin) , this mutagen-
icity aslessement is organized as follows: A discussion and
evaluation (for regulatory purposes) of the data base located by
both mljor" sources (DYNAMAC; EMIC/GENE-TOX PROGRAM) , tabulated
according to the FIFRA categories of genetic effect (gene mutation,
chromosomal damage, primary DNA damage/repair, and other mechanisms),
lonoweSby our conclusions, as well as recommendations to satisfy
both regulatory requirements ("data gaps") and to indicate
farther testing the results from which would be useful for risk
characterization.
Although a definitive genetic toxicology profile cannot be
established from the individual published studies ^viewed here,
collectively they permit the following preliminary conclusions.
1. Neither heptachlor nor heptachlor epoxide appears to be
mutagenic in microbial or mammalian cells.
2
3.
Althouah negative for chromosome damage in germinal
assays? thS potential for somatic cytogenetic activity
has not been adequately evaluated.
The combined results of DNA repair assays indicate that
heptachlor is not genotoxic in bacterial, yeast, or
mammalian cells.
genotoxic mechanisms.
In addition to our previous cursory assessment (Appendix I),
a listing of pSSliShed articles cited is attached as Appendix II.
Attachments
^ ..r-hlordang/Heptachlor Mutagenicity , " memorandum: Mayer to
Spencer, December lb, "
*** Carcinogen Assessment
Carcnogen ssessm
for Chlordane and H^phaehlor/Heptachlor Epoxide (DRAFT,
December, 1985) .
B-2
-------
MUTAGENICITY OF HEPTACHLOR
The Data Base
Genetic
Endpoint
Gene
Mutation
Chromosome
Aberration
DNA Damage/
Repair
Other
Mechanisms
ssays with techni
ormulations) , dis
Test
System
Bacterial
Mammalian cell
in vivo
in vitro
in vivo
in vitro
in vivo
in vitro
in vivo
cal heptachlor/heptach
tributed according to <
Chemical
JHeptachlor
12
1
2
4
6
1
2
|H. epoxide
'' 5
2
2
'
1
_
-
DiT
r,M»^ (47%) of these reports are inadequate (judged
UNACCEPTABLE) to support the negative results reported, because
of one or more of the following deficiencies:
- No primary data presented, and/or only qualitative
assessments (+/-);
- Preliminary screening surveys or new techniques, with
insufficient details/procedures; .-
- Only one dose or insufficient (nontoxic) doses tested in
incomplete assays;
- Assays with no positive controls to assure sensitivity of
the test system to respond; and L
- Inappropriate procedures/test systems.
However, these studies were considered to corroborate the
conclusions from the minority of sufficiently adequate assays
B-3
-------
(10, or 31%) judged ACCEPTABLE. An equal number of assays were
judged "inconclusive" because they reported unconfirmed presump-
tively) positive results which could not be satisfactorily
interpreted, due to inadequate procedures or controls, and/or
the reporting of qualitative assessments in assays compromised by
coSfliSinJ Sariabies. Some of these studies are useful, however,
in directing attention to further testing necessary to establish
the mechanism(s) of action possessed by these organochlorines,
discussed more fully below.
Since the available data base is incomplete in some of the
genetic endpoints (as indicated in the above summary table ,
further testing is recommended (see below) in order to satisfy
both the regulatory requirements for continued registration of
heptachlor formulations and to provide approaches to risk
characterization.
Discussion and Evaluation of Assay Results
The results of all published studies conducted with technical
heotachlor/heptachlor epoxide, as well as other formulations of
this^rganochlorine insecticide, are summarized below in tabular
form (following this discussion).
A. Survey Studies
As indicated in the summary table, a number of these reports
are considlrel technically inadequate and unacceptable in meeting
Regulatory requirements, but tend to support the overall conclusions
drawn from fully adequate studies discussed below.
Thus as reported by Moriya et al. (1983), concentrations up
to 5000 Si/plate Sf heptachlo/andTtKe- oxidation product, heptachlor
eooxide, were negative for mutagenicity in both the Salmonella
tvgnimurium/mammalian microsome (Ames test) a"g theEsch^richia
coli trvotophan reversion assays. Similarly, qualitative negative
limits were reported over a onefold to 10,000-fold nonactivated
Ind S9 activated dose range of heptachlor in the concentration
agar gradient test with eight S. typhimurium and two E. £oli
trains (Probst et al. 1981). In an unactivated spot test,
II ug/disc of hepTachlor was also negative with four S. typhimurium
and~two E. coli strains (Shirasu et al. 1976).
Gentile et al. (1982) reported ^.technical but not
commercial gride"eptachlor induced a 2.3-fold and 1^ fold
^-^^^l^^^ * " and
'is r sish
B-4
-------
doses lower than 10 ug/mL to support a dose-related increase;
moreover, the increase with TA100 was only slightly higher than
2-fold at the highest dose, and the background spontaneous frequency
for TA98 (112) was excessive. < is
The same authors also reported that 889 uM technical
heptachlor induced a 2.3-fold increase in revertents in S.
typhimurium TA1535 cultures exposed to a IS fraction derived from
corn microsomes; this finding was also considered inconclusive
evidence for a positive response for the same reasons as above.
In the same article, Gentile and associates also reported
positive results in a reverse mutation assay using commercial '
grade heptachlor on Zea mays at the waxy (wx) locus. In this
study, 1.12 kg/hectare of the formulation was sprayed in situ on
mutant seeds (wx~/wx~) prior to the emergence of corn seedlings.
Plants were allowed to grow 12 to 14 weeks and pollen grains were
harvested, dehydrated, and stained. The authors reported a
significant increase (p < 0.05) in back mutations of wx~ to wx*.
However, because essential primary data were not present, the
results are considered inconclusive, but heptachlor is listed as
a presumptive mutagen in this study.
Four survey articles evaluated the potential of heptachlor
to interact with and damage DNA (Shirasu e_t al. 1986; Gentile et al.
1982; Griffin and Hill 1978; Seller 1977). When 20 ug/disc of~~n~otv^
activated heptachlor were placed on a Petri dish, Shirasu and
associates recorded no preferential inhibition of Bacillus subtilis
repair-deficient M45 relative to the repair-proficient wild-type,
H17. Similarly, Griffin and Hill reported that 0.1 mg/mL hepta-
chlor did not induce singlestrand breaks or alkali-labile sites
in purified Col El DNA derived from _E. coli K12, and the Gentile
group reported that four doses of technical or commercial grade
heptachlor over a threefold to hundredfold dilution range, with
or without rat liver S9 or corn IS microsomal fractions, were not
recombinogenetic in Saccharomyces cerevisiae D4.
Finally, Seiler (1977) reported that there was no increased
inhibition of testicular DNA synthesis in mice exposed by gavage
to 40 mg/kg heptachlor. Although the author provided data that
indicate a high rate of detection of mutagens/carcinogens in the
inhibition of testicular DNA synthesis assay, several well docu-
mented genotoxic/carcinogenic agents gave a negative response in
this test system (ethylenethiourea, aminotriazole, maleic hydrazide,
and isoniazid). Hence, interpretation of a negative result in
this assay is unclear.
Although individually the data provided by these survey
articles were insufficient to form definitive conclusions and
thus unacceptable in meeting regulatory requirements, nevertheless
the data collectively provide evidence that heptachlor is not
B-5
-------
mutagenic in bacteria. In addition, apparently heptachlor does
not cause other genotoxic responses in bacteria or yeast. On
the other hand, the ability of heptachlor to induce a mutagenic
response in corn is not clearly established.
B. Primary Studies
A respectable number of genotoxic assays with heptachlor did
contain sufficient primary data for review and evaluation according
to FIFRA genetic points.
1. Gene Mutation
a. Bacteria
Heptachlor/heptachlor epoxide were assayed in three Ames
tests. Marshall et al. (1976) showed that both heptachlor and
heptachlor epoxide were cytotoxic at doses that exceeded 2500
uq/plate in the absence of metabolic activation provided by rat
S9, and 1000 ug/plate in the presence of rat S9 activation, but
neither agent~was mutagenic over decreasing twofold dilutions
ranging from 1000 to 50 ug/plate. Although the more sensitive
S. typhimurium TA98 and TA100 were not included in the assays,
the study is acceptable, since the study was properly controlled
in that heptachlor and the epoxide product were adequately tested
in S. typhimurium TA1535, TA1536, TA1537, and TA1538.
Glatt et al. (1983) evaluated six half-log dilutions
(3000-10 ug/plate) of heptachlor epoxide with or without rat S9
activation" in the Ames assay (strains TA1535, TA1537, TA1538,
TA98, and TA100) and found that the test material was insoluble
at concentrations of 300 ug/plate or greater. Their results
further showed that the epoxidation product of heptachlor was
negative. By contrast, the authors clearly demonstrated the
sensitivity of the test system to detect epoxides of several
potent polycyclic aromatic hydrocarbon carcinogens.
In an ancillary assay reported in the same publication,
Glatt and associates assayed the same six selected doses of S9-
activated heptachlor epoxide (10 to 3000 ug/plate) using TA93 in
the presence of the epoxide hydrolase inhibitor and glutathione
depletor, 1,1,l-trichloropropene-2,3-oxide (TCPO). Under these
conditions, heptachlor epoxide was not mutagenic. Since the study
was well designed, properly controlled, and heptachlor epoxide
was assayed up to the limit of solubility, the study is acceptable.
\s part of the National Toxicology Program, heptachlor
was selected for evaluation in the preincubation modification to
the Ames test with strains TA1535, TA1537, TA98, and TA100 (NTP,
1983) Preliminary studies showed that this compound precipitated
at doses exceeding 1000 ug/plate. Two independent assays were
conducted with five nonactivated doses (0.3 to 1000 ug/plate in
-------
experiment 1 and 0.1 to 10 ug/plate in experiment 2). No increase
in His4" colonies of any strain was reported for either study.
Five doses (100 to 10,000 ug/plate) were used for the SB-activated
preincubation assays; samples were preincubated for 20 minutes-
with the appropriate strain and either 10 percent rat or hamster
S9 cofactor mixes. In two independent assays, S9-activated
heptachlor was not mutagenic. Thus, heptachlor appears to have
been adequately tested up to an insoluble level in a properly
controlled study. Therefore, this study is acceptable, and we
conclude that heptachlor is nonmutagenic in this preincubation
modification of the Ames assay.
Finally, van Dijck and van der Voorde (1976) found that
isomer A of technical heptachlor epoxide was negative in seven
Ames strains of £3. typhimurium capable of detecting base-substitution,
frameshift, and differential toxicity, when tested up to cytotoxic
doses in the absence and presence of mouse mammalian activation
(S9) systems.
t^,
b. Mammalian Cells
Telang et al. (1982) exposed 4 x 10^ primary adult rat
liver epithelial cells (ARL) to six concentrations of heptachlor
(10~3 to 10~6 M) in two independent experiments. Heptachlor was
cytotoxic at 10~3 M but at no dose was there an increase in the
frequency of 6-thioguanine-resistant mutants (6-TGr). By contrast,
the positive controls, benzo [a]pyrene (B[a]P) or 7,12-dimethylbenz-
[a]anthracene (DMBA), induced definitive mutagenic responses.
This study was judged acceptable and the results demonstrated ,
that heptachlor is not mutagenic in mammalian liver cells.
c. In Vivo
The negative sex-linked lethal result reported for both
heptachlor and its epoxide in Drosophila (Benes and Sram, 1969)
is discounted because of the low dosage necessary in testing this
organochlorine insecticide.
d. Conclusions for Gene Mutation
Heptachlor and/or the oxidation product, heptachlor
epoxide, were extensively investigated in £. typhimurium using
nonactivated and rat and/or hamster S9-activation conditions.
Uniformly, the results indicated that heptachlor was not mutagenic
in bacteria. In the only published mammalian cell gene mutation
study available for review, the results were also negative. No
mammalian, cell assays were conducted using the cell lines for
which large data bases exist (mouse lymphoma, Chinese hamster
ovary cells, or V79), and we recommend that at least one such
assay be conducted to confirm the negative result found in primary
ARL cells.
B-7
-------
2. Structural Chromosome Aberrations
a. Somatic Cells
Only -two studies assaying the potential clastogenic
(chromosome-breakage) effects of heptachlor on somatic cells were
located for review, both considered inconclusive, although
reporting presumptively positive results.
In a study reported only as an abstract of a presentation
at a meeting of the European Environmental Mutagen Society, Cerey
and associates (1973) claimed that 1 and 5 mg/kg/day heptachlor
(of unstated purity and source) caused increased "abnormal mitoses"
in the mouse bone marrow cells of the F2 and F3 generations of a
dietary reproduction study, but provided no other details. The
same incompletely reported study also stated that "increased
resorptions" ("dominant lethals"?) were found. In the second
study, Markaryan (1966) reported that the intraperitoneal injection
of "0.0052 mg/g heptachlor" (stated to represent "4% of the LD50"
for mice) produced "nuclear disturbances" and unspecified "breakage"
in bone marrow cells of an unstated number of mice. In addition
to inappropriate sampling techniques employed by this investigator,
no attempt was made to differentiate the purely cytological
effects of treatment from the standard categories of cytogenetic
damage, hence the positive result claimed cannot be properly
interpreted.
b. Germinal Cells
In the mouse dominant lethal assay performed by Arnold
et al. (1977) a 25:75% blend of heptachlor:heptachlor epoxide was
administered once by gavage (7.5 and 15 mg/kg) or i.p. (7.5 and
15 mg/kg) to eight male mice per experimental dose; two control
groups (eight males/group) were used for each treatment group.
The authors reported no significant increase in dominant lethal
indices over a 6-week consecutive mating sequence (three untreated
females/male/week). The parameters measured were pregnancy rates,
total implants, number of early deaths, and the embryonic index.
No toxic signs were seen during the main study; however,
preliminary acute toxicity studies performed by these investigators
showed that the heptachlor blend administered either by gavage or
i.p. was toxic at 30 and 100 mg/kg. Although the study was
performed without a concurrent positive control, the study is
acceptable and showed that the maximum nontoxic dose of the
heptachlorrheptachlor epoxide mixture did not induce a dominant
lethal effect.
In an earlier study Epstein et al. (1972) tested a total
of 174 materials, which included heptachlor and heptachlor epoxide,
in mouse dominant lethal assays. Groups of seven and. nine male
B-8
-------
mice received 4.8 and 24 mg/kg heptachlor, respectively, by i.p.
injection and other groups of 12 animals each were dosed by
gavage for 5 consecutive days with 5 and 10 mg/kg/day. The
concurrent negative control group consisted of 10 males. Following
dosing, the males were sequentially mated (three untreated females)
for 8 weeks. Females were sacrificed 13 days after mating and
were scored for pregnancy, total implants, and early fetal deaths.
Based on the detailed criteria developed by the authors to evaluate
assay validity and positive responses, heptachlor was assessed to
be negative. Although no pharmacotoxic responses were reported
for the i.p. dosed groups, two of the males that received either
5 or 10 mg/kg of test material by gavage died. Therefore,
heptachlor did not induce dominant lethal effects in mice when
assayed up to a toxic dose level; and since all appropriate
controls were included, the study is acceptable. :
Heptachlor epoxide (6 and 30 mg/kg, i.p. and 8 mg/kg,
P.O. x5) was also investigated by Epstein and associates. No ;-
toxic response was reported for the i.p.-dosed animals; howeve'r,
4 to 9 males exposed by gavage died. No dominant lethal effects
among offspring of females mated with the heptachlor epoxide-
treated males were observed. Since heptachlor epoxide was also '
assayed up to a toxic dose, the study is acceptable.
c. Conclusions for Structural Chromosomal Aberrations
Only inconclusive evidence for heptachlor to induce
chromosomal aberration in somatic cells was available for review.
On the other hand, in two adequate mouse dominant lethal assays,
neither the parent nor epoxidation product induced major chromosome
aberrations in male germinal cells.
3. Other Mutagenic Mechanisms
a. DNA Repair in Mammalian Cells
Maslansky and'Williams (1981) assayed 10"1 to 10~6 M
heptachlor for unscheduled DNA synthesis (UDS) in rat, mouse, and
hamster primary hepatocytes. In three separate experiments, 30
morphologically normal cells/experiment/species were scored for
nuclear grains at the highest noncytotoxic dose only. Heptachlor
at _> 10"^ M was cytotoxic in all of these cell systems and there
were no significant increases in net nuclear grain counts at 10~5 M
heptachlor. The positive control, DMBA, adequately demonstrated
the sensitivity of the test system to detect UDS in rat, mouse,
and hamster hepatocytes. The study is acceptable and showed that
heptachlor assayed up to a cytotoxic level was not genotoxic in
rodent hepatocytes.
Probst et al. (1981) confirmed the lack of heptachlor-
induced UDS esponse in rat hepatocytes. No significant increase
B-9
-------
in net nuclear grain counts was seen in 20 morphologically normal
hepalocytes exposed to the maximum noncytotoxic dose of heptachlor
(10 nmol/mL).
In contrast to the negative results in primary rodent
hepatocytes, Ahmed et al. (1977) reported «ualj;tativ;.?^?"°! °f
a UDS response in a virus (SV-40) transformed human fibroblast
cell line (VA-4) exposed to S9-activated doses of heptachlor
UOO and 1000 uM) aSd heptachlor epoxide (10, 100, and 1000uM).
it should be noted that hydroxyurea treatment was used to block
cell entry into scheduled (S-phase) DNA synthesis. The -lack of
priLry data and the inability to determine if the response was
dose related, however, precludes acceptance of these data as
definitive evidence of a genotoxic response. These data are
suspect for the following reasons:
1.
2.
3.
The SV-40, VA-4 is a transformed cell line; a panel
of experts1 has recommended that only nontransformed
diploid human cells be used in this assay.
The cells used in this study were not grown under
reduced serum or arginine deprivation conditions in
conjunction with hydroxyurea to ensure complete
blockage of S-phase synthesis, increasing the
possibility of a false positive response.
The length of exposure in the presence of S9 activation
(8 hours) was twice the recommended exposure time;
hence, the increases in UDS may have been related to
the cytotoxicity of the S9 fraction.
Several agents that were also demonstrated to induce
strong responses in this study, i.e., Diquat, 2,4-D
fluid, and carbaryl, have been reported elsewhere . to.
be nongenotoxic or weakly genotoxic.
5. The results were not confirmed.
6. The slides were not reported as being coded to
eliminate bias.
4.
but
363-410.
B-10
-------
inconclusive presumptive genotoxins' for SV-40 transformed human
cells. ..,.,;
'» W i*r .
-"»
b. Inhibition of Spindle Apparatus
Markaryan (1966) reported that anaphase and early telophase
bone marrow cells harvested from male mice exposed to 0.0052 mg/g
heptachlor for 21 hours had increased frequencies of "nuclear
disturbances, total chromosome rearrangements (chromosome and
chromatid brides and fragments), and adhesions." Based on ,
standard procedures practiced by experts in this assay, however;,
the study should be considered inconclusive as a positive response
for the following reasons: . ? «,
(1) The method used to prepare anaphase and telophase - ...-
plates (slices of bone marrow stained with acetocar-
mine) has a tendency, to increase the number of arti-
facts and lends itself to a planar effect (causing
chromosomes to be excluded from a field being viewed);
this makes it difficult to distinguish between true
chromosomal rearrangements and artifacts.
(2) The number of animals investigated (three male mice)
is an inadequate sample population.
(3) The inclusion of cytotoxic effects (vacuolation,
karyopyknosis, and karyolysis) in the total percent
nuclear disturbances is an indication that severe
cytotoxicity occurred at the assay dose, which was
stated to be only approximately 4 percent of the LD5Q.
(4) The authors did not report that the slides were coded.
Since the study results cannot be properly interpreted, the study
is considered inconclusive, but presumed positive until refuted
in an adequate assay.
c. Conclusions for Other Genetic Mechanisms
Published studies that evaluated the potential of heptachlor
to interact with and damage DNA provides evidence that the test
material appears to be inactive in rat, mouse, or hamster primary
hepatocyte cultures. Based on a single inconclusive and unconfirmed
positive in VA-4 cells, both heptachlor and heptachlor epoxide
are listed, with reservations, as presumptively genotoxic in
human cells. The results of the spindle mechanism/inhibition;
assay performed in mouse bone marrow cells with heptachlor coujld
not be interpreted. .--.. ;
B-ll
-------
4. Epigenetic Effects
''" Telang et al. (1982) evaluated six concentrations of heptachlor
(10~4 to 10~7 M) for the potential to inhibit intercellular
communication in mixed cultures of ARL (HGPRT~) 6-TGr mutant and
normal ARL (HGPRT+) cells. In a preliminary dose-selection test,
heptachlor was cytotoxic at 10~4 for ARL (HGPRT~) cells. In the
presence of HGPRT+ cells, 1 x 10~6 to 1 x 10~7 M heptachlor markedly
inhibited the metabolic cooperation between the HGPRT-deficient
and HGPRTcompetent cells. At 1 x 10~6 M, an almost complete
blockage of phosphoribosylated TG transfer between the cells was
observed as indicated by the 140 percent and 95 percent recovery
Of 6-TGr mutants cocultivated with 0.75 and 1.25 x 106 HGPRT+
cells as compared to the controls. Increased survival of 6-TGr
mutants was recorded at the remaining doses.
The specificity of the assay to detect only agents which
interfere with cell-to-cell communication in contrast to chemicals
which induce a genotoxic effect was demonstrated with the lipophilic
procarcinogen/promutagen B[a]P. Four doses of B[a]P up to a
cytotoxic dose did not increase the recovery of TGr colonies.
To support the conclusion that heptachlor interferes with
intercellular communication, the authors also evaluated the
effects of heptachlor on guanine metabolism of ARL cells, dosed
at both the cytotoxic level and at those concentrations that
profoundly affected cell-to-cell communication in the primary
assay (10~4-10~6 M). Heptachlor did not cause appreciable reductions
of [l4C]guanine conversion to its principal metabolites, and thus
such conversion is apparently not related to intercellular
communication.
5. Transformation Assays
No studies assaying the potential for heptachlor or heptachlor
epoxide to induce neoplastic transformation in mammalian cells
were located.
Overall Conclusions/Recommendations
Although adequate studies are still required in some areas,
the evaluation of the available data in published literature on
heptachlor and its epoxide indicates that neither directly inter-
feres with DNA or chromosomes, i.e., not mutagenic in the sense
of initiating genetic effects likely to be transmitted. Thus,
sufficient evidence exists to conclude that neither possesses
mutagenic activities in bacteria but further testing is needed to
confirm the negative result for heptachlor in other than the
single mammalian cell system tested, as well as to assay the
B-12
-------
epoxide in at least one sensitive test system. Additional studies
are also necessary to resolve presumptively positive mutagenic
activities under specialized conditions, e.g., in plant systems.
At least two adequate germinal assays indicate that neither
organochlorine poses serious risks of transmissible chromosomal
aberrations, but the positive results in inadequate somatic
chromosomal studies mandate further testing for this endpoint.
Finally, while confirmation of the negative results for DNA
damage/repair in microbial and mammalian cells is available,
comparable assays with the epoxide are recommended to assess its
potential, especially for UDS in mammalian systems.
On the other hand, consistent with genetic toxicology
assessments prepared for other members of this class (e.g^,
aldrin/dieldrin, lindane), these organochlorines probably act by
epigenetic mechanisms likely to promote/sustain cellular processes
initiated by other agencies, thus indirectly affecting primary
genetic mechanisms. Such activity is indicated by inadequate but
presumptively positive unconfirmed studies for mitotic spindle
effects, inhibition of both cell-to-cell communication and DNA
synthesis, as well as the apparent potentiation of repair processes
in transformed (i.e., initiated) mammalian cells.
In order to affirm the absence of a potential for direct
genotoxic activity, and to complete regulatory requirements, the
following additional testing is required:
1. Mammalian cell gene mutation assay with established test
systems, e.g. mouse lymphoma (L5178Y/TK), or Chinese
hamster (CHO/V79/HGPRT) cells inter alia, specifically
comparing activation systems (S9) derived from rat vs.
mouse (or hamster) liver microsomes; and
2. Somatic cell cytogenetic assays, either in vitro or jji
vivo.
B-13
-------
Further, to confirm the potential activity of these chemicals
in indirect (epigenetic) processes2'5 the following are recommended:
1. Adequately controlled promotion assays, e.g., in cell
lines already initiated (by viral transformation), or
exposed to known active chemical initiators.
2. Mammalian cell transformation in systems with an
established data base, e.g., C3H 10 Tl/2, BALE 3T3,
inter alia.
3. Assays for mitotic spindle effects (in vitro or jln.
vivo), and/or involving other cellular mechanisms
(e.g. , oncogene activation), inter alia.
4. Assays which can distinguish effects on replicative
S-phase (scheduled) DNA synthesis from UDS, e.g., in
primary hepatocytes from several species (mouse vs.
rat/hamster) .
2 Malcolm, A.R.; Mills, L.J.; McKenna, E.J. Inhibition of
metabolic cooperation between Chinese hamster V79 cells by
tumor promoters and other chemicals. In: Cellular Systems for
Toxicity Testing, eds. Williams, G.M. ; Dunkel, V.C.; Ray, V.A. ;
Ann., N.Y. Acad. Sci . 407 ( 1983 ); 448-450.
3 Williams, G.M. Classification of genotoxic and epigenetic
hepatocarcinogens using liver culture assays. Ann. N.Y. Acad.
Sci. -349(1980) ; 272-282.
4 Williams, G.M. Genotoxic and epigenetic carcinogens: Their
identification and significance, Ann. , N.Y. Acad. Sci_ 407(l9bJ).
328-333.
5 Weisburger, J.H; Williams, G.M. Bioassay of carcinogens: In
vitro and in vivo in Chemical Carcinogens, Vol. 2, Univ. of
Bl^mTngham7~uirA₯. Chem. Soc. Monograph 192 (1984) pp 1323-1373.
B-14
-------
»J
H
£
EH
2EI
EH
CO
B
(J)
e
e
o
0
o
H
4J
CO IB
EH 3
rH
(8
H
V
, 01
E- >.
>,
c
o>
IH
O
01
^
HI
>
3
co
01
XI
3
a
0>
o
o
IB
B
°
2
4J
IB
cn
oi
z
B
r-f
IB
JJ
01 ^^
m
(0 CO
>, cn
-H TT
Ll
o
X
cn
o to
4-> 1
r- +
o"
O
wlo
=1
c
01
01
u
01
^,
01
>
3
CO
01
3
(8
JJ
CU
§
IB
B
D
g
H
OJ
cn
z
^
,-H
IB
JJ
D ~
4J CO
01 cn
XI .-
O
0.
,
O
o
o
0*
cn
i co
- i
X
JJ
IB
E d)
(11 01
Q) O
tl "O
O
01 ai
B
>i 0
(U
Ll fH
3 B
CO O
11
XI
as
4-1
8-
§
B
D
>
H
JJ
(8
en
01
z
rH
IB
JJ
dl
^^
3 vO
w r~
nj cn
iH ^*
_f*
CO
o
01 >,
'O C
^~ o
o
B
H O
H >,
01 r-t
O B
CU O
01
.*
(0
3
i-H
O
B
O
O
E
H
rH >1
10 rH
0 O E
o «- o cn
E > CO
H -H 4J cn i
D1 IB CO
01 01 +01
> co >
'H * a\ . -H
4J 0 < rH 4J
H 0 EH 6 IB
O < E Cn 01
ft. EH -H 3 Z
E
O
H
JJ
(B
(8
Oj
01
Li
a.
rH
IB
H
O
g
8
o
01
XI
2
81
8
IB
B
a
g
4J
IB
cn
0)
Z
^
IB
4-1
01
f~.
01 .
E rH .,* IB
O "8 ,E 01
._i .-, a si
C 4J 3! Ll >J<
o
01
4J
IB
JJ
0!
B
3
4J
(B
a
ai 01
JJ 01
01 01
0) O
4J -0
JJ
18
ei
01
jj
O 01
§8
t< -a
o cy
> E
H O
4J
H >1
8-3
CU O
01
H
III
3
rH
O
§
O
B
w
C
H
JJ
0) (8
4J m en
H in 3
8 < o
0. EH r-
jj
o >
o
01 01 cn
0) JJ co
§18
4-> 1
Q 01 \
>,
rH
0-0
E
a co
W 01 i-
O 4J
01 IB E
O 4J IH
Q 01 O
o
x|
s
o
H
JJ
IB -a
14 01
IB JJ
a, IB
01 4J
01 3
rH
(B 4J
H IB
0
Ll 13
01 01 01
E 01 01
O 01 O
O 4J -o
01
rH
XI
&
0
o
18
B
a
§1
H
4J
(B
en
01
z
+
>,
= 4J ^ C
rH O -O O
IB C 01
-J 4J CO
O 01 Ll r-
Ll 01 O
01 01 CH B
e o 01 LI
E a LI c
O " O
o
0)
rH
XI
3
s-
o
o
01
H
4J
IB
0) O
0} *iH
O X
o o
o'&
o
Cn o 01
0) - 18
z ^ s
-a
B
IB
J^
o
r-i
H
a
E
(B
^
<
0)
E
0
01
^»
u
X
01
a
o
o
^^
al r-
D cn
E
IB
Cn
3|cn
CO
o
O 01
O 01
Q
a e
c
IB 1
- (-
c
3
JJ
co
4J 31
(fl
o
EH
Ll
01
>
K
tl
1'
JJ
o
IB
XI
>
JJ
^;
w
col i
~
"*
,l
51
B-15
-------
o
o <
o
Ql M 0)
u o ci
CH
ce a
o a)
IB o o
OCA
O O 3
C -H r-(
3 4J O
H M
a) >o c
Z 4J
JJ O - .C
o en - 10
i|
= 1
-o c
T- Ol HI
. 01 O E
t --- 4J 0)
= 1
al)
U
O 10 t/3| E O
0) O
^j
cl nl
O (0 -P
H > « Q4
X 0) J 0
Q) 0) Ul U
co o a
B-16
-------
a
EH
_3
M
W
6-
«C
EH
|3
4J
C
E
e
o
cj
c
o
H
4J
m io
E-i 3
CO
^
u
a
0) 0!
4-1 4J
iJ iH
0 3
CM 01
01 01
OS OS
c
o
r-4
4-1
4J
H
U
u
O 01
C O1
o c
Oi 1 10
\1 10
01 IH
E 4J E
C Cu
v a.
CJ 0 -
a c 01
O 0 3
Q U
\ e
01 11
04 4J
>, in
CO
13 4J
3 01
4J CJ)
CO EH
^
O 1-
-> o
o 3
C rJ
O (0
0 0
1
1
1
01
iH
XI
10
4J
04
01
0
o
"*
4->
CO
01
H
CO
DI
01
z
1 1
CO
4J
01
o
l-H
o
c
*
f ,
m
(-»
.,
Lfl
CN
ft]
X
X
**
o
in
3
O
g
4J
C
01
o
o
o:
1
o
_
^)
C^
CO t!
C E
O .-0
.-, Q
4-1
1
1
1
01
rH
10
Qi
0>
0
o
<
o
4J
a,
3 01
in
01 O
H
4J 0
(0 1
DI X
0) O
Z 4->
10
4-1
01
C CN
.-4 r-
0) Ol
4J ,-
in
Cd
00
XI O
x *~
i 73
^ C
' CN CO
CO iH
>- T3 in CO
C X U
co in o
i'l
1
1
1
0)
1-1
CO
4J
Oi
o
o
o
Qj
3 0)
> T3
H
4J CJ
10 -H
Oi X
01 O
Z 4J
c
10 -~
1,0
X rH
,- . 10
~ . o
10 -H
- CD
O X
n m
u
X
o
(0
4J
JS
Ifl
c
(O
01
CO
o
0>
4J
i-)
a^,
(-H
0) C
OS 0
01
H
01
3
rH
O
c
o
o
c
iH
in
O 01
U-l C
a o
01 01 -H
> 01 4J
H (0 Qi
4-1 01 IJ
H ^4 O
01 O 01
O C 01
CU -H X4
f
cfl
O ro
>i Ol
01 ^~
ij '
01
0
1
V4 10
01 M
> 01
m o c
01
c c
Ifl -H CI 01
T3 01 C
,- 01 V4 0
01 .C -H
y-i 4-» 4J
i'l
4-1
o
CO
4J
JS
CO
c
CO
01
CO
a
0)
M *
o >,
Pi r 1
01 C
OS O
01
.H
0)
3
r-4
O
S
0
CJ
c
tH
s
i in
XI -H
V4 Ifl 01
0=0
UH 4-1
T3 -H
0) 01 S
> 01
H CO rH
4-> 01 10
01 U M
O C O
Qj -H C
CN
Cl4
- C
m -H
*O Oi
C c n
CO -H fc,
a
>- 01 T5
01 C
UJ CO
i'i
c
H
01
S 0
o --^
.W 4J
Ij 01
CO C
E 01
01 O 01
C 4J O
m O"'E=
D>
C 113
H 1 01
M -H -a
i* o" IH
CO tH O
01 O C
4J -H
4-1 O 01
CO 4J
H > O
^4 01 01
frg-a
>J -H 01
a< c
CO U CO
C 01 .H
M 4-> CJ
01
H
01
3
iH
O
C
0
o
c
=
4-1 O
co m
a
^
H H-l
4J 0
in *>
O ^1-
04 =
c
CO ~
>i VO
co cn
^4 " *
S
Oi
CM .
in .H
0
o =
O Di
= \
Ol
E
i'l
c
01
01
Jj
o
01
^
01
^
3
CO
01
rH
XI
10
4-1
04
01
o
o
cfl
c
D
C
H 01
4-1
01 01
> 01
H 4J
4-1
CO 4-1
DI O
01 Q4
Z 01
rH
Ifl
4J
01
3 ^O
01 r-
(0 CTl
V4 ^
H 1vr
CO
^
0
in >,
H iH
a c
CM CTl
co
i
i'l
rH -^
CO 0
H c ml 01
4J .H >H
c
01 >, CO 01
it 4J .H .H
01 -H >J rH
UJ O 01 -rH
14-1 -H 4J 4J
U-t X O XI
H O Cfl 3
Q 4J XI 01
V.
o<
DI
Ifl 1-4
E -H
co ifl
Q 04
01
< cs
z
Q
2
01
U
J
01
^
u
^
3
CO
01
rH
S
*^.
u
o
o
CO
c
0)
c
o
4J 13
CO 01
4-1
01 01
H 4J
4->
(0 01
Di 01
01 O
Z 13
^
H
X
C CO
U-l Ol
U-4 T
i-4 *^
U
g
^s^
D!
3 1
O
=C|
1 0
01 14
CO tH
rH *-^
04 *- 01 1-1 O lH
01 Ji 01 ^ O
CO 4J CJ
<: o> o IQ
Z >H CO -H
Q XI XI E Id
B-17
-------
ca <8
w
a
o
C. Q C)
0>
o<
(8 4J
18 -! bl
03 -» O 6
, -- .rf jj -a o
i'l
a,
I
> c
O 01
'O 111
Q< IM
a) in >,
I 0)
o in
01 -P O
P Q) <
3 C 4J
O nj ,H
3 - 10 C
H d
o -e 5)
u - c
C -O -^
a o 3 >
I V 3 XI O
o o e -H ij
S 15 0) O -El
IB I* O -H C J 2i
4J Q) i *J O 2 M
, a) a o m i < o
E O 4J O 4-> ==
s s. o
0 O
X 0)
U E
-------
APPENDIX I
B-19
-------
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
OFFICE OF
PESTICIDES AND TOXIC SUBSTANCES
MEMORANDUM
SUBJECT: Chlordane/Heptachlor Mutagenicity
FROM: Irving Mauer, Ph.D., Geneticist
Toxicology Branch
Hazard Evaluation Division (TS-769C)
TO:
THRU:
Henry Spencer, Ph.D., Pharmacologist
Section 7, Toxicology Branch
Hazard Evaluation Division (TS-769C)
Jane E. Harris, Ph.D.
Section Head, Toxicology Branch
Hazard Evaluation Division (TS-769C)
This is in response to your request for an expeditious
appraisal of the positive mutagenicity studies for chlordane
and heptachlor/heptachlor epoxide, as reported in the GAG
document ("Carcinogenicity Risk Assessment for Chlordane and
Heptachlor/Heptachlor Epoxide"). The negative reports listed
in that document were also scanned.
Based upon recent Velsicol submissions (Accession Nos.
254320 and 254324) and other information available to me (NTP,
EMIC), two general conclusions are warranted from this preliminary
assessment (see below for a summary of available data):
1. Although the adequacy of the data base as reported
could not be undertaken (e.g., DER's) because of the
timely response requested, it appears that all the
genetic end-points we require to be assayed (gene
mutation, chromosomal aberrations, DNA damage/repair)
have been addressed.
B-20
-------
2. Consistent with the results of mutagenicity testing
previously reported for other agents of this chemical
class (organochlorines such as Lindane, chloroform,
inter alia), the mutagenic potential of these chemicals
are low to unsubstantial. Hence, from the available
data it would appear their oncogenic potential derives
from mechanisms other than "genetic initiation" (direct
interaction with DNA). [There is suggestive evidence
in mammalian systems (the few promotion assays available)
of an "epigenetic" mode of action for these chemicals.]
Survey of Available Literature
1.
Chlordane-technical (but not reagent-grade alpha-
chlordane, gamma-chlordane, and gamma-chlordene) was"
positive in adequate bacterial (Ames) assays (three
positive, one negative), but consistently so only in
the most sensitive strain (TA-100), and only at very
high concentrations (5000 ug_/plate and above). Testing
for mutagenicity in mammalian cells in culture (four
reports) revealed inconsistent results (two positive,
two negative), due in part to the different test systems
employed. The.two negatives were reported for HGPRT
in ARL (adult rat liver) and for both thioguanine and
diphtheria-toxin resistance in V79 (Chinese hamster
lung) cells. One of the positives was for ouabain
resistance in V79 cells, but reported for only a single
concentration of an unstated formulation, at less, than
50 percent cell survival; the second was found in non-
activated mouse lymphoma cells (L5178Y-TK.) , but the
study is incomplete since metabolic activation was not
employed.
The plant systems may activate chlordane to mutagenic
derivatives is suggested by a single article reporting
both gene conversion in Saccharomyces cerevisiae D4
cultures exposed to a reagent grade (presumably alpha-
chlordane according to the CAS Number stated), and
reversion to wild type in pollen grains from the
homozygous "waxy" strain W22 of Zea mays (corn) exposed
to a "commercial-grade formulation." However, too few
procedural details were reported to adequately interpret
these results.
Although reportedly negative for gross chromosomal
damage in vivo (two mouse DLT' s) and ir± v i t rg (CHO
cells), technical chlordane or one of its principal
components (alpha-chlordane) apparently has DNA-
damaging activity, as revealed in a single study
reporting increased dose-dependent sister-chromatid
exchanges (SCE) in intestinal cells sampled from
B-21
-------
exposed central mudminnows (Umbra limi)/ as well as
in CHO cells in culture and in a human lymphoid cell
line (one report each). Whereas the positive SCE in
fish was recorded at nonlethal concentrations (added
to aquarium water), both in vitro studies were only
marginally positive (less than twofold above controls)
at toxic concentrations. Only one of the four studies
assaying for unscheduled DNA synthesis (UDS) in vitro
was positive (in VA-4 cells, an SV-transformed human
cell line), but only in the absence of metabolic
activation (negative with MA). Technical chlordane
was negative for UDS in primary hepatocyte cultures
from rats, mice, and hamsters (two reports) as well
as in a human fibroblast cell line (D-550). Finally,
gene conversion was reported in yeast cells
(S. cerevisiae D4) exposed to an activation system
(negative without), but too few data are included in
the article to properly interpret the results.
2. Heptachlor/Heptachlor Epoxide. In contrast to the
positive results for (gene) mutagenicity reported for
chlordane (both technical and reagent grades), the
available data for heptachlor and its epoxide is
consistently negative (three Ames and/or E_. coli, one
IB. subtilis rec assay, 1 ARL-HGPRT, two Drosophila
SLRL, 1 HPC/UDS). Reversions to histidine prototrophy
in Salmonella TA-1535 and 100 under activation conditions
to an unstated dose range, as well as to the wild-type
(nonwaxy) phenotype in corn pollen grains were reported
by the same investigator who found "commercial" chlordane
positive.
Adequate reports on dominant-lethal assays in mice
were negative, but an abstract from a meeting reported
positive results for both germinal (DLT) and somatic
(bone marrow) chromosome damage in rats fed 1 and 5
ppm of an unstated formulation of heptachlor for
three generations. A Russian study also reported
positive chromosome damage in bone marrow cells from
"white male mice" treated i.p. with "heptachlor"
(also of unstated source and purity) at a single dose
level stated to be "4% of the LD50." Too few procedural
details are included in these "positive" studies to
interpret the results reported.
As with chlordane, negative UDS results have been
reported for heptachlor technical in primary rodent
hepatocytes (two reports), but a positive recorded in
VA-4 cells (virus-transformed human fibroblasts) for
both the technical and epoxide, but only with metabolic
B-22
-------
activation (in contrast to chlordane, positive only
in the absence of activation). [The mutagenicity
data base for heptachlor epoxide is less than adequate
to satisfy FIFRA guidelines.]
Discussion
Recent studies have suggested that organochlorines
(Lindane, chlordane, heptachlor, inter alia) do not interact
directly with DNA (i.e., are not "genotoxic"), but rather act
"epigenetically" by mechanisms affecting cell membrane
permeability and/or following an irreversible "initiating
event" (i.e., are "promoters" ensuring survival of preexisting
transformed cells). The following citations employing chlordane
heptachlor available for this "quick-and-dirty" review (and
listed in the CAG document) are consistent with this suggestion:
1.
Inhibition of metabolic cooperation in miked cultures
consisting of thioguanine-resistant and TG-sensitive
cells (several reports from both Williams'
labs).
and Trosko's
2.
3.
The putative positive UDS results in cells already
"initiated" (e.g., the SV-40 transformed cell line,
VA-4).
Inhibition of DNA synthesis and/or cell cycle mechanisms
by severely toxic concentrations leading to .perturbation
of repair (increased UDS, SCE).
cc: Dr. Amy Rispin
Science Intergration Staff
Hazard Evaluation Division (TS-769C)
B-23
-------
PRELIMINARY EVALUATION OF REPORTED POSITIVE STUDIES
Compound
CHUHOANE
Test
Material
Technical
Technical
Technical
Reagent
(alpha)
"Cctimerclal"
(Unstated)
(Unstated)
Technical
Reagent
(alpha)
(Unstated)
Reagent
(gamma)
Reagent
(alpha)
Reagent
Assay
Gene Mutation
Gene Mutation
Gene Mutation
Gene Mutation
Gene Mutation
Ouabaln
resist.
Gene Mutation
In vivo SCE
In vitro SCE
In vitro UOS
Cell cycle
Inhlb.
In vitro CA/
SCE
[Gene Mutation
Test
System
Ames (all)
Ames (all)
Ames (TA 98,
100)
Sacch. - 04
Z. mays
Po 1 1 en
V79 cells
ARL/HGPRT
Mudmlnnow
LAZ-007 eel Is
VA-4 ce M s
L5178Y cells
CHO eel Is
1 L5178Y/TK
Dose/Cone.
Range
Reference | Evaluation
1
20- 1 00 | S 1 mmon et al. | ACCEPT.
(77)
Comments
POS. only In TA 100
a) 10-5000 | Simmon and ACCEPT. |a> EQUIV In TA 98
b) 1000-50, OOOJ Tanaka (77) |b> POS. In TA 100
5-10,000
(Unstated)
(Unstated)
(Only 1 dose
reported )
10"3-10"6 M
-12 -9
5.4/10 -10
10"6-10"3 M
Maruyama (80) ACCEPT. |POS. In TA 100; NEG.
| In TA 98
i
|Gentl le et al.
(82)
1
| Ibid
1
1
| Ahmed et al.
| (77b)
|Telang et al .
I (82)
1
Ivigfusson et al .
| (83)
|Sobtl et al.
| (83)
I
1 , 10 , 100 , 1 000 | Ahmed jt al.
uM | (77a)
I
4_ug/ml [Brubaker et al.
| (70)
1
(Unstated)
(Multiple)
|NTP (85)
1
1
INTP (85)
INCONCL.
INCONCL.
1 NCONCL.
ACCEPT.
ACCEPT.
INCONCL.
ACCEPT.
INCONCL.
INCONCL.
INCONCL.
POS. only + S9; too
few data
1
[Tech not tested; too
| few data
1
|"Weak» at < 50)5
| survival; few data
I
1
INEG. for HGPRT, but
j POS. for promotion
loose-dependent POS.
< 2-fold Increase;
no dose response
POS. only without S9
NEG. for DNA, POS. for
G2 arrest; only 1 dose
|NEG. for CA; POS. tor
SCE
1
(Not tested with S9
B-24
-------
PRELIMINARY EVALUATION OF REPORTED POSITIVE STUDIES (cont'd)
Compound
HEPTACHLOR
1
1
1
Test
Material
| Technical
(Unstated)
(Unstated)
Epoxlde
(Unstated)
Assay
[Gene Mutation
Gene Mutation
in vitro UDS
In vitro UOS
in vivo CA
(Unstated) JDLT/BM-CA
Tecnnica 1
1
1
in vitro CA/
SCE
Test
System
| Ames (all)
ARL/HGPRT
VA-4 eel Is
VA-4 eel Is
Mouse BM
Rat rep ro.
study
CHO eel Is
Dose/Cone.
Range
(Unstated)
10~7 to 10~4 M
100,1000 uM
10,100,1000 uM
5 mg/kg
1 , 5 ppm
(Multiple)
1 TB
Reference (Evaluation
Genti le et al .
(82)
Telang et a 1 .
(82)
Ahmed et a 1 .
aiat
ibid.
Markarjan (66)
Cerey <74)
NTP (85)
INCONCL.
ACCEPT.
1 NCONCL
INCONCL.
1 NCONCL
INCONCL.
1 NCONCL .
Comments "'-
POS. In TA 1535/100,
Out only witn S9
NEG. for HGPRT; POS.
for promotion
no va 1 ues for UDS
POS. only with S9 Out
no values for UOS
detai Is; 1 dose
[Abstract!
POS for both CA and
SCe, but too few
detai 1 s aval labl e
B-25
-------
APPENDIX II
B-26
-------
REFERENCES
*F;E"" Hartf R'W-; Lewis' N-J- d977) Pesticide induced
DNA damage and its repair in cultured human cells. Mutation
Res. 42:161-174.
Arnold, D.W. ; Kennedy, G.L.; Keplinger, M.L.; Calandra, J.C.;
Calo, C.J. (1977) Dominant lethal studies with technical
chlordane, HCS-3260, and heptachlor: heptachlor epoxide. J.
Toxicol. Environ. Hlth. 2:547-555.
Benes, V.; Sram, R. (1969) Mutagenic activity of some pesticides
in Drosophila melanogaster. Indust. Med. 38:442-444.
Cerey, K.; Izakovic, V.; Ruttkay-Nedecka, J. (1973) Effect of
heptachlor on dominant lethality and bone marrow in rats
(Abstract No. 10). Mutation Res. 21:26.
Epstein, S.S.; Arnold, E.; Andrea, J.; Bass, W.; Bishop, Y.
(1972) Detection of chemical mutagens by the dominant lethal
assay in the mouse. Toxicol. Appl. Pharmacol. 23:288-325.
Gentile, J.M.; Gentile, G.J.; Bultman, J.; Sechriest, R.; Wagner,
E.D.; Plewa, M.J. (1982) An evaluation of the genotoxic
properties of insecticides following plant and animal
activation. Mutation Res. 101:19-29.
Glatt, H.; Jung, R.; Oesch, F. (1983) Bacterial mutagenicity
investigation of epoxides: drugs, drug metabolites, steroids,
and pesticides. Mutation Res. 11:99-118.
Griffin III, D.E.,; Hill, W.E. (1978) In vitro breakage of
plasmid DNA by mutagens and pesticides. Mutation Res.
52:161-169.
Markaryan, D.S. (1966) Cytogenetic effect of some chlororganic
insecticides on mouse bone marrow cell nuclei. Genetika
.2:132-137.
Marshall, T.C.; Dorough, H.W.; Swim, H.E. (1976) Screening of
pesticides for mutagenic potential using Salmonella typhimurium
mutants. J. Agric. Food Chem. 24:560-563.
Maslansky, C.J.; Williams, G.M. (1981) Evidence for an epigenetic
mode of action in organochlorine pesticide hepatocarcinogenicity:
A lack of genotoxicity in rat, mouse, and hamster hepatocytes.
J. Toxicol. Environ. Hlth. 8_:l21-130.
Moriya, M.; Ohta, T.; Watanabe, K.; Miyazawa, T.; Kato, K.;
Shirasu, Y. (1983) Further mutagenicity studies on pesticides
in bacterial reversion assay systems. Mutation Res. 16:185-216.
B-27
-------
National Toxicology Program (1983) NTP mutagenesis (Salmonella
typhimurium) test results: Heptachlor (No. 59). NTP Bulletin
No. 9 (April 1983), p. 6 (Table 3).
Probst, G.S.; McMahon, R.E.; Hill, L.E.; Thompson, C.Z.; Epp,
J.K.; Neal, S.B. (1981) Chemically-induced unscheduled DNA
synthesis in primary rat hepatocyte cultures: A comparison
with bacterial mutagenicity using 218 compounds. Environ.
Mutag. 3:11-32.
Seiler, J.P. (1977) Inhibition of testicular DNA synthesis by
chemical mutagens and carcinogens: Preliminary results in
the validation of a novel short-term test. Mutation Res.
46:305-310.
Shirasu, Y.M.; Moriya, K. Kato, Furuhashi, A.; Kada, T. (1976)
Mutagenicity screening of pesticides in the microbial system.
Mutation Res. 40:19-30.
Telang, S.; Tong, C.; Williams, G.M. (1982) Epigenetic membrane
effects of a possible tumor promoting type on cultured liver
cells by the nongenotoxic organochlorine pesticides chlordane
and heptachlor. Carcinogenesis 3:1175-1178.
Van Dijck, P.; Van de Voorde, H. (1976) Mutagenicity versus
carcinogenicity of organochlorine insecticides. Meded. Fak.
Landbouwwet. R.U. Gent. 41:1491-1498.
B-28 *U.S. GOVERNMENT PRINTING OFF ICEj-1987 -748-121 <40727
-------
-------
CO
- c
.E
O T3
UJ T3
X W
(J 0)
Is
Q. c
0) t
1"
s s ^
(U O Q)
» U ^ 3 O £
: § j; a: -S 3
o
CD
co
o
o
to
Q-
UJ
8
a"
m
0)
o ffl
*S m
OrS
------- |