ERA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA 600 1 78 060
September 1 9/8
Research and Development
Toxaphene
Composition and
Toxicology
Lir? -
U.S. L.v '
'. :..:-» AGENCY
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4 Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-78-060
September 1978
TOXAPHENE COMPOSITION AND TOXICOLOGY
by
John E. Casida and Mahmoud A. Saleh
Pesticide Chemistry and Toxicology Laboratory
Department of Entomological Sciences
University of California, Berkeley, California 94720
Grant No. R-803913
Project Officer
Ronald L. Baron
Environmental Toxicology Division
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
LIB,
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products constitute endorsement or recommendation for use.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of existing and new man-made environmental hazards is necessary
for the establishment of sound regulatory policy. These regulations
serve to enhance the quality of our environment in order to promote the
public health and welfare and the productive capacity of our Nation's
population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.
These studies address problems in air pollution, non-ionizing
radiation, environmental carcinogenesis and the toxicology of pesticides
as well as other chemical pollutants. The Laboratory participates in
the development and revision of air quality criteria documents on
pollutants for which national ambient air quality standards exist or
are proposed, provides the data for registration of new pesticides or
proposed suspension of those already in use, conducts research on
hazardous and toxic materials, and is primarily responsible for providing
the health basis for non-ionizing radiation standards. Direct support
to the regulatory function of the Agency is provided in the form of
expert testimony and preparation of affidavits as well as expert advice
to the Administrator to assure the adequacy of health care and surveillance
of persons having suffered imminent and substantial endangerment of
their health.
As part of its overall mission, the U.S. Environmental Protection Agency
is concerned with the effects of pesticides on mammals including man. One
area of specific concern is the composition and toxicology of toxaphene. The
following report deals with analytical methodology for determining toxaphene
composition, preparation and identification of major toxaphene components
including those most toxic on an acute basis, and the metabolic fate and
mutagenic activity of certain toxaphene components.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
iii
\
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PREFACE
The Federal Insecticide, Fungicide, and Rodenticide Act designates
the Environmental Protection Agency as the governmental body responsible
for the safety of all pesticides used in the United States. More recently,
the Federal Environmental Pesticide Control Act (PL 92-516) strengthened
EPA's regulatory responsibilities in the area of pesticides to include intra-
as well as inter-state commerce.
To be federally registered, a pesticide must be determined to not be
hazardous to health or to the environment when used according to its labeling
restrictions. Thus, relative to the new law as well as to specific directives
included in Public Law 93-1355 1973, EPA now is conducting a thorough review
of the implications of using alternate chemicals for pest control, including
older registered pesticides. The University of California at Berkeley is
contributing to these goals through studies on toxaphene composition and
toxicology. This report summarizes and discusses the findings on toxaphene.
IV
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2,5-endo>,6-exp,8,9,lO-hexachlorotiornan-2,3-ene in the triphenyltin hydride
and reduced hematin systems and in each of the organisms examined. Reduced
hematin and the tin hydride system also convert the heptachlorobornane to
2 35-j2njk>,8,9,10-pentachlorotricyclene. Fat from chickens and mammals treated
orally with toxaphene contains products similar in GLC characteristics to
toxaphene itself whereas liver and feces contain toxaphene-derived products
of greatly altered GLC properties.
Toxaphene preparations and related chlorinated terpenes are mutagens
in the histidine-requiring SalJtnonella typhimurium assay. The most potent
mutagenic components, which are not identified, reside in the polar fractions
on crystallization or column chromatography.
VI
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ABSTRACT
The composition and metabolism of toxaphene are examined, to aid in
understanding the conditions under which this major insecticide can be most
effectively and safely used. The consistency of composition of toxaphene
and related chlorinated terpenes are evaluated by open tubular column GLC
analyses with an EC detector. Each of 8 toxaphene samples manufactured by
Hercules from 19^-9 to 1975 shows the same 29 major peaks and in almost
identical ratios. About 85$ of the total peak area is accounted for by these
29 peaks which individuality vary from 1 to 8% of the total. The 8 toxaphene
samples from Hercules are easily differentiated by open tubular column GLC
from 12 samples of related chlorinated terpenes from other manufacturers in
the United States and abroad and from [ \l]- and [^ Clltoxaphene prepared by
Hercules. A more detailed analysis of toxaphene composition is provided by
open tubular column GLC of toxaphene components in each of 5 TLC regions
which are precisely defined by the use of selected fluorene marker dyes.
Despite large composition differences between some of the samples, there is
surprisingly little variation in their mouse IP and housefly topical LD,-n
values.
Five major toxaphene components [2,2,5-endo, 6 -exo,8,9?10-heptachloro-
bornane (l) and its 3-exo-chloro-, 8-chloro-, 9-chloro- and 10-chloro-
derivatives] collectively account for up to 23% of the GLC-EC properties of
chlorinated technical grade camphene (jl.e_., toxaphene insecticide) and up to
3^ of those of chlorinated 2-exo,10-dichlorobornane. Chlorination of 2-exo,
10-dichlorobornane provides a convenient source of J^, which on further
chlorination gives the indicated octachlorobornanes and the 5-exo-chloro-
derivative of ^ plus two nonachlorobornanes, one with the introduced chlorines
at C-8 and C-10 and the other with these chlorines at the 3-exo-position and
at C-10. On dehydrochlorination J^ yields two hexachlorobornenes and the 3-
exo-chloro derivative of I gives one heptachlorobornene and one hexachloro-
bornadiene. The toxicity to mice, houseflies and goldfish of the octa-
chlorobornanes formed by introducing chlorine substituents into J^ relative
to L itself, generally decreases in the order: 9-chloro > 8-chloro > no added
chlorine (i-.e_. ^} > 3-exo-chloro, 5-exo-chloro or 10-chloro.
Heptachlorobornane I undergoes reductive dechlorination at the geminal-
dichloro group to yield 2-endo,5-endo,6-exo,8,9?10-hexachlorobornane and 2-
exo,5-endo,6-exo,8,9,10-hexachlorobornane in the following systems: photolysis
in hexane solution with UV light; triphenyltin hydride in hexane containing
2,2'-azobis(2-methylpropionitrile); reduced hematin in glacial acetic acid-
N-methyl-2-pyrollidone; bovine rumen fluid; sewage primary effluent; rat
liver microsomes under anaerobic conditions with NADPH as the critical co-
factor; houseflies, chickens, guinea pigs, hamsters, rabbits, mice, rats and
monkeys in vivo. This heptachlorobornane is also dehydrochlorinated to give
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ABBREVIATIONS
CI-MS chemical ionization-mass spectrometry
DMF dimethylformamide
DMSO dimethylsulfoxide
GLC gas-liquid chromatography
GLC-CI-MS GLC coupled with CI-MS
IP intraperitoneal
lethal dose for 50$ of the animals
nuclear magnetic resonance
PB piperonyl butoxide
Rf ratio for distance moved by compound to that moved
by developing solvent on TLC. ^Ef refers to three TLC
development s
TLC thin-layer chromatography
T or tR GLC retention time
UV ultraviolet light
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ILLUSTRATIONS
1. Open tubular column GLC analysis of the toxaphene standard and
related chlorinated terpenes. The 29 peaks making up ^ 1$, of
the total peak area in the toxaphene standard are designated by
numbers as in Table 1. The same numbers designate peaks in the
other samples with identical T values to those in the toxaphene
standard. Additional peaks making up S 1% of the total peak
area in the related chlorinated terpenes are designated by
asterisks. No additional components are eluted at times later
than those indicated
Open tubular column GLC analysis of the toxaphene standard and
fractions j^,-j^ obtained from this standard by TLC. The 29 peaks
making up ^ 1% of the total peak area in the toxaphene standard
are designated by numbers as in Table 1. The same numbers
designate peaks in the TLC fractions with identical T values
to those in the toxaphene standard. Additional peaks making
up § 1% of the total peak area in the TLC fractions are
designated by asterisks. Each peak is designated only for
the TLC fraction in which it appears in maximum amount. The
first major peak in each chromatogram (T 25-9 min) is aldrin
used as an internal standard. Structures are given for
toxaphene components 8-Cl-j., plus 9-Cl-J^ (peak l6), 1 (peak 9),
and a nonachlorobornane (peak 27) (see also Table 1)
3. Conversion of camphene and 2-exo,10-dichlorobornane to 2,2,5-
endo,6-exo,8,9,10-heptachlorobornane (l) and several octa- and
nonachlorobornanes, hexa- and heptachlorobornenes, and a
hexachlorobornadiene ih
k. Chromatography of I and its chlorination products on a silicic
acid column (265 gT developed with hexane. The elution
position of two unidentified nonachlorobornanes is designated
by ? 17
5. Conversion of heptachlorobornane I to various hexachlorobornane,
hexachlorobornene, and pentachlorotricyclene derivatives. ... 30
6. Chromatography of I and its reaction products with reduced
hematin on a silicic acid column developed with hexane .... 30
x
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CONTENTS
FORWARD iii
PREFACE iv
ABSTRACT v
ABBREVIATIONS ix
LIST OF ILLUSTRATIONS x
LIST OF TABLES xli
ACKNOWLEDGMENTS xiii
INTRODUCTION 1
SECTION I. COMPOSITION OF TOXAPHENE AND RELATED CHLORINATED
TERPENES 3
MATERIALS AND METHODS 3
Samples 3
Chromatography 3
Open tubular column GLC 3
TLC and TLC-GLC k
Bioassays k
RESULTS k
Open Tubular Column GLC and TLC-GLC Analysis of Hercules
Toxaphene Standard k
Open Tubular Column GLC Analysis of Various Hercules
Toxaphene Samples 10
Open Tubular Column GLC Analysis of Related Materials 10
Other Criteria for Intercomparison of Samples 10
DISCUSSION 10
SECTION II. RELATION OF STRUCTURE TO BIOLOGICAL ACTIVITY OF
TOXAPHENE COMPONENTS 13
MATERIAL AND METHODS 13
Chromatography 13
Spectroscopy and Elemental Analyses 13
Bioassays 13
EXPERIMENTAL PROCEDURES 13
I and 3-exo-Cl-I, from Chlorination of 2-exo,10-Dichloro-
**" bornane . . T 15
Octa- and Nonachlorobornanes from Chlorination of I 15
Hexa- and Heptachlorobornenes and a Hexachlorobornadiene
from Dehydrochlorination of ^ and 3-exo-Cl-j.. .-. 18
vii
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RESULTS AND DISCUSSION 18
Preparation of Polychlorobornanes and Polychlorobornenes. . . l8
Chromatographic Properties 20
Identification of New Compounds 20
Relationship of Chemical Structure and Biological Activity. . 25
SECTION III. METABOLISM AND ENVIRONMENTAL DEGRADATION OF TOXAPHENE
AND ITS COMPONENTS 2?
MATERIALS AND METHODS 27
Analyses 2?
Chemicals 28
Reactions of Heptachlorobornane ^ 28
Photolysis 28
Triphenyltin hydride 28
Reduced hematin 28
Bovine rumen fluid 29
Sewage primary effluent 29
Rat liver microsome-NADPH system 29
In Vivo Studies 29
Treatment of chickens and mammals and analyses of their
feces and tissues 29
Treatment and analysis of houseflies 31
Bioassays 31
RESULTS AND DISCUSSION 31
Identification of New Compounds 31
Reaction Products of Heptachlorobornane ^ in Various Chemical,
Photochemical and Metabolic Systems 35
Metabolites of Heptachlorobornane ^ 35
Products Derived from Toxaphene in Fat, Liver, and Feces of
Rats 39
Products Derived from Toxaphene in Fat, Liver, and Feces of
Other Mairanals and Chickens 39
Degradation of 2,2,5-endo,6-exo,8,8,9,10~Octachlorobornane
(8-Cl-I^ by Reduced Hematin 42
Biological Activity 42
DISCUSSION 42
SECTION IV. MUTAGENIC ACTIVITY OF TOXAPHENE AND SOME OF ITS
COMPONENTS 46
MATERIALS AND METHODS 1*6
RESULTS AND DISCUSSION . . . 46
Mutagenic Activity of Toxaphene and Related Chlorinated
Terpenes 46
Other Observations 46
RECOMMENDATIONS 48
REFERENCES 49
vni
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7. Open tubular column GLC analysis of toxaphene and of toxaphene-
derived products in fat, liver and feces of rats 72 hr after
oral administration of toxaphene. The 29 arable numerals
refer to toxaphene components present in greater than 1%
amounts as designated in Section L. The chromatographic
positions of toxaphene components I (peak 9) and ®~^~jf*
plus 9-Cl-J^ (peak l6) and of metabolites j^-J^, of hepta-
chlorobornane I are designated "by structural formulae.
Letter designations (A-D) refer to toxaphene -derived products
in liver, some or all, of which may be toxaphene components.
Asterisks designate interfering materials of biological
origin
8. Open tubular column GLC analysis of toxaphene and of toxaphene-
derived products in the fat of chickens and mammals 72 hr
after oral administration of toxaphene. The 29 arabic numerals
refer to toxaphene components present in ^ 1% amounts as
designated in Section I. The chromatographic positions of
toxaphene components J, (peak 9) and 8-C1-L plus 9-Cl-^L (peak 16)
are indicated. Asterisks designate interfering materials of
biological origin
9. Open tubular column GLC analysis of toxaphene and of toxaphene-
derived products in the liver of chickens and mammals 72 hr
after oral administration of toxaphene. The 29 arabic numerals
refer to toxaphene components present in S 1% amounts as
designated in Section I. The chromatographic positions of
toxaphene components _£ (peak 9) and 8-Cl-vJ, plus 9-Cl-^L, (peak 16)
are indicated. Letter designations (a^u,) refer to toxaphene-
derived products in liver, some of which may be toxaphene
components. Asterisks designate interfering materials of
biological origin ........................ lj-3
10. Open tubular column GLC analysis of toxaphene and of toxaphene-
derived products in the feces of chickens and mammals 72 hr
after oral administration of toxaphene. The 29 arabic numerals
refer to toxaphene components present in § 1$ amounts as
designated in Section I. The chromatographic positions of
toxaphene components^ (peak 9) and 8-Cl-j^ plus 9-Cl-^J. (peak l6)
and of metabolites II-IV of heptachlorobornane Jj, are indicated.
Asterisks designate^LriUerfering materials of biological
origin ............................. 44
XI
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TABLES
1. Open Tubular Column GLC Analysis, Elemental Composition, and
Biological Activity of Various Samples of Toxaphene and Related
Chlorinated Terpenes ...................... 5
2. Products From Chlorination of Camphene, 2- exo , 10-Dichloro-
bornane and Heptachlorobornane __£ ................ 16
3. NMR Spectra of Hepta-, Octa-, and Nonaehlorobornanes , Hexa- and
Heptachlorobornenes , and a Hexachlorobornadiene ........ 21
k. Biological Activity of Heptachlorobornane^, and Related Octa-
and Nonachlorobornanes , Hexa- and Heptachlorobornenes , and
a Hexachlorobornadiene ..................... 2.6
5. Properties of Heptachlorobornane J^. and Its Reaction Products
in Various Chemical, Photochemical and Metabolic Systems .... 32
6. WMR Spectra of Heptachlorobornane ^ and Its Reaction Products
With Reduced Hematin ..................... 3^
7. Percent of Heptachlorobornane I and Its Reaction Products in
Various Chemical, PhotochemicaT and Metabolic Systems ...... 36
8. Amount of Heptachlorobornane I and Its Metabolites in Fat and
Liver at 7 and 72 Hr After Oral Administration of ^ to Rats
at 3-1 Mg/Kg .......................... 37
9. Percent of Heptachlorobornane I and Its Metabolites in Feces
Within 72 Hr After Oral Administration of J, at ~ 3 Mg/Kg .... 38
10. Mutagenic Activity of Hercules Toxaphene and Related Chlorinated
Terpenes in the TA100 Histidine-Requiring Mutant Strain of
Salmonella typhimurium ..................... 1^-7
11. Mutagenic Activity of Hercules Standard Toxaphene and Its
Fractions in the TA100 Histidine-Requiring Mutant Strain
of Salmonella typhimurium .................... k7
Xll
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ACKNOWLEDGMENTS
This study was supported in part by EPA grant R 803913 and by NIH
grant P01 ES00049 and a grant from Hercules Inc. We thank several colleagues
of this laboratory for advice and assistance as follows: Walter V. Turner
for the studies considered in Section II; Roy L. Holmstead for determinina-
tion-s of mass spectra; Judith L. Engel for performing the bioassays; Virginia
Schwan for technical assistance; Robert F. Toia for advice. Robert H. Wohleb
of J & W Scientific, Inc. (Orangeville, Calif.) assisted initially in pre-
paring the open tubular columns. Charles L. Dunn of Hercules Inc.
(Wilmington, Del.) supplied samples of toxaphene and related preparations
and of [l^C]- and P°C1]toxaphene. James N. Seiber of the Department of
Environmental Toxicology (University of California, Davis, Calif.) provided
the sample of a nonachlorobornane toxaphene component (referred to as peak
27). Herbert M. Moorefield of Union Carbide Corp. (South Charleston, W. Va.)
supplied the fluorene derivatives. The 3&0 MHz NMR spectra were obtained
at the Stanford Magnetic Resonance Laboratory (Stanford University, Stanford,
Calif.) by arrangement with W. W. Conover and as supported by NSF grant GR
23633 and NIH grant RR 00711. X-ray crystallography analyses were performed
by Rosalind Y. Wong (Western Regional Research Center, U. S. Department of
Agriculture, Albany, Calif.). Mutagenic studies were carried out in coopera-
tion with N. Kim Hooper and Bruce M. Ames of the Department of Biochemistry
(University of California, Berkeley).
XI11
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INTRODUCTION
Toxaphene is produced by chlorination of camphene to yield a chlorine
content of 67-69$ and an overall composition approximating C^QHiQClg
(Buntin, 1951). In contrast to most pesticides, toxaphene is not a single
major chemical entity but rather is a complex mixture of related chlorinated
terpenes. About 60-70 million pounds of this insecticide are used each year,
in large part combined with methyl parathion for cotton pest insect control.
The cumulative use of toxaphene since it was introduced by Hercules Inc. in
the late 19^4-0's exceeds one billion pounds. Several other companies in the
United States and abroad have produced and marketed similar insecticides
prepared by chlorination of camphene and related terpenes. Knowledge of the
metabolic and environmental fate of toxaphene has developed rapidly in recent
years with associated advances in identification of components and improve-
ments in analytical procedures.
The purpose of the present investigations is to define the composition,
structure-activity relations, and the metabolic and environmental fate of
toxaphene. The studies are described in four sections as indicated below.
Section I focuses on the composition of toxaphene and related
chlorinated terpenes. Food and feed containing residues of toxaphene and
related materials are regulated on the basis of tolerances derived from
analytical data using methods developed for toxaphene (Guyer et al., 1971;
Zweig and Sherma, 1972) and from dietary no-effect levels in chronic feeding
studies with toxaphene from Hercules (Lehman, 1965). These residue methods
and toxicology data are only suitable for use with materials that closely
approximate the composition of Hercules toxaphene. It is therefore important
to intercompare the composition of toxaphene samples manufactured by Hercules
since the 19^O's and of related commercial materials. Toxaphene is a complex
mixture of at least 177 components revealed by a combination of liquid
adsorption column chromatography followed by GLC-CI-MS analysis on a packed
column of the resulting fractions (Holmstead et al., 197^-)• An improved
procedure for separation and quantitative analysis of toxaphene components
is needed to critically intercompare the composition of toxaphene samples
and related materials. This section gives an open tubular column GLC
method for toxaphene analysis and applies this procedure to 8 samples of
toxaphene manufactured by Hercules from 19^9 until 1975, to 12 samples of
toxaphene-like materials from other manufacturers, and to samples of [-'•Gl-
and [^Cl]toxaphene. It also evaluates a TLC-GLC method for more complete
separation and analysis of toxaphene components and the effect of composi-
tion on the acute toxicity of toxaphene-like materials.
Section II deals with the relation of structure to biological
activity of toxaphene components. Two of its most toxic components are
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2,2,5-endo,6-exo,8,9,10-heptachlorobornane (compound Jp and a mixture of
the 8-chloro and 9-chloro derivatives of I (referred to here as 8-Cl-Jj. and
9-C1-J,, respectively) (Casida et al., 1975; Khalifa et al., 197^; Matsumura
et al., 1975; Palmer et al., 1975; Seiber et al., 1975; Turner et al., 1975).
A probable precursor for these toxicants is 2-exo,10-dichlorobornane, a major
product at an early stage in chlorination of camphene (Jennings and
Herschbach, 1965; Richey et al., 1965). This dichlorobornane has been used
to prepare trichlorobornanes as models for identification of toxaphene
components (Parlar et al., 1977) and to obtain a product from which the
mixture of 8-C1-L and 9-C1-L can be isolated by TLC and preparative GLC
(Nelson and Matsumura, 1975)' Section II gives a procedure for chlorination
of 2-exo, 10-dichlorobornane to prepare I on a gram scale and to obtain 3-j~xo-
Cl-^ Additional octa- and nonachlorobornanes, hexa- and heptachlorobor-
nenes, and a hexachlorobornadiene are obtained on chlorination or dehydro-
chlorination of JJ, or dehydrochlorination of 3-exo-Cl-^.. These products are
identified by NMR and MS and used in comparative toxicity studies to deter-
mine the relationship of chemical structure and biological activity.
Section III examines the metabolism and environmental degradation of
toxaphene and its components. Toxaphene undergoes rapid dechlorination in
rats (Casida et al., 197^; Growder and Dindal, 197^5 Ohsawa et al., 1975)
and is metabolized in houseflies (Hoffman and Idndquist, 19527 and in a
cotton leafworm enzyme preparation (Abd El-Aziz et al., 1965, 1966). No
toxaphene metabolite other than chloride ion was identified in these studies,
in large part because of difficulties in examining such a complex mixture
of polychlorobomanes and other materials (Holmstead et al., 197*0- One
toxaphene component, heptachlorobornane ^, constitutes up to 8$ of the
technical grade insecticide (Palmer et al., 1975; see Section l)
and four octachlorobornanes, each derivable by addition of one chlorine
atom to ^ make up an additional ~ 15$ of toxaphene (Matsumura et al.,
1975; Turner et al., 1975; see Sections I and II). Heptachlorobornane _£,
has relatively high biological activity and is one of the most easily iso-
lated components of toxaphene. It is therefore a suitable model compound
for use in studies to gain an understanding of reactions involved in
detoxication of several polychlorobornane components of toxaphene. An
aqueous reduced hematin system degrades this heptachlorobornane to un-
identified products by reductive dechlorination and dehydrochlorination,
and it also dechlorinates many other toxaphene components (Khalifa et al.,
1976). Section III considers the degradation and metabolic chemistry of
heptachlorobornane I in several systems, selected to emphasize reductive
dechlorination reactions, and the nature of toxaphene-derived products in
a variety of organisms.
Section IV is a preliminary report on the mutagenic activity of toxaphene
and some of its components. Toxaphene is a purported carcinogen in rats
and mice (National Cancer Institute, 1977) and a related polychloropinene
preparation is reported to give chromosomal abberations in humans (Samosh,
197*0- The mutagenic activity of toxaphene and related insecticides and
some of their components was therefore examined to expand the available
knowledge on potential side effects from use of this insecticide.
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SECTION I
COMPOSITION OF TOXAPHENE AND RELATED CHLORINATED TERPENES
MATERIALS AND METHODS
Samples. Charles L. Dunn (Hercules Inc., Wilmington, Del.) provided
the following samples: standard toxaphene and toxaphene batches manufactured
by Hercules in 19*4-9, 195^, 1957, I960, 1963, 1970, and 1975; t1^]toxaphene
(1.35 mCi/g) and [3°ci]toxaphene (^3.6 MCi/g) prepared by Hercules; Hercasa
product from the Hercules owned plant at Managua, Nicaragua; two samples
from Vicksburg Chemical Co. (Vicksburg, Miss.); two samples from Bison
Chemical Co. and one from Sonford Chemical Co. (each at Fort Natchez, Texas);
one sample from Procida (Paris, France). Strobane T-100 was supplied by
Roy T. Gottesman (Tenneco Chemicals, Piscataway, N.J.). Kenneth R. Hill
(Agricultural Environmental Quality Institute, United States Department of
Agriculture, Beltsville, Md.) provided four samples: Flit & Fontaine manu-
factured in South Africa; Melipax manufactured in the German Democratic
Republic; two East European samples (light and dark) from one producer in
Eastern Europe but of different manufacturing periods. One of the Vicksburg
samples and the Bison and Sonford samples were obtained as 90% solutions in
xylene, from which the solvent was removed under reduced pressure. The
Melipax sample was provided as a 9-10% dust from which the desired material
was recovered in 7% yield on extraction with hexane. The physical properties
of the samples were as follows: yellow viscous liquid - Flit & Fontaine;
yellow-brown and black viscous liquids - East European light and dark,
respectively; white waxy solid - [l^CJtoxaphene; yellow or yellow-brown waxy
solids - the remaining samples. Elemental analyses of these samples were
carried out by the Department of Chemistry, University of California,
Berkeley, Calif.
The following three toxaphene components were used as chromatographic
standards:^- 2,2,5-endo,6-exo,8,9,10-heptachlorobornane (Palmer et al.,
1975); 8-C1-I plus 9-C1-I - mixture of 2,2,5-endo,6-exo,8,8,9,10-octachloro-
bornane and ?,2,5-endo,6^exo,8,9,9,10-octachlorobornane (Matsumura et al.,
1975; Turner et al., 1975); 2-endo,3,3,5-exo,6-exo,8,9,10,10-nonachloro-
bornane (for structure see Figure 2 given later) (Anagnostopoulos et al.,
197*0.
Chromatography
Open tubular column GLC. The Hewlett-Packard Model 5830A gas chromato-
graph was used with a linear electron capture °3]>ji detector with extended
dynamic range and an open tubular column (0.25 mm i.d. x 30 m) coated with
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SE 30 (k |Ug/ml). The operating conditions were: injection temperature 210°C;
oven temperature maintained at 170°C for 60 min followed by programming from
170 to 200°C at 0.5°C/min and finally a constant temperature of 200°C for 30
min; detector temperature 255°Cj split ratio 1:120; helium carrier gas and
argon-methane (95:5) makeup gas for the detector; 1 (jg sample injected in
2 (jil hexane. An on-line computer provided the t-^ of each peak and its
normalized area as percentage of the total peak area from the chromatogram.
The linear electron capture detector used provided excellent propor-
tionality of the amount of compound injected (examined with component I and
aldrin) to peak area over the entire range of peak areas involved in the
present study. This linear response also appears to hold for most if not
all of the other GLC peaks on chromatography of toxaphene.
TLC and TLC-GLC. Silica gel 60 chromatoplates (20 x 20 cm, 0.25 mm
layer thickness, EM Laboratories, Inc., Elmsford, N. Y.) were spotted with
500 |ag of standard toxaphene divided equally among 11 spots and, in addi-
tional spots, with 1 ng/spot of the appropriate fluorene marker dyes. The
chromatograms were developed three times in the same direction with hexane
saturated with DMF. Gel regions from the toxaphene chromatograms correspond-
ing in Rf values (Stahl, 19&9) ~k° "the appropriate marker dyes (detected by
their yellow color or UV-absorbing property) were scraped free from the
glass support and extracted with acetone. The acetone was evaporated to
dryness, the residue redissolved in 300 |ol acetone, and a 2 \jH aliquot of
the extract, fortified with aldrin as the GLC marker, was analyzed by open
tubular column GLC.
Bioassays. Male albino mice (18-20 g, Horton Laboratories Inc.,
Oakland, Calif.) were treated IP with the test sample dissolved in DMSO
using 100 (ol of DMSO per mouse. Adult female houseflies (Musca domestica
L., SCR susceptible strain, 3-^ days after emergence, 18-20 mg) were treated
topically on the dorsum of the abdomen with acetone solutions of the test
sample, using 1 jol of acetone per fly. The 2^-hr LD,-O values are based on
8-12 mice for each dose and a 1.It-fold dose differential and 70 flies per
dose and a 2-fold dose differential.
RESULTS
Open Tubular Column GLC and TLC-GLC Analysis of Hercules Toxaphene
Standard. Open tubular column GLC of standard toxaphene reveals 29 peaks
(Figure l) that individually make up 1.0 to &.k% of the total peak area
and collectively account for about 88$ of the total peak area (Table l).
The contribution of each of these peaks to the total peak area is highly
reproducible on repeated analyses (Table l).
Several of these open tubular column GLC peaks consist of multiple
components as shown most clearly by combined TLC-GLC analysis (Figure 2).
The five TLC fractions were selected with marker dyes to recover the precise
TLC regions of component^ (TLC region J^, -1L, 0.35-0.39, bifluorenylidene
marker), of components 8-C1-I plus 9-Cl-^ (TLC region d, 3p 0.45-0.1+9,
benzylidenefluorene marker)fand of regions above, below, and between those
of components J., and 8-Cl-j, plus 9-Cl-jN GLC analysis of the TLC fractions
k
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9-ci-i.
HERCULES
STANDARD
28
BISON
i i iji;-.»y".v.,w
^-j^^-'1 '••"" ••
SONFORD
HERCASA
60 MINUTES 80
100
120
Figure 1. Open tubular column GLC analysis of the toxaphene
standard and related chlorinated terpenes. The 29 peaks making
up a 1% of the total peak area in the toxaphene standard are
designated by numbers as in Table 1. The same numbers designate
peaks in the other samples with identical Tr values to those in
the toxaphene standard. Additional peaks making up s 1% of the
total peak area in the related chlorinated terpenes are
designated by asterisks. No additional components are eluted
at times later than those indicated.
(Continued)
-------
PROCIDA
FLIT a
FONTAINE
25
24
;s I i
26 '
MELIPAX
4 Ml
7
I
6 ,1
24
619
•>22 i
25
27
c ' 28
29
EAST
EUROPEAN *
TOXAPHENE
[36d]-
TOXAPHENE
3 89 i
j,JjUXW^:^X
j_^a!
41
JLjuiJU1
24
24
Jt
23^
24
25
27
28
T
'uJ'J
2?
J'-JJv.
"S MINUTES S"
00
•So"
Figure 1. (Continued)
-------
Figure 2. Open tubular column GLC analysis of the toxaphene
standard and fractions _a,-e obtained from this standard by
TLC. The 29 peaks making up § 1% of the total peak area in
the toxaphene standard are designated by numbers as in
Table 1. The same numbers designate peaks in the TLC fractions
with identical Tr values to those in the toxaphene standard.
Additional peaks making up a 1% of the total peak area in the
TLC fractions are designated by asterisks. Each peak is
designated only for the TLC fraction in which it appears in
maximum amount. The first major peak in each chromatogram
(Tr 25.9 min) is aldrin used as an internal standard.
Structures are given for toxaphene components 8-C1-I plus
9-Cl-_J, (peak 16), T^ (peak 9)> and a nonachlorobornane (peak 27}
(see also Table l).
-------
reveals that several peaks from the toxaphene standard consist of two or
more components since GLC peaks of similar or identical tR values appear
in different TLC regions which are not adjacent to each other (Figure 2).
Some of these multicomponent peaks are as follows: ^, Jjj,, $,, JL2, Ik,, 1^ _19,
21^, 22^, 2%, and 2^. On an overall basis, the TLC-GLC analysis of the toxaphene
standard serves to detect more than 7^ components as unique and relatively
major peaks (designated by numbers and asterisks in Figure 2).
The proportion of the standard toxaphene components that appear in the
five TLC regions is not known. However, studies with [^G]- and P°Cl]-
toxaphene establish a fairly similar radioactivity distribution for the five
TLC regions and that the acetone extraction procedure recovers 99-100$ of
the radioactivity from the gel in each of the TLC regions.
Open Tubular Column GLC Analysis of Various Hercules Toxaphene Samples.
The seven samples manufactured from 19^9 "to 1975 are not distinguishable by
open tubular column GLC, each showing the same 29 major peaks and in almost
identical ratios to those observed in the toxaphene standard (Table l).
Open Tubular Column GLC Analysis of Related Materials. Very similar
results were obtained with each of the two samples designated as Vicksburg,
Bison, and East European so only the average results are presented for
these materials. Some of the samples (Strobane T-100, Hercasa, and [3°C1]-
toxaphene) are very similar to Hercules toxaphene while others (Procida,
Melipax, East European, and [l^Cltoxaphene) are considerably different
(Figure 1, Table l). Several GLC peaks appear in 5 1% amount in samples
other than those manufactured by Hercules in the United States but are
minor or almost absent in Hercules toxaphene. These components, designated
by asterisks in Figure 1, are useful in recognizing samples originating
from a particular manufacturer. Another criterion which is adequately
reproducible and characteristic in comparing various samples is the time
required for 25 and 50% of "the total peak area to elute. On this basis,
the [3"Cl]toxaphene most closely reproduces the Hercules toxaphene samples
(Table 1).
Other Criteria for Intercomparison of Samples. An almost identical
elemental composition, approximating CioH^QClgj is obtained with each of
the Hercules toxaphene samples, and with Strobane T-100 and the Hercasa
sample (Table 1). The other samples are less heavily chlorinated, ranging
from 6l.O to 6^.9% in chlorine content.
The bioassay results are of little value in differentiating between
the various samples. However, the East European samples are slightly less
toxic and the Melipax and Procida samples are distinctly less toxic than
the others.
DISCUSSION
Open tubular column GLC has been used previously for determination of
the purity of toxaphene components (Khalifa et al., 197*0 and for quali-
tative analysis of toxaphene composition (Seiber et al., 1975). In the
present investigation, this method is optimized for quantitative analysis
10
-------
of toxaphene and related materials. Particular attention is given to five
factors: a suitable column, as to length and liquid phase; the lowest possi-
ble temperature to minimize thermal decomposition of the components; a
suitable temperature program to maintain a constant baseline and reasonable
overall time for analysis; an electron capture detector linear over the
range of amount of individual peaks to be analyzed; an on-line computer
to normalize the peak areas and provide precise tR values. The standard
toxaphene reveals the following numbers of peaks exceeding the indicated
percentages of the total peak area: 29 peaks at the 1% discriminating level,
51 peaks at 0.3$, 66 peaks at 0.1%, and 10*f peaks at 0.03$.
The TLC-GLG method provides a more complete analysis than GLC alone of
components in toxaphene and related materials. The two major GLC peaks
(jg, and 16) in Hercules toxaphene contain components I, and 8-C1-I plus 9-C1-
J^ respectively. Marker dyes were sought for the precise TLC positions of
these components in a TLC system that provides near optimal separation of
toxaphene components. After examining many TLC systems (mostly based on
Khalifa _et al., 197*+) and potential marker dyes, benzylidenefluorene was
selected as the marker for components 8-Cl-^ plus 9-Cl-^ and bifluorenyli-
dene for component I in the hexane-DMF system. The fact that the marker
dyes cochromatograpn* with components 8-Cl-jI, plus 9-C1.-I and I in the hexane-
DMF system does not mean that they are necessarily useful on 'the same basis
with other chromatographic conditions. Thus, on three TLC developments with
hexane, the corresponding markers fall 0.05-0.15 ^R units below the posi-
tions of these components, individually or with these components in mixture
with the normal toxaphene constituents. Although two-dimensional TLC
(hexane x 3 and then hexane-DMF x 3) provides better separation of toxaphene
components than one-dimensional development, this two-dimensional procedure
negates the use of the marker dyes to exactly locate components 8-C1-I plus
9-C1-J, and JC,. **
The present methodology is not adequate for quantitative analysis of
each individual component in toxaphene and related materials. Thus, some
of the GLC peaks including 9 contain multiple components so quantitative
analysis by open tubular coluimn GLC alone overestimates the amount of
component I in toxaphene. This problem is not completely overcome by the
TLC-GLC method in the case of component _£. Also, there is no evidence that
the same components are present in each GLC peak over the wide range of
samples analyzed although TLC-GLC analyses should be suitable to evaluate
this point.
Criteria useful in critically intercomparing toxaphene and related
materials are the GLC peaks exceeding 1$ of the total peak area, the percent
of GLC peaks Q and 1^ since they reflect major components that vary con-
siderably among the samples, and the time required for elution of 25 and
50$ of the total GLC peak area. Based on each of these criteria, the
samples of Hercules tcxaphene are essentially identical with each other
even though they were manufactured at intervals over a period of 26 years.
Thus, residue and toxicology data obtained with any one of the Hercules
samples are probably applicable to any of the other Hercules samples but
not necessarily to certain of the remaining chlorinated terpenes examined.
11
-------
The toxicity of the toxaphene samples and related materials to mice
and houseflies does not clearly correlate with their chlorine content, with
the amount of components (including I and 8-Cl-vI, plus 9-Cl-vjp appearing in
GLC peaks £ or 16^ or with the amount of any individual GLC peak. This
suggests that the toxicity of such diverse samples may be due to many com-
ponents which could vary with the manufacturing method or that it is due to
relatively minor components not easily differentiated on examining such
complex mixtures.
Methodology is now available to distinguish between toxaphene and
related materials. These procedures may be useful in evaluating the chemical
and environmental degradation of these insecticides and their residues.
12
-------
SECTION II
RELATION OF STRUCTURE TO BIOLOGICAL ACTIVITY OF TOXAPHENE COMPONENTS
MATERIALS AND METHODS
Chromatography. The composition of reaction mixtures and the purity
of individual products were determined by open tubular column GLC (see
Section I), assuming the same response for each component with the electron-
capture detector. TLC utilized 20 x 20 cm silica gel 60 F-25^ chromato-
plates of 0.25 mm layer thickness (EM Laboratories) for analysis and silica
gel F-25^- chromatoplates of 0.5 mm layer thickness (EM Laboratories) for
product isolation on a 5-10 mg scale. The products on the chromatoplates
were detected by spraying with diphenylamine (10$ w/v) in acetone and
irradiating with UV light. Column chromatography utilized silicic acid
(AR-100 mesh, Mallinckrodt Inc., St. Louis, Mo.; column length ~ 10 times
the diameter) packed in technical grade hexane or hexane saturated with DMF
and developed with the same solvent under 7-9 lb of N2 pressure to increase
the elution rate. In most cases, bifluorenylidene (~ 10 mg) was added as
a marker dye (see Section l), since it elutes in almost the same position as
2*
Spectroscopy and Elemental Analyses. 360 MHz NMR spectra were obtained
on the Bruker HXS-360 spectrometer at the Stanford Magnetic Resonance
Laboratory. 90 MHz spectra were run on the Perkin-Elmer R-32 spectrometer.
CI-MS and GLC-CI-MS determinations utilized the Finnigan Model 1015D mass
spectrometer as previously described (Holmstead et al., 197*0 but with iso-
butane as the ionizing gas. Samples for elemental analyses were prepared by
evaporating hexane solutions and drying at pump vacuum for 14 hr.
Bipassays. Procedures for determining the 2U-hr LD,-,-, values with male
albino mice, adult female houseflies and goldfish were as previously report-
ed (see Section I; Turner et al., 1975) except that the volume of water
for the goldfish was 3 liters rather than k liters. In synergism studies,
PB was applied topically to the houseflies at 250 Ug/g 30 min prior to the
chlorinated hydrocarbon or administered ip to the mice at 150 mg/kg one hr
prior to the test compound. The carrier solvent for PB was 1 |^1 acetone for
the houseflies and 50 >tl DMSO for the mice.
EXPERIMENTAL PROCEDURES
Synthesis routes used to obtain the various chlorinated hydrocarbons
are illustrated in Figure 3 which also gives the compound designations.
13
-------
H!
-------
I and.3-exo-Cl-I from Ghlorination of 2-exo,10-Dichlorobornane. 2-exo,
lO-Dichlorobornahe Q?2 g) (Richey et al., 1965T"containing up to 25% 2-exo,
10,10-trichlorobornane (Parlar _et al., 19?6a, 1977) was dissolved in CCl^
(1 liter) which was then heated to boiling to drive out G>2 and cooled under
NO- Chlorine was bubbled into the solution at ~ 0°C, the amount added being
determined by weight gain. Stirring the solution under a sunlamp resulted
in rapid HC1 evolution (probably with some loss of Cl?) and disappearance of
the yellow color within a few min. GLC monitoring revealed that addition
of 105-5 g C12 gave the optimal yield of J, (12$) (Table 2). Half of the
product mixture was combined with the marker dye and chromatographed on a
silicic acid column (l kg) with hexane. Details on this column chromato-
graphic procedure are given in Section III. Elution of the yellow dye and
_£ began after 10.6 liters of hexane had been eluted and was essentially
complete after an additional 3-8 liters of hexane. The crystals obtained
on concentration of the fractions weighed 1.2 g (2.5$ yield of I from 2-exo,
10-dichlorobornane) after washing with hexane and 0.7 g after recrystalliza-
tion from hot acetone (> 98$ pure I; mp 222°C).
In a second preparation, pure 2-exo,10-dichlorobornane (20 g) was
chlorinated as above, using 52 g Clg, yielding 6% I, ^% 3-exo-Cl-I. and 19$
8-C1-I plus 9-C1-I (Table 2). Fractions from chrdmatography as atTove con-
taining 65-71$ I were processed in the usual manner to obtain 220 mg T.
Those containing 22-25$ 3-exo-Cl-j. were concentrated, and on standing yielde
ed crystals which were washed with hexane and recrystallized from hot ace-
tone to give 3-exo-Cl-^ (220 mg, > 98$ pure, mp > 2^0°C). Fractions contain-
ing kk-55% of 8-C1-I plus 9-C1-I were combined (7.2 g) and rechromatographed
on a silicic acid column (265 gT with DMF-saturated hexane as eluent to
yield 8-C1-J,, plus 9-C1-J, in ~ 70$ purity.
Qcta- and Nonachlorobornanes from Chlorination of I. A solution of I
(1.63 g; 99$ pure) in CCl^ (100 ml) was heated to boiloSg, cooled and- —— ~
stirred under N^. To this solution was added C12 (0.2 g) in CC1. previous-
ly boiled and stored under Ng. On stirring the solution under a sunlamp,
the dp color disappeared within 3 min. Conversion of L was 30$ to the
products shown in Table 2. Most of the unreacted L and a small portion of
3-exo-Cl-I were removed as crystals on recrystallization of the chlorina-
tion product mixture from hexane and then acetone. Chromatography of the
supernatant from recrystallization on a silicic acid column (265 g) with
hexane separated the octachlorobornanes and two of the nonachlorobornanes
as shown in Figure h. The fractions richest in each component were
combined. The 5-exo-Cl-j^ obtained (25 mg; 70$ purity containing 9$^
2% 8-Cl-^t, plus 9-Cl-I^ and 1^$ 10-Cl-jp was not further purifiable "by TLC
or recrystallization. Early fractions of the mixture of 8-Cl-^ and 9-Cl-j^
were found to be > 90$ 8-C1-I (MMR). Recrystallization of one fraction gave
relatively pure 8-Cl-£ (23 mg"; < 2$ 9-01-^ 5$ 3-exo-Cl-jp. The last
fractions of the 8-C1-J., and 9-Cl-J^ mixture gave an 8-Cl-I:9-Cl-_I^ ratio of
1:1.5 but they were already rich in 10-C1-I. The next editing portion was
used to obtain 10-C1-J after recrystallization from hot hexane (32 mg; 98$
purity). The small amount of S-exc-jlO-Cl^-L obtained (12 mg) was > 86$
pure (containing no I, 8-C1-I or 9-Cl-lJ and was not further purified.
8,10-Clp-_I^ from this preparation (22 mg) was combined with that from another
preparation and subjected to recrystallization from hot hexane, further
15
-------
TABLE 2. PRODUCTS FROM CHLORINATION OF CAMPHENE, 2-exo,10-DICHLOROBORNANE
AND HEPIACHLOROBORNAWE I.
Hepta- and octachlorobornanes
Compound or mixture analyzed and
elemental composition of mixture, %
***
3-exo-C\-l_
5-exo-
C1'I
8-Cl-l,
9-C1-J,
10-Cl-j,
Individual polychlorobornanes
Chlorination of tech. grade camphenec
; C, 28.7; H, 2.4; Cl, 68.9
Chlorination of 2-exo,10-dichlorobornane
C, 30.90; H, 2.62; Cl, 66.68
(C10H10-1Cl,-3)
C, 27.43; H, 2.15;C1, 70.27
(C,oH,.3Clg ,)
Chlorination of J[.
C, 29.03; H, 2.67; Cl, 67.92
No elemental analysis
GLC (R, min
(peak no. designation)0
63.8(9) 79.4(14) 95.8
Composition, %b
3
8
12
6
70
22d
4
4
12
<1
<1
<1
2
82.2(16) 82.2(16) 90.7(23)
12
11
27
7
5
13
5
6
13
a Procedure described in Section I. The identity of 3-exo-Cl-I with Ik and
of 10-C1-I with 23 is based on GLC cochromatography and the TLC-GLC method
of Section* I. Additional t^ values (min) for four nonachlorobornanes are
10U.O for S-exOjlO-Clg-^ 107.6 for S^O-Clg-^I. and 102.7 and 10U.il- for two
unidentified compounds.
Based on GLC analysis. The yields of I and possibly some other components
include other materials not adequately separated by GLC, particularly in
the Chlorination products of tech. grade camphene and 2-exo,10-dichloro-
bornane. The 8-C1-I plus 9-C1-I content is based on GLC and their ratio
on NMR (Turner ejt aT., 1975 for toxaphene; this study for the Chlorination
products of 2-exo,10-dichlorobornane and I) .
c Data for standard toxaphene from Section I.
Additional components are 3-exo,10-Clp-I (2$), SjlO-Cl^-I (5$) and two
unidentified nonachlorobornanes (2 ana 3%)• These nonach^Lorobornanes also
appear in small amounts on Chlorination of I to 3°% conversion but they
are not detected (< 1%) in toxaphene or in The Chlorination products of
2-exo,10-dichlorobornane.
16
-------
c
o
o
*-
c
o
c
o
o
-------
purification by preparative TLC with hexane and another crystallization (>
1% purity containing no ^, 8-C1-I, or 9-Cl-J^).
Hexa- and Heptachlorobornenes and a Hexachlorobornadiene from Dehydro-
chlorination of I and 3-exo-Cl-I. A solution containing X (1^-3
purity) and KOH *t~ 2 g) IrfethdJfol (?0 ml) was held at 25°C for 28 hr,
yielding I (10%) and two dehydrochlorination products [90%; ^-HCl(5,6) and
^-^1(3,2*7 in a 2.k:l ratio]. The GLC tR values (min) at 200°C isothermal
"are 20.2 for I; 11.6 f or J^-HCl ( 3 , 2 ) and 10.5 f or J..-HC1 ( 5 , 6 ) . Their TLC
Rf values with hexane for 'development are 0.08 f or J^ 0.12 for ^-HCl(332)
and 0.19 for ^-HCl(5,6). Water was added to the residue after ethanoj.
evaporation, and the hexane- soluble products were purified by TLC as above
or by chromatography on a silicic acid column (36 g) with hexane. The
column separation yielded L-HCl(5,6) (39 nig) eluting in fractions from 370-
U80 ml total volume and J,-HCl(3j2) (31 mg) in the 620-780 ml region. Re-
crystallization from hot hexane gave j-HCl(3,2) in 95% purity, while re-
crystallization from a small volume of hexane gave I-HCl(556) in > 98%
purity. On dehydrochlorination of ^ by dissolving Tt in n-propylamine and
holding for several days, the product mixture consisted of 10% I and 90%
_£-HCl(5,6) plus I-HCl(3,2) in a 6:1 ratio. Both of the dehydrochlorination
products, when exposed to air and light, became discolored by a brown,
hexane-insoluble material.
3-exo-Cl-I (51 mg) in ethanol (kO ml) containing KOH (250 mg) reacts
rapidly~T> 99%*xn 1 hr) to give 3-exo-Cl-^-HCl(5,6) (> 97%) and 3-exo-Cl-jr
2HC1 (1%). The GLC tR values at 20015C isothermal are 25.1, 12.7, and 7-3 m
respectively, for 3-exo-Cl-I and its mono- and didehydrochlorination pro-
ducts. After preparative TLC with hexane (Rf 0.17 for 3-exo-Cl-X and 0.26
for both dehydrochlorination products), 3-exo-Cl-j.-HCl(5,6) was recrystalliz-
ed (> 99% purity) from a 2:1 mixture of tetramethylsilane and hexane, the
tetramethylsilane serving to reduce the solubility in hexane. On treatment
with more concentrated KOH, 3~ exo-Cl-J,-HCl ( 5 , 6 ) eliminates another HC1 to
give 3-exo-Cl-I-2HCl. This hexachlorobornadiene was also obtained directly
from 3-exo-Cl-T (10 mg) on treatment with KOH (~ 100 mg) in ethanol (3 ml)
for one hr (> 99% conversion). Several such preparations were combined
and subjected to preparative TLC to obtain 3-exo-Cl-I-2HCl [17 mg; oil; 95%
with 5% 3-exo-Cl-I-HCl(556)]. A brown impurity, removable by preparative
TLC, was formed on exposure of its CClr solutions to light and air.
RESULTS AND DISCUSSION
Preparation of Polychlorobornanes and Polychlorobornenes . On photo-
chlorination , camphene and 2-exo,10-dichloro'bornane yield relatively large
amounts of I and its 3-exo-Cl,8-Gl, 9-C1, and 10-C1 derivatives (Table 2).
This is the"*tirst report of 3-exo-Cl-I and 10-Cl-J^ as toxaphene components.
Since all these octachlorobornanes , along with 5-exo-Cl-J^, are also formed
on phot ochlorinat ion of I (Table 2), it appears likely that I is their major
precursor in both toxaphene and chlorinated 2- exo , 10 -dichlorobornane .
There is remarkable selectivity in chlorination of 2-exo ,10-dichloro-
bornane to I, since this product is one of 9^3 isomers derivable by addition
of five chlorine atoms to the dichlorobornane. Considerable site selectivity
18
-------
also exists in photochlorination of I, ±._e., C-8 > C-9 = C-10 ^ C-3 > C-5-
The two endo hydrogens (H-3W and H-wf) and the bridgehead hydrogen (E-k)
appear not to be substituted at all. The greater resistance of position
5-exo to substitution relative to position 3-exo may be due to the de-
activating effect of the 5-endo chlorine or to steric protection of this
site by chlorines at positions 5-endo, 6-exo, and 8, as observed in com-
parison of a space-filling model with the crystal structure of I (Palmer
et al., 1975). The 3-endo position of £ is considerably protected by the
two endo chlorine atoms, and the 6-endo position is almost completely
occluded by these chlorine atoms and the one on C-10. Studies on norbor-
nane also indicate that the bridgehead hydrogen is very unreactive, pre-
sumably because the dihedral angle at the bridgehead does not lend itself
to stabilization of a radical, and that the endo positions are substituted
less readily than the exo positions, possibly because of steric effects on
chlorine approaching the radical (Kooyman and Vegter, 1958j Poutsma, 19&9J
Walling and Mayahi, 1959)-
Chlorination of 2-exo,10-dichlorobornane with GLC monitoring of the
product composition provides a convenient source of ^ an. a gram scale. At
an optimal I content of 12$, about one-fifth of this amount can be isolated
pure by a single column chromatography, followed by recrystallization. The
easiest products to isolate are ^ and 3-exo-Cl-I. because of their low solu-
bility in hexane, acetone, and some other solvents relative to most toxaphene
components. On chlorination of the dichlorobornane to a higher chlorine
content, the amount of ^ is reduced to 6% and that of 3-exo-Cl-T, 8-Cl-j^, and
9-C1-J, is multiplied by*a factor of 2 to k (Table 2). Chromatography of this
product yields a mixture containing J0% 8-C1-I and 9-Cl-^, but recrystalliza-
tion does not provide further purification. IPhus,chlorination of 2-exo,10-
dichlorobornane provides convenient access to J., and 3-exo-Cl-j, but not to
the other individual octachlorobornanes. Fortunately, direct chlorination
of J", yields 8-C1-I and 9-Cl-J^ in a purity such that 8-C1-I can be obtained
by chromatography and recrystallization. In addition to "the five identified
octachlorobornanes and two identified nonachlorobornanes which are easily
separated by column chromatography, two unidentified nonachlorobornanes are
also formed, but they are not separated by this chromatographic technique
(Table 2, Figure 4).
Dehydrochlorination of J^, and 3-exo-Cl-I with KOH or propylamine yields
polychlorobornenes. An alternative dehydrochlorination of either compound
via the equivalent of a Wagner-Meerwein rearrangement might give cis and
trans isomers of two 8-chlorocamphene derivatives. An analogy for this
rearrangement is the dehydrochlorination of 2-exo,10-dichlorobornane with
dimethylaniline (Jennings and Herschbach, 1965T- However, the MMR spectra
of the dehydrochlorination products of JC, and 3-exo-Cl-I are not appropriate
for the 8-chlorocamphene derivatives which would resuit from such a re-
arrangement. The preponderance of HC1 elimination from the 5?6 positions
of jr_ over the 3?2 positions is expected from the greater acidity of the
5-exo-hydrogen over the 3~exp-hydrogen, due to the electron-withdrawing
properties of the 5-endo-chlorine. The $-exo-hydrogen is more easily re-
moved than the 6-endo-hydrogen. This is predictable from the almost
exclusive exo-cis dehydroehlorinalions observed in trans-2,3-dihalonor-
bornanes (LeBel _e_t al. , 1964). Lacking an exo hydrogen in position 2 or 3,
19
-------
3-exo-Cl-I can form a. 2,3-olefin only by some mechanism other than exo-cis
dehydrochlorination, and less than 1$> of such a product is obtained in the
initial deny drochlorinat ion. With more concentrated base, however, the mono-
dehydrochlorination product from 3-exo-Cl-I, undergoes further loss of HC1 to
form a hexachlorobornadiene.
Chromatographic Properties . Consistent chromatographic patterns
related to the number of chlorine atoms are evident for I and its poly-
chlorobornane derivatives on a silicic acid column (Figure U) and on open
tubular column GLC (Table 2). With the silicic acid column, the sequence
of elution is four nonachlorobornanes first, then five octachlorobornanes ,
and finally the heptachlorobornane (jp . The GLC t^ values of these bornane
derivatives decrease in the same sequence. In addition, the GLC t-p values
decrease further in the sequence of two heptachlorobornenes , two hexachloro-
bornenes, and one hexachlorobornadiene. These patterns are probably restrict-
ed to compounds within a closely related series, since many exceptions are
evident in the variety of components in toxaphene (Casida et al. , 1975;
Holmstead et al. ,
Identification of New Compounds . Mass spectrometry confirmed that the
major products of chlorination of ^ are C-[_oH-|_QClg and C-j_QHgClg compounds
and that base treatment of ^ and 3-exo-Cl-^ leads initially to CioHlOG16 and
C-^QHgClry derivatives, respectively. In CI-MS with methane as the ionizing
gas , toxaphene components generally give no M + 1 ions, but instead have
their highest masses at M-C1 (Holmstead et al. , 197^)- In the present study
using isobutane as the ionizing gas , the identified products from chlorina-
tion of ^ conform to this rule, as do ^-HCl(5,6) and 3- exo-Cl-I.-HCl ( 5 , 6 ) .
However, r-HCl(3,2) gives a weak molecular ion (m/e 3^0) and a weak M-l ion
(m/e 339) as well as the base peak at m/e 305 (M-Cl). The fragmentation
patterns of 1^01(5,6) and I-HCl(332) are almost identical in other respects,
with major fragment ions at**m/e 305, 30*4-, 269, 2^, 209, 195, 173, 159, and
125. 3-exo-Cl-^,-2HCl has a molecular ion at m/e 338 and an M+l ion at m/£
339, as well as prominent fragment ions at mfe 303, 302, 26?, 266, 231, and
193-
NMR spectroscopy, employed previously to assign the structures of 8-C1-
1 and 9-C1-I (Matsumura _et al. , 1975; Turner et al. , 1975 ), also proved
in structural assignments for the new compounds in the present study
(Table 3). The structure of each compound, except the dehydrochlorination
products of 3-exo-Cl-j^ is readily evident from the number and coupling
patterns of protons at low and high fields. However, some difficulty is
encountered in resolving and assigning all the resonances, particularly the
chloromethyl and chloromethylene protons. Determining the spectra in
several different solvents and in some cases at 3&0 MHz allowed the chemical
shifts of protons incompletely resolved in CClr solution to be estimated.
These structural assignments proceeded from the assumption that the
bornane skeleton of 2-exo,10-dichlorobornane and I does not undergo any
rearrangement on chlorination. While rearrangement during photochlorina-
tion is reported for bicyclic compounds with considerably strained three-
and four-membered rings, it is not expected in less strained systems
(Poutsma, 1969) such as bornane derivatives. The resemblance of observed
20
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coupling constants in the new compounds to analogous coupling constants in
I (Palmer et al., 1975) supports their proposed bornane skeletons. A
"departure from the coupling patterns of J^., however, is the absence of some
long-range coupling between protons on the bridge chloromethyl groups G-8
and C-9. In I, each of the chloromethyl group protons, in addition to
geminal coupling, shows ~ 1.8 Hz four-bond coupling across the bridge to one
other proton. The fact that none of these protons shows long-range coupling
to two protons suggests the importance of the relative conformations of the
chloromethyl groups in 1 (Palmer et al., 1975)- It is thus not surprising
that addition of chlorine to the molecules should alter these conformations
and thus the long-range coupling. Addition of chlorine is less likely to
affect angles of protons attached to the relatively rigid six-membered ring
of bornane, and thus four-bond coupling of H-3X to H-5X, as observed in ^
is expected in related compounds which contain both of these protons.
10-C1-I has a singlet at 6 6.8l, indicating the presence of one di~ •
chloromethyl group. Two of the three possible isomers with one dichloro-
methyl group are already identified (8-C1-J, and 9-C1-.L), so the dichloro-
methyl group in the third isomer must be C-10. The absence of long-range
coupling to this dichloromethyl proton is in accord with this structure.
The coupling constants of the ring protons are essentially the same as in I.
Although H-5X is isochronous with chloromethyl group proton 9b, its distinc-
tive coupling pattern can be observed in the 3&0 MHz spectrum, and it can
be decoupled from H-3X, H-4, and H-6W. Several resonances of this compound
are broad, probably reflecting long-range coupling: 6N and 8b are broadened
doublets, while 8a (and probably the obscured 9b) appears as a broad singlet.
3-exp-Cl-^, was examined in several solvents and solvent mixtures, but
complete resolution of the resonances was not achieved in any individual
spectrum; a benzene-CHCl,. mixture (2:1) gave the best resolution. The C-3T"""
methylene group is not present, since there is only a single proton resonat-
ing at high field (a doublet, R-k), compared with three in JC,. If the
remaining H-3 were exo, H-4 would be an apparent triplet (overlapping doub-
let of doublets), rather than a simple doublet, since it would be coupled
to both H-3X and H-5X; the absence of long-range coupling to H-5X also
indicates that H-3X is lacking. Irradiation of H-5X collapsed the doublets
for E-k and H-6N to singlets. In this compound, only one proton on C-8 is
coupled to one on C-9-
5-exo-Gl-l was the most difficult of these octachlorobornanes to
- jj^p
identify, because neither the three protons at higher field nor the six
chloromethyl group protons are fully resolved, even at 360 MHz. After
identification of 3-exo-Cl-I, 8-Cl-^ 9-Cl-J^ «"* ^~^~^ however, there
remain just four positions (3-endo, 4,5-exo, and 6-endo) to which a
chlorine can be added to I. Only addition of chlorine to position 5-exo
would yield a compound with three protons at higher field (H-3N, H-3X, and
E-k) and an isolated singlet at 6 5-78 (H-6N). Support for the structure
was obtained by computer simulation of the complex multiplets for the three
upfield protons as they appear in two different solvents, CCl^ and benzene,
using the same set of coupling constants, but different chemical shifts.
As expected, H-3X lacks the four-bond coupling to H-5X anticipated
if the latter proton remained in the molecule.
23
-------
8,10-C1 -I has resonances of two dichloromethyl group protons, one of
which has 2 Hz coupling to one of the protons of the remaining chloro-
methyl group. This must be four-bond coupling, indicating that the chloro-
methyl group and coupled dichloromethyl group are on the "bridge. Thus,
8,10-Clp-I is related to 10-Cl-J^ in the same way in which 8-Cl-I^ or 9-C1-I,
is related to T. so the assignment of the dichloramethyl group to C-8 or
C-9 is similar to its earlier assignment in 8-C1-I and 9-Cl-I^. In the
latter two compounds, relative to I, there is significant deshielding of
the 3-exo or 5-exo proton to which the dichloromethyl group is syn, without
significant change in the chemical shift of the exo proton to which it is
anti (Matsumura et al. , 1975; Turner et al. , 1975)- In benzene solution,
where this effect is seen most dramatically, protons 3X and 5X of 8,10-Clp-I^
are shifted downfield 0.05 and ^ 0.58 ppm, respectively, relative to their'*'
chemical shifts in lO-Cl-^ thus, the dichloromethyl group must be C-8,
:i.e_. , syn to the 5-exo proton. In 8,10-Clg-^, H-k resonates 0.75 ppm down-
field of its chemical shift in 10-C1-I, and this effect also has an analogy
in the chemical shift of H-U of 8-Cl-^,and 9-Cl-v£, relative to ^.
3-exo,10-Cl2-I. has an NMR spectrum similar to that of 3-exo-Gl-I, in
that both have a doublet for H-H- as the single proton at high field, indicat-
ing that chlorine has been added to position 3X. The broadness of the
dichloromethyl proton might suggest that it is on C-8 or C-9 with small four-
bond coupling, but this is not likely, since H-U is not significantly shifted
downfield relative to H-U- of 3-exo-Cl-j. as noted above in compounds with C-8
and C-9 dichloromethyl groups. As in lO-Cl-v^, other resonances (H-8a and
H-9b) are broad. The resonances of H-5X, although isochronous with those of
chloromethyl proton 8b at 6 ^.73> are apparent in the 360 MHz spectrum;
absence of coupling of H-5X to a 3-exo proton is confirmed by an IHDOB
experiment involving monitoring the resonances of H-^ or H-6N.
The dehydrochlorination products of I have either one proton [j
(3,2)] or three protons [j.,-HCl(5,6)] at high field. Thus the methylene
group at C-3 of I is preserved in J^-HC1(5,6) but not in J..-HC1 ( 3 , 2 ) . The
vinyl proton of X-HC1(5,6) appears as a singlet. If it were at C-55 it
would be expectea to show 3 to k Hz coupling to H-^, as H-3 does in ^-HCl-
(3,2). The broadness of H-U and H-6 suggests small allylic coupling in
^,-HCl(5,6). In comparison with J^ protons 3N in j.r-HCl(556) and 6N in
I-HCl(352) are shifted considerably to higher field. This effect is probab-
±y due to decreased deshielding by chlorine atoms on C-2 and C-5 in ]^ which
are lost or repositioned on formation of the double bond; this chemical
shift change is not expected to arise from mere introduction of the double
bond into the bornane system (Jackman and Sternhell, 1969).
3-exo-Cl-I-HCl(556) has a vinyl proton, indicating that the double
bond is in posTtion 5,6 rather than 2,3. Although this vinyl proton
is coupled to H-*4-, the observed coupling of 1.2 Hz (confirmed by a de-
coupling experiment) is more likely to be allylic JY /• than vicinal J,
(see above). The broadness of the resonances for H-4 and H-6 in the '
analogous ^-HC1(5,6) is attributed to small allylic coupling. The re-
maining endo proton (H-3N), like those of the dehydrochlorination products
of 1^ resonates at higher field than in its saturated precursor.
-------
3-exq-Cl-1-2HC1 also has a single vinyl proton coupled to the bridge-
head proton, and here the coupling constant is increased to 1.7 Hz. The
protons of the three chloromethyl groups fall in the range of 6 < O.k.
Although a 3&0 MHz spectrum in C/-D/- separated the protons on C-8 and C-9,
those on C-10 were isochronous and thus appeared as a singlet.
Relationship of Chemical Structure and Biological Activity. Toxaphene
is moderately toxic to mice and houseflies and highly toxic to goldfish
(Table ^). The synergist PB increases its housefly toxicity but not its
mouse toxicity. Compound I (> ^Q% purity) is less toxic to mice than toxa-
phene, but with PB-treated mice it is more toxic than toxaphene, as a
result of the 7.9-fold synergism. The previous finding that J^ is more toxic
than toxaphene to mice (Khalifa et al., 197*0 suggests the presence of a
minor impurity (< 10$) of high toxicity in the sample used in the earlier
studies. This impurity is not removed by column chromatography or prepara-
tive GLC, but is minimized on recrystallization. With houseflies, in the
presence or absence of PB, and with goldfish, ^ is 1.6 to 7 times as toxic
as toxaphene. Addition of a chlorine atom at the 3-exo or 5-exo position
generally reduces the toxicity of I to each species. A large toxicity in-
crease results in introducing the tf-chloro substituent into ^ except with
houseflies in the presence of PB. The mixture of 8-Cl-^ and 9-C1-J, is 1.2
to 2.0 times as toxic as 8-C1-I alone, so 9-C1-I is as much as four times
as toxic as 8-C1-I. Introduction of chlorine aT C-10 greatly reduces the
toxicity of L andits 3-exo-chloro- and 8-chloro derivatives. The toxicity
of I and 3-exo-Cl-I is reduced by dehydrochlorination, particularly when
the olefin is formed at the 5>6 position. Three of the samples assayed
(5-exo-Cl-I, 3-exo, lO-dp-^, and 8,10-Cl2-l) were of only moderate purity
(70 - > 86%). It is not known to what extent the reported potency values
for these compounds are due to the assigned structures as opposed to im-
purities. However, it is clear that each of these compounds is of low
toxicity relative to j^, 8-Cl-^,, and 9-Cl-J^.
In general, the potency of compounds formed on introducing one
chlorine substituent into I decreases in the order: 9-chloro > 8-chloro
> none > 3-exo-chloro or 5-exo-chloro or 10-chloro. It appears that PB-
sensitive mechanisms detoxify ^ more readily than its 8-chloro- and 9-chloro
derivatives.
25
-------
TABLE k. BIOLOGICAL ACTIVITY OF HEPTACHLOROBORNANE I AMD RELATED OCTA- AND
NONACHLOROBORNAWES, HEXA- AND HEPTACHLOROB^RNENES, AND A HEXA-
CHLOROBOKNADIENE
LD,0 , 24 h
Mouse ip, mg/kg
Compound
Purity, %
-PB
+ PB
-PB/ + PB
Housefly topical, Mg/g
-PB
+ PB
-PB/ + PB
Goldfish,
PPb
Comparison Standard
Toxaphene
47
42
1.1
18.0
9.5
1.9
20
Heptachlorobornane
I
>98
75
9.5
7.9
11.5
2.4
4.8
2.9
Octachlorobornanes
3-exo-C\-I.
5-exo-Cl-l
8-C1-I *"
i 8-Cl-T(57%)
+ 9-C1-I (43%)
lO-CM.*'
>98
70
>93
>92
98
>100
-24
3.3
2.5
MOO
MOO
-28
3.1
1.9
48
-0.9
1.1
1.3
>2.1
18.5
26
5.5
3.1
80
3.2
7.5
2.2
1.9
34
5.8
3.5
2.5
1.6
2.4
43
13
1.1
0.55
36
Nonachlorobornanes
3-exo,10-Cl2-I
8,10-C1 -I """
>86
>75
95
60
65
22
1.5
2.7
MOO
44
Hexachlorobornenes
L-HC1(3,2)
j I-HC1(5,6)
95
>98
-65
-50
-1.3
36
225
11
85
3.3
2.6
27
MOO
Heptachlorobornene
I 3-exo-Cl-Jt-
' HC1(5,6)
>99
MOO
MOO
105
35
3.0
MOO
Hexachlorobornadiene
95
105
29
3.6
MOO
26
-------
SECTION III
METABOLISM AND ENVIRONMENTAL DEGRADATION
OF TOXAEHENE AND ITS COMPONENTS
MATERIALS AND METHODS
Analyses
The composition of reaction mixtures and the purity of individual
products were determined by open tubular column GLC using conditions identi-
cal to those in Section I for studies on toxaphene and its metabolites
whereas in analyses of heptachlorobornane ^ and its reaction products the
column temperature was isothermal at 200°C. To assist in quantitation in the
latter investigations, mirex was used as an internal standard with correc-
tions for differences in detector response for mirex and other products
under consideration.
The sulfuric acid-celite column procedure of Zweig and Sherma (1972)
was used for cleanup of biological samples prior to GLC. The column (2 x
20 cm) was packed with 2 g celite 5^5 powder (Sargent-Welch Scientific Co., _-
Anaheim, Calif.), then with a sulfuric acid-celite mixture prepared by
thorough blending with a mortar and pestle of 10 g celite with 10 ml of a
1:1 (v/v) mixture of concentrated sulfuric acid (98.6%) and fuming sulfuric
acid (115$). The following solutions were then added to the column in
sequence, allowing the solvent each time to completely enter the column: 10
ml hexane; 2 ml biological extract in hexane containing 5 Mg mirex; three
portions of 2 ml each of hexane; 100 ml hexane. Once the biological extract
completely entered the celite column, the total eluate was collected up to
a volume of 100 ml. This procedure elutes all toxaphene components without
detectable alteration in their ratios (GLC). It provides > 98^ recovery
with [-^C]toxaphene and essentially quantitative recoveries of the hepta-
chlorobornane, hexachlorobornane and hexachlorobornene derivatives discussed
later (Figure 5); however, the acid treatment decomposes the pentachloro-
tricyclene derivative.
TLC involved the use of bifluorenylidene and benzylidenefluorene as
marker dyes (See Section I). Components or derivatives of [-^CJtoxaphene
and unlabeled toxaphene were detected by radioautography and the diphenyl-
amine reagent (See Section II), respectively.
Spectroscopy and elemental analyses were carried out as in Section II.
27
-------
Chemicals
Sources for the chemicals used were the same as in Sections I and II
except as noted below.
Heptachlorobornane I was obtained by chromatographing a mixture of
crystallized toxaphene (35 g> obtained in 6l% yield as a white crystalline
material on crystallization from isopropanol) and bifluorenylidine (10 mg)
(a yellow marker dye for the elution position of jj in hexane (25 ml) on
a 7 x 100 cm column containing 1 kg of silicic acid packed with hexane. The
column was developed with hexane under 20 psi BL pressure. Elution of the
yellow dye and I (GLC monitoring) began after ID liters of hexane had been
eluted and was "Essentially complete after an additional 1.25 liter of hexane.
Heptachlorobornane £ (360 mg, 1.03% yield; > 99% purity) was obtained on
evaporation of this 1.25 liter of eluent to 2 ml, washing the resulting
crystals several times with ice cold hexane to remove traces of the yellow
dye and recrystallization twice from hexane.
Hexachlorobornene rv, [the same as I-HCl(3,2) in Figure 3] from dehydro-
chlorination of heptachK>robornane _£ wi^h ethanolic KOH and 8-Cl-J^ (see
Section II) from chlorination of ^ were obtained as reported in Section II.
Reactions of Heptachlorobornane
_
Photolysis. Heptachlorobornane ^ at 1.3 x 10 JM in hexane was irradiat-
ed with UV light (\ > 220 nm; ^50 watt medium pressure lamp with quartz fil-
ter; Conrad-Hanovia, Inc., Newark, N.J.) using GLC to monitor the reaction.
The major product was isolated as with the hematin reaction (see below) for
examination by MMR and GLC-CI-MS.
Triphenyltin hydride. A mixture of heptachlorobornane ^ (80 mg, 0.21
mrnol), triphenyltin hydride (120 mg, 0.3U mmol; prepared from triphenyltin
chloride and LiAlHi according to Kuivila and Beumel, 1961) and 2,2'-azobis-
(2-methylpropionitrile) (AIBN) (2 mg) in hexane solution (100 ml) was re-
fluxed for 3 hr. The products were analyzed by GLC, then isolated by column
chromatography (see below) for examination of the three major components by
NMR and GLC-CI-MS. Each of these products was contaminated with ~ 10%
triphenyltin chloride even after chromatographic purification.
Reduced hematin. A preparative scale reaction was carried out by the
general procedure of Wade and Castro (1973) as follows. A solution of
hematin (500 mg, 0.79 mmol) in 500 ml of glacial acetic acid-N-methyl-2-
pyrrolidone (1:1) was mixed with washed (glacial acetic acid and ether) iron
powder (50 mg) in a one liter round bottom flask. Argon was flushed through
the flask for a few min to displace the air and the mixture was then sub-
jected to magnetic stirring for 1 hr, resulting in a color change for the
solution from brown to red indicating the presence of reduced hematin. Hepta-
chlorobornane I (280 mg, 0.7^ mmol) in 250 ml of glacial acetic acid-N-
methyl-2-pyrrbTidone (1:1) was then added through a dropping funnel and the
reaction wag allowed to proceed under argon with continuous stirring for
72 hr at 25 C. The products were extracted into hexane (500 ml x k) which
was then washed twice with each of water, saturated NaHCO- and saturated
28
-------
NaCl and dried over anhydrous MgSCK . Evaporation of the hexane gave 220
mg crude product (~ 86% yield considering the degree of dechlorination)
which was chromatographed on a silicic acid column (2.5 x ^0 cm) with hexane
as the eluting solvent and pressure as above. GLC analysis of relevant
fractions revealed heptachlorobornane 1^ four major products (££r£) and an
unknown (Figures 5 and 6). The first e'luting compound is an unidentified
C10H11C:15 derivative (GLC-CI-MS) which, although in very minor amount, is
not separable by GLC from compound V. Compounds III and IV are easily obtain-
ed pure by evaporating the hexane from fractions GL> 80^*purity and re-
crystallizing from hexane. Compound j£ is essentially pure in the last
fractions eluted. nomnounds j and JJjj- when present in the same fractions
are separated by first crystallizing j, from hexane and then crystallizing
III. When compounds II and iyx are present in mixtures, compound JjJ- is
"removed first on crystallization from hexane then jj. is recrystallized from
hexane. Selective crystallization is not appropriate to separate mixtures
of II and _T£. The amount and purity of each product were as follows: 11-25
mg, > 90%; JJJL-106 mg, > 99& j£-28 "S* > 99& V-10 mg, > 99$. Structures
of these compounds were assigned by HMR and CI-MS.
The reaction rate was monitored (GLC) in a small scale reaction involv-
ing 3 ing heptachlorobornane _£, 15 mg hematin, 2 mg iron powder and 25 ml
total reaction volume but otherwise as above.
Bovine rumen fluid. Rumen fluid from a fistulated cow at the University
of California at Davis was used immediately after filtration through four
layers of cheese cloth to remove large particles. Heptachlorobornane ^
(66 (Og) in ethanol (l ml) was added to the fresh fluid (~ 500 ml) completely
filling a flask which was then stoppered and incubated at 37 C. The reaction
mixture was acidified to pH ~ 1 by adding sulfuric acid and extracted with
ether containing mirex (5 Mg5 internal standard). The ether was dried
(MgSO. ), evaporated and the resulting residue was dissolved in hexane (2 ml)
and subjected to cleanup on the fuming sulfuric acid-celite column prior to
GLC analysis.
Sewage primary effluent. The incubation and analysis procedures used
for the rumen fluid were also employed with the primary effluent (anaerobic)
from the sewage treatment process (Richmond Field Station, University of
California, Richmond).
Rat liver microsome-MADPH system. Reaction mixtures in 0.1M pH 7.4
phosphate buffer (2 ml) consisted of rat liver microsomes (U mg protein),
NADPH (0 or 3 Mg) and heptachlorobornane^ (10 Hg) added last in ethanol
(50 |ol). After one hr incubation at 37°C in air or argon with shaking, each
mixture was extracted with hexane (5 ml x 3) containing mirex (5 Hg, internal
standard) and the extract was subjected to cleanup and analysis as in the
rumen fluid studies.
In Vivo Studies
Treatment of chickens and mammals and analyses of their feces and
tissues. The following test animals were used: female white leghorn chickens
(1.1-1.5 kg) and male rabbits (^78-610 g) from Western Scientific Supply Co.
29
-------
Figure 5. Conversion of heptachlorobornane ^ to various hexachloro-
bornane, hexachlorobornene, and pentachlorotricyclene
derivatives.
100
i.o
3.5
Figure 6.
1.5 2.0 2.5 3.0
Eluent Volume, liters
Chromatograpliy of I and its reaction products with reduced
hematin on a silicic acid column developed with hexane.
Conditions are given in the text.
-------
(West Sacramento, Calif.); male Swiss-Webster mice (18-20 g), male albino
Sprague-Dawley rats (150-165 g), male Hartley guinea pigs (224-260 g), and
male hamsters (70-85 g) from Simonsen Laboratories, Inc. (Gilroy, Calif.);
male long-tailed monkeys (Macaca fascicularis) (~ 8 kg) (colony born and
handled at the Primate Research Center, University of California, Davis).
Chickens, mice, rats and monkeys received either toxaphene (~ 13 mg/kg)
or heptachlorobornane _£ (~ 3 mg/kg) using soybean oil as the administration
vehicle and rinse for the stomach tube. Hamsters, guinea pigs and rabbits
received the same mg/kg dose as above but the compounds in soybean oil were
applied to lettuce which was quickly consumed by the animals. Feces were
collected in each case for 72 hr then the toxaphene-treated animals, except
the monkeys, were sacrificed for removal of the liver and a sample of fat.
Liver and fat samples were removed in sei arate experiments from rats 7 nr
after treatment with heptachlorobornane I. These tissues were also obtained
by biopsy at 72 hr after treatment of monkeys with toxaphene and heptachloro-
bornane I.
An additional study with rats involved administration of compounds
JjJ, and 11^ (3-1 mg/kg each) or a mixture of compounds J£, and jj^. (0.95 and
0.52 mg/kg, respectively) as above, collecting the 0-72 hr feces.
The fat, liver and feces, as appropriate, were extracted with acetone
(~ 10 ml/g; containing a total of 5 Mg mirex as internal standard in studies
with heptachlorobornane^.), the acetone evaporated and the products in hexane
solution were subjected to cleanup on the sulfuric acid-celite column and
GLC analysis.
Treatment and analysis of houseflies. Adult female houseflies (Musca
domestica L., SCR susceptible strain, 3-4 days after emergence, 18-20 mg)
were treated topically on the abdomen with heptachlorobornane I (4.5 Mg/g)
in acetone. After 24 hr the flies and their feces were extracted with ace-
tone containing mirex (5 Mg) and subjected to cleanup and GLC analysis.
Bioassays
Procedures for determining the 24-hr LDSO values using adult female
houseflies (with and without PB) and goldfisn were as in Section II.
RESULTS
Identification of Mew Compounds. The products under consideration and
their designations are shown in Figure 5. Compounds j^ are crystalline materials
that are partially resolved by TLC and completely by GLC (Table 5). They
vary in sensitivity of detection by EC depending on the number of chlorines
and, with IT and III, the configuration of the chloro substituent at C-2
(Table 5).
Each of compounds J£-%, was isolated from the reduced hematin system in
sufficient amount for identification by I\MR and CI-MS. Compound IV was
identical in all respects with an authentic standard [l>-HCl(3,2) of Section
II)] so its structure is not considered further here. **"
31
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The CI-MS data provide the elemental compositions of compounds
(Table 5). Under CI conditions saturated chlorobornanes give no [M+l]+ peak
but instead give [M-Cl] as the base peak (Holmstead _et al. , 197^-; see
Section II). Hexachlorobornanes _I^ and J.££, conform to this relationship.
In contrast, hexachlorobornene P£ and pentachlorotricyclene V_ give small
[M+l]+ peaks with [M-Cl]+ and fM-HCl]+as the base peaks, respectively.
The structural assignments for compounds jj.., Hjk, and J£, are based on
MMR spectral data given in Table 6. Each compound contains six chloromethyl
group protons with the typical geminal coupling of ~ 12 Hz observed with
heptachlorobornane ^.
The assignment of the bornane skeleton for compounds IJ, and J^^ is based
on the resemblence of their observed coupling constants to the analagous
coupling constants of heptachlorobornane J... Compounds ^ and LI.JL each give
signals for six ring protons, establishing that they are formeoby reductive
dechlorination. The similarity of coupling patterns of protons k} 5 and 6
in compounds I -III reveals that I undergoes reductive dechlorination at the
2 position to form J^. and LLjL. The new proton on C-2 of _££. and TjJ. can be
assigned as endo or exo by its coupling with the protons on C-3- In this
ring system, syn coupling is typically larger than anti (Williamson, 1963).
The C-2 proton in jj^ is assigned to the exo position since it is coupled with
the 3-endo proton with a. coupling constant of 5-0 Hz and with the 3-exo pro-
ton with a coupling constant of 10.5 Hz. In compound III, the coupling con-
stant of the proton on C-2 with H-3N (9.0 Hz) is larger than that with H~3X
(^.7 Hz), so this isomer must have H-2 in the endo position. The relative
chemical, shifts of H-5X and H-6W in compounds I-III are consistent with these
assignments. In ^^ as in J^, H-6N is farther ITownTi eld than H-5X whereas in
I1Z H-6N has been shifted to considerably higher field by introduction of the
endo proton at C-2. Changes in the long-range coupling of protons on the
geminal chloromethyl groups with addition or removal of chlorine atoms on
the ring have been observed before (see Section II).
Compound %, does not retain the bornane skeleton and instead is assigned
a tricyclene structure to accommodate the C^oK--.^!^ composition and the MMR
spectral features. There are five ring protons, as in Jf,, but two chlorines
are removed. The absence of any vinylic proton (see Section II ) and the
presence of a single proton at higher field (b 1.9^-) a^e consistent with
a cyclopropane ring formed on elimination of chlorine atoms at positions 2
and 6. Suitable literature data do not appear to be available for the
coupling patterns in tricyclene derivatives, and unfortunately the spectrum
of 3k is inadequate for accurate measurements of the small coupling constants
involved (~ 1.3 Hz). However, the coupling constants of the ring protons
are considerably different from those of bornane derivatives j^-TTT. The
geminal coupling of the protons on C-3 is reduced from 16.2 Hz in I to 12.0
Hz in V, and long-range coupling appears to be introduced between $-k and
H-6. "The geminal protons on C-3 have similar small (~ 1.3 Hz) coupling with
E-k, as expected in a tricyclene derivative, where they are symmetrical with
regard to H-4. The same small coupling is also evident for H-k with H-5.
The structures of hexachlorobornanes ^ and jjjr are confirmed by
X-ray crystallography (Wong et al. , 1978) but suitable crystals of penta-
33
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chlorotricyelene V. have not been obtained for X-ray examination.
Products from reaction of heptachlorobornane J^ in other systems are
identified or tentatively identified by comparison with the standards from
the reduced hematin system and with authentic hexachlorobornene J^£, (see
Section II) using GLC cochromatography in each case. Supporting WMR and
GLC-CI-MS evidence is available on .identifications in the following cases: II
from photolysis; II-IV from the triphenyltin hydride system.
^Wd^ - 4^i'^
Reaction Products of Heptachlorobornane I in Various Chemical, Photo-
chemical, and Metabolic Systems. Quantitative data on the products from
reaction of heptachlorobornane Jj, in various systems are given in Table 7.
These data are based on at least three analyses in each case and standard
errors are reported when three to five independent experiments were involved.
Photolysis of heptachlorobornane Jj, in hexane irradiated with UV light
proceeds rapidly and gives only two products detected by GLC-EC, hexa-
chlorobornanes r^j. and ^J, in a ~ 0.12 ratio. The major products with tri-
phenyltin hydride "are hexachlorobornanes Lj^ and _.££. in similar yields, and
there are small amounts of hexachlorobornene _TV, and a compound tentatively
identified as pentachlorotricyclene V. Reduced hematin gives excellent
yields of products .£%.-£. with minimal'*difficulty in their isolation. The
reaction proceeds rapidly at 25°C (^/2= ~10 min) ajl<3- the Protiuc'fc ratio does
not significantly change during the course of the reaction, with hexachloro-
bornanes JjTjL and II appearing in a ~ 3-8 ratio . Bovine rumen fluid and
sewage primary effluent form only two products detected by GLC-EC after
cleanup, _i._e. , hexachlorobornanes !££, and H, in a ~ 2.h ratio. The con-
version rate is rapid in rumen fluid (t.,/p= ~ 2 hr)and significant in sewage
primary effluent considering that it lacks the more potent degrading organ-
isms of sewage sludge.
Rat liver microsomes do not metabolize heptachlorobornane I unless
fortified with NADPH. They apparently carry out different reactions under
aerobic and anaerobic conditions in the presence of cof actor. The identi-
fied products under anaerobic conditions are hexachlorobornanes 13.J. and II
in a 2.0 ratio, but these compounds are not detected on incubation in air
where metabolism of I proceeds at a greater rate.
Metabolites of Heptachlorobornane 1^ Rats treated with this heptachloro-
bornane contain moderate levels of the parent compound and low levels of
metabolites I£-J-3£. ^n *ne fa* ant^ -Low levels of each of these compounds in
the liver (Table 8). A monkey treated with heptachlorobornane I at 3-0 mg/kg
contains the following ppb levels of j^, j^, ^f, and IQfa respectively^ in
tissues at 72 hr: 255, 0, 0 and 0 in fat; 50, 0, ^-50 and 0 in liver.
Metabolite yields in feces are given in Table 9- Each species examined
excretes metabolites I^-jT^.. Chickens excrete large amounts of ^ whereas
mice, guinea pigs and rabbits excrete intermediate amounts and rats, hamsters
and monkeys excrete little or no unmetabolized compound. Metabolite P^ is
minor relative to JX and Ijf^ in each species. The yields of ^jL + jjjj. are
highest with rabbits and monkeys, intermediate with chickens, rats and guinea
pigs, and lowest with mice and hamsters. The ratio of metabolites III/ II
35
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TABLE 8. AMOUNT OF HEPTACHLOROBORNANE _£ AND ITS METABOLITES IN FAT AND
LIVER AT 7 AND 72 HE AFTER ORAL ADMINISTRATION OF I TO RATS AT
3.1 mg/kg ***
Tissue
Fat
Liver
Time, h
7
72
7
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I
453 ± 285
335 ± 44
8.6 ± 4.1
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10.2 ± 2.3
9.2 i 7.0
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15 ± 9
14 ± 2
3.4 ± 0.9
0.8 t 0.6
Metabolite
ratio, III/II
1.8
2.6
1.7
4.6
Q
Average and standard error based on experiments with 3 rats at 7 hr and
5 rats at 72 hr.
37
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-* CD
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0 I 43
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S4 o3
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-------
is nearly constant (l.O-l.U) for all species except rats where metabolite
definitely predominates.
Studies with rats reveal that the yields noted for metabolites IT and
•&gF&
1IT, are minimal values. Thus, when rats are administered either hexachloro-
bornane JJ, or JQ£, their f eces contain the administered compound as the only
GLC-EC detectable product. On administering a mixture of hexachlorobornanes
J.J. and IIj., the f eces excreted within 72 hr contains ^5-^7$, of the un-
metabolized compounds and no metabolites are detected by GLC-EC. These
findings clearly establish that reductive dechlorination at a geminal di-
chloro group is a major pathway in metabolism of heptachlorobornane I in rats,
other mammals and chickens .
Houseflies treated with heptachlorobornane j^, contain more hexachloro-
bornane J^ than its isomer LI^L. (Table 7). The relatively high yield of
hexachlorobornene IV suggests that dehydrochlorination may be more important
in housefly than in mammalian or chicken metabolism of £,.
Products Derived from Toxaphene in Fat, Liver, and Feces of Rats.
Figure 7 compares the GLC-EC pattern of toxaphene with those of toxaphene-
derived products in fat, liver, and faces 72 hr .after oral administration of
toxaphene. The pattern of products in fat is similar to that of toxaphene
itself although there are minor changes, e,.g_. , reduced importance of peaks
16 (3-C1-I + 9-Cl-vp and 2k relative to 9 (containing j^). The liver chromato-
gram is characterized by: peaks corresponding to each of the 29 designated
toxaphene components, the major one appearing at the position of 9 (other
criteria not used to confirm identity as ^); a large change in ratio for
peaks 9 and 16; four major late-eluting peaks (A-D), some or all of which
are possibly toxaphene components undergoing slow metabolism and selective
concentration; appearance of a new peak between those designated as 26 and
27; a major peak at ~ hO rain (the general position of hexachlorobornane II
or III - possible identity with this hexachlorobornane not further examined).
The patterns of fat and liver chromatograms at 7 hr are intermediate be-
tween those of toxaphene and the corresponding 72-hr samples. The feces
shows peaks corresponding to each of the 29 designated toxaphene components
but in contrast to the tissues there is a predominance of short tR compounds.
Peaks 9 and 16 are barely detectable with feces and three of the major peaks
correspond to compounds .El- IV (metabolites of heptachlorobornane JjJ,
TLC-GLC evidence is available that L-3X are present in feces of rats
receiving [^C]toxaphene orally. The acetone-soluble metabolites include
compounds at the origin on TLC as well as materials spread over the normal
broad TLC region for toxaphene. Cleanup on the sulfuric acid-celite column
removes the materials at the origin on TLC and subsequent TLC-GLC reveals
compounds ^I,-,!^. each at its expected TLC position (Table 5; see Section I).
Products Derived from Toxaphene in Fat, Liver and Feces of Other
Mammals and Chickens. The fat of each species gives a GLC product pattern
very similar to that of toxaphene with each of the 29 designated toxaphene
components clearly evident although with some alterations in peak ratios
(Figure 8). The liver chromatogram patterns differ with each species and
39
-------
40
60 80
MINUTES
100
120
140
Figure 7. Open tubular column GLC analysis of toxaphene and of
toxaphene-derived products in fat, liver, and faces of rats
72 hr after oral administration of toxaphene. The 29 aratdc
numerals refer to toxaphene components present in greater than
1$ amounts as designated in Section I. The chromatographic
positions of toxaphene components ^ (peak 9) and 8-C1-I +
9-Cl-£ (peak 16) and of metabolites jfe^L of heptachloro-
bornane J,, are designated by structural formulae. Letter
designations (A-D) refer to toxaphene-derived products in
liver, some or all of which may be toxaphene components.
Asterisks designate interfering materials of biological
origin.
-------
BO 80
MINUTES
140
Figure 8. Open tubular column GLC analysis of toxaphene and of
toxaphene-derived products in the fat of chickens and
mammals 12 hr after oral administration of toxaphene.
The §9 arabic numerals refer to toxaphene components
present in fe 1$ amounts as designated in Section I .
The chromatographic positions of toxaphene components
I (peak 9) and 8-Cl-j^ plus 9-C1-I (peak 16) are
^5idicated. Asterisks designate interfering materials
of biological origin.
Page
-------
vary from relatively few major peaks (chicken, rat and monkey) to many major
peaks (the other species) (Figure 9)- Several products retained for 72 hr
in liver give similar tR values in two or more species (A -^). It
is not established which of the designated liver peaks ar"e* toxaphene com-
ponents and which are metabolites. The feces chromatograms differ greatly
depending on the species with chickens and guinea pigs showing the closest
similarity to toxaphene and rats and monkeys the largest difference (Figure
10). This relationship parallels in the most part the amount of unmetaboliz-
ed heptachlorobornane I excreted by each species (Table 9)- Fecal products
chromatographing in the positions of metabolites II, JJI. and JJ£, are detected
with monkey and several of the other species (Figure 10J but their identity
is not confirmed by other analytical methods.
Degradation of 2,2,5-endo,6-exo,8,8,9?10-Octachlorobornane (8-Cl-l)
by Reduced Hematin.This oct^Cttlor'olSOrnane reacts rapidly with reduced-**
hematin in a small scale reaction to give five major products. Two of these
have t-n values similar to hexachlorobornane III and hexachlorobornene IV and
a third chromatographs as anticipated for a Tieptachlorobornane. The differs
have shorter tR values. Heptachlorobornane I is found in < 1% amount.
Biological Activity. Heptachlorobornane I is more toxic than its
metabolites or derivatives H-%, to houseflies and goldfish (Table 5)- A
greater loss in toxicity occurs on tricyclene formation (j^) or removing the
exo chlorine at C-2 to form jy^, than on removing the endo chlorine or on de-
hydrochlorination to give ^J^. and JT^., respectively. Each of the products
is probably metabolized by a microsomal cytochrome P-^-50 system in houseflies
since they are synergized by PB.
DISCUSSION
Figure 5 gives the metabolic and chemical pathways established for
heptachlorobornane I. In most cases examined, the major reaction is reduc-
tive dechlorination at the geminal dichloro group yielding isomeric hexa-
chlorobomanes J.£ and ]^^> but in some systems there is also dehydrochlorina-
tion to hexachlorobornene i and formation of pentachlorotricyclene J£.
Photochemical reductive dechlorination of I leads preferentially to
the product with chlorine at the 2-endo position/*^.. js. , hexachlorobornane
II. However, there are large amounts of unidentified products which are
not detected by the usual GLC-EC method. Similar findings are reported in
the photochemistry of 2-endo ,3,3,5- exo , 6- exo , 8 , 9 ? 1° ? 10-nonachlorobomane
(Parlar et al. , 1976 b). Triphenyltin hydride gives more n£ than J£ and also
yields some hexachlorobornene jjf. and possibly pentachlorotrocyclene j^. It
is likely that a radical intermediate is involved in these photochemical
(Parlar _et al. ,1976b) and tin hydride (Kuivila, 1968) reductive dechlorina-
tions of £, to Jkk and JO.I .
Reduced hematin provides a convenient system to study the various
dechlorination reactions since it rapidly gives j^Jrjf. in high yields and the
products are more resistant to reaction than starting heptachlorobornane I.
A radical intermediate is likely to be involved in reductive dechlorina-
tion of I to hexachlorobornane s II and III (Wade and Castro, 1973). Tri-
»M -^ -* --"^--' ^ ' •* "* f
-------
TOXAPHENE
CHICKEN
* GUINEA PIG
:JL
HAMSTER
c MONKEY
IK
iao i4o
MINUTES
Figure 9- Open tubular column GLC ajaalysis of toxaphene and of
toxaphene-derived products in the liver of chickens
and mammals 72 hr after oral administration of
toxaphene. The 29 arabic numerals refer to toxaphene
components present in g 1% amounts as designated in
Section I. The chromatographic positions of toxaphene
components ^ (peak 9) and 8-C1-J, plus 9-Cl-^ (peak 16)
are indicated. Letter designations (A-J£) refer to
toxaphene-derived products in liver, some of which may
be toxaphene components. Asterisks designate interfering
materials of biological origin.
Page 43
-------
TOXAPHENE
CHICKEN
"it
GUINEA PIG
HAMSTER
\ *t» a a
MINUTES
Figure 10. Open tubular column GLC analysis of toxaphene and of
toxaphene-derived products in the feces of chickens
and mammals 72 hr after oral administration of
toxaphene. The 29 arat>ic numerals refer to toxaphene
components present in g 1$ amounts as designated in
Section I, The chromatographic positions of toxaphene
components j, (peak 9) and 8-C1-J, plus 9-Cl-j^ (peak 16)
and of metabolites ^J^-J^ of heptachlorobornane ^ are
indicated. Asterisks designate interfering materials
of biological origin.
Page
-------
cyclene formation to give V may also proceed via the same radical intermediate
whereas dehydrochlorination to IV probably involves a different pathway.
Reduced hematin reacts with octachlorobornane 8-Cl-_I, to give products formed
by both reductive dechlorination and dehydrochlorination (this study; Khalifa
_et al., 1976). It also acts in aqueous medium to cleave about half of the
carbon-chlorine bonds in toxaphene (Khalifa et al., 1976).
The finding of extensive reductive dechlorination of heptachlorobornane
I in bovine rumen fluid and sewage primary effluent suggests that this and
other toxaphene components may undergo significant reductive dechlorination
in the bovine rumen prior to absorption and in microbial systems under anae-
robic conditions.
Metabolism of heptachlorobornane j, by rat liver microsomes requires
NADPH but proceeds by a different mechanism in air, where the products are
not identified, than in an inert atmosphere, where hexachlorobornanes II and
III are the major products. It was therefore of considerable interesf*£b
'rind hexachlorobornanes _££, and J^., in the feces of chickens and six mammalian
species orally administered heptachlorobornane "^ The hexachlorobornane
ratio (HJ/jjp is similar in the fat, liver and feces of rats to that found
in the microsomal system, indicating that reductive dechlorination in vivo
may occur in the liver microsomes. This ratio also is similar to those found
in the hematin, bovine rumen fluid, and sewage primary effluent reactions,
suggesting similar mechanisms of reductive dechlorination in each case on
reaction with reduced porphyrins. Chickens, guinea pigs, hamsters, rabbits,
mice and monkeys give more similar amounts of jj, and IO than observed in
rats and these in vitro systems. In contrast, houseflxes give a. greatly
different ratio of hexachlorobornanes III and IX, possibly due to varying
rates in their further metabolism rather than to different mechanisms in
their formation since synergist studies suggest the involvement of cytochrome
P-450 in detoxification of heptachlorobornane ^ and its derivatives.
Metabolite identification is more difficult following administration of
toxaphene compared to an individual toxaphene constituent because of the
likelihood that many toxaphene components undergo reductive dechlorination
and dehydrochlorination to products that fall within the same GLC tR range.
However, some findings with toxaphene itself are of interest. The liver of
several species contains an unusual proportion of toxaphene-derived products
of very high t^ values appropriate for heavily chlorinated compounds.. The chroma-
tographic pattern of the rat fecal products is characterized by short tg
compounds, suggesting extensive dechlorination, a conclusion supporting
previous studies with [-^C]- and [3°Cl]toxaphene (Ohsawa et al., 1975). The
rat fecal products appear to include metabolites II-iy
-------
SECTION IV
MUTAGENIC ACTIVITY OF TOXA.PHENE AND SOME OF ITS COMPONENTS
MATERIALS AND METHODS
Samples of Hercules toxaphene manufactured in various years (see Section
l) or of toxaphene components or fractions and samples of related chlorina-
ted terpenes from other manufacturers (see Section I) were dissolved in
DMSO and added to cultures of the TA100 histidine-requiring
mutant strain of Salmonella typhlmurium (Ames et al., 1975)- Potency is
expressed as revertants per mg of test chemical.
RESULTS AND DISCUSSION
Mutagenic Activity of Toxaphene and Related Chlorinated Terpenes.
Toxaphene manufactured by Hercules from 19^9 to 1975 has a mutagenic potency
averaging 728 revertants/mg and ranging from 310 to 1270 revertants/mg (Table
10). This potency range is similar to that for other samples of related
chlorinated terpenes from various manufacturers (Table 10).
Other Observations. Preliminary studies were made on the nature of the
mutagenic components in standard Hercules toxaphene (Table 11). Hepta-
chlorobornane ^ has no significant mutagenic activity. Crystallization of
toxaphene from isopropanol concentrates the mutagenic activity in the mother
liquor fraction but does not completely remove mutagenic agents from the
crystalline portion. Passage of toxaphene in hexane through a celite column,
with or without fuming sulfuric acid, removes some of the mutagenic activity,
thereby decreasing its overall potency. A highly mutagenic fraction (> 17,000
revertants/mg) is. obtained on chromatographing toxaphene on celite or silicic
acid columns using various solvents, with methanol for final elution of the
mutagenic fraction. The identity of the mutagens in toxaphene has not been
established.
-------
TABLE 10. MUTAGENIC ACTIVITY OF HERCULES TOXAPHENE AND RELATED CHLORINATED
TERPENES IN THE TA100 HISTIDINE-REQUIRING MUTANT STRAIN OF
SALMONELLA TYPHIMURIUM
Revertants/
mg ^^
Sample
Hercules toxaphene by year
Sample
Revertants/
mg
Related chlorinated terpenes
195^
1969
1973
1957
19^9
1970
1960
1975
1974
1963
310
530
575
685
725
7^5
750
760
930
1270
Procida
Vicksburg A
Vicksburg B
Hercasa
Flit & Fontaine
East European dark
East European light
Strobane T-100
Melipax
Sonford
Bison A
Bison B
230
380
620
394
440
465
580
515
635
945
1530
420
TABLE 11. MUTAGENIC ACTIVITY OF HERCULES STANDARD TOXAPHENE AND ITS
FRACTIONS IN THE TA100 HISTIDINE-REQUIRING MUTANT STRAIN OF
SALMONELLA TYPHIMURIUM
Sample
Revertants/
mg
Heptachlorobornane I
Standard toxaphene
Crystallization of toxaphene
Crystalline fraction
Mother liquor
Column chromatography of toxaphene,,
methanol fraction
< 30
550
116
950
> 17,000
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RECOMMENDATIONS
The findings provide a portion of the needed information on toxaphene
composition and toxicology but there are continuing research needs in several
aspects of the overall problem. The open tubular column GLC method can be
further perfected by coupling it with CI-MS for peak analysis. It is evident
that further toxaphene components can be prepared and identified for toxicological
evaluation. The metabolism studies deal with only one major toxaphene component
and provide only partial information in this case on its metabolism and
environmental fate. These studies should be continued using mammals, plants,
soils and various environmental systems. Mutagenic components in toxaphene
should be further defined and possibly identified and ways and means sought
to remove them from the commercial insecticide.
Difficulties in defining the composition and toxicology of toxaphene
illustrate the dilemma created when complex and poorly-defined mixtures of
polychlorohydrocarbons are introduced into the environment in enormous amounts.
While alternatives are being developed to minimize dependence on chemicals for
pest management, it is preferable where possible to use pesticides whose
structures and metabolites are well characterized and which have short-half
lives and target organism specificity.
-------
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Abd El -Aziz, S. A., M. T. Shafik and S. A. El-KMshen. 1965. The Separation
of the In Vitro Breakdown Products from Toxaphene Using Paper
Chromatography. Alexandria J. Agr. Res. 13:37-
Abd El -Aziz, S. A., M. T. Shafik and S. A. El-Khishen. 1966. The In Vitro
Breakdown of Toxaphene by the Cotton Leafworm Using the Colorimetric
Methods of Analysis. Alexandria J. Agr. Res. 14:13.
Ames, B. W. , J. McCann and E. Yamasaki. 1975- Methods for Detecting
Carcinogens and Mutagens with the Salmonella/Mammalian -Mi cros ome
Mutagenicity Test. Mut. Res. 3~
Anagnostopoulos , M. L. , H. Parlar and F. Korte. 1974. Beitrage Zur
Okologischen Chemie. LXXI. Isolierung, Identifizierung und Toxikologie
Einiger Toxaphenkomponenten. Chemosphere 3:65.
Buntin, G. A. 1951- Insecticidal Compositions Comprising Chlorinated
Camphene. U. S. Patent No. 2,565,14-71.
Casida, J. E., R. L. Holmstead, S. Khalifa, J. R. Khox, T. Ohsawa, K. J.
Palmer and R. Y. Wong. 1974. Toxaphene Insecticide: A Complex
Biodegradable Mixture. Science 183: 520.
Casida, J. E., R. L. Holmstead, S. Khalifa, J. R. Khox and T. Ohsawa. 1975.
Toxaphene Composition and Metabolism in Rats. Environ. Qual. Saf.
Suppl. 3 -.365.
Crowder, L. A. and E. F. Dindal. 1974. Fate of Cl-Toxaphene in Rats.
Bull. Environ. Contam. Toxicol. 12:320,
Guyer, G. E. , P. L. Adkisson, K. DuBois, C. Menzie, H. P. Nicholson,
G. Zweig and C. L. Dunn. 1971. Toxaphene Status Report. US EPA,
Washington, D. C.
Hoffman, R. A. and A. W. Lindquist. 1952. Absorption and Metabolism of
DDT, Toxaphene, and Chlordane by Resistant House Flies as Determined
by Bioassay. J. Econ. Entomol. 45:233.
Holmstead, R. L. , S. Khalifa and J. E. Casida. 1974. Toxaphene Composition
Analyzed by Combined Gas Chromatography- Chemical lonization Mass
Spectrometry. J. Agr. Food Chem. 22:939=
-------
Jackman, L. M. and S. Sternhell. 1969- "Applications of Nuclear Magnetic
Resonance Spectroscopy in Organic Chemistry", p. 231. Second Ed.,
Pergamon Press, New York, N. Y.
Jennings, B. H. and G. B. Hersch'bach. 1965. The Chlorination of Camphene.
J. Org. Chem. 30:3902.
Khalifa, S., T. R. Mon, J. L. Engel and J. E. Casida. 1971)-. Isolation of
2,2,5-endo,6-exo,8,9,10-Heptachlorobornane and an Octachlorobornane
from Technical Toxaphene. J. Agr. Food Chem. 22:653-
Khalifa, S., R. L. Holbnstead and J. E. Casida. 19?6. Toxaphene Degradation
by Iron(II) Protoporphyrin Systems. J. Agr. Food Chem. 2^:277-
Kooyman, E. C. and G. C. Vegter. 1958. Bicyclanes-I. The Halogenation of
2:2:l-Bicycloheptane (Norbornane). Tetrahedron 4:382.
Kuivila, H. G. 1968. Organotin Hydrides and Organic Free Radicals. Ace.
Chem. Res. 1:299.
Kuivila, H. G. and 0. F. Beimel,Jr. 1961. Reduction of Some Aldehydes
and Ketones with Organotin Hydrides. J. Am. Chem. Soc. 83:12^4-6.
LeBel, W. A., P. D. Beirne and P. M. Subramanian. 1964. The Mechanism
of Elimination Reactions II. Kinetic Preference for exo-cis Biomolecular
Elimination with trans-2,3-Dihalonorbornanes. J. Am. Chem. Soc.
86:4144.
Lehman, A. J. 19&5- "Summaries of Pesticide Toxicity". P. 36. Association
of Food and Drug Officials, U.S., Topeka, Kan.
Matsumura, F., R. W. Howard and J. 0. Nelson. 1975- Structure of the
Toxic Fraction A of Toxaphene. Chemosphere 4:271.
National Cancer Institute. 1977. Experimental Design and Status Report,
and Individual Animal Histopathologic Report:Toxaphene. Completed
October 1, 1977-
Nelson, J. 0. and F. Matsumura. 1975- A Simplified Approach to Studies of
Toxic Toxaphene Components. Bull. Environ. Contam. Toxicol. 13:464.
Ohsawa, T., J. R. Knox, S. Khalifa and J. E. Casida. 1975. Metabolic
Dechlorination of Toxaphene in Rats. J. Agr. Food Chem. 23:98.
Palmer, K. J., R. Y. Wong, R. E. Lundin, S. Khalifa and J. E. Casida. 1975.
Crystal and Molecular Structure of 2,2,5-_endo,6-exo,8,9,10-Hepta-
chlorobornane , Ci0H-]jLCl7, a Toxic Component of Toxaphene Insecticide.
J. Am. Chem. Soc. 97:408.
50
-------
Parlar, H. , S. Ga'b, A, Michna and F. Korte. 19?6a. Beitrage Zur Qkologischen
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Vergleichssubstanzen fur die Struckturanalyse von Toxaphen-Komponenten.
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Parlar, H. , S. Nitz, S. Gab and F. Korte. 1977. A Contribution to the
Structure of the Toxaphene Components. Spectroscopic Studies on
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Poutsma, M. L. 1969. Free-Radical Chlorination of Organic Molecules.
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Ed. Marcel Dekker, New York, N. Y.
Richey, H. G. , J. E. Grant, T. S. Garbacik and D. L. Dull. 1965. Chlorina-
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Samosh, L. V. 197^-. Chromosome Aberrations and the Character of Satellite
Associations in Accidental Exposure of the Human Body to Polychloro-
camphene. (Transl. from Russian). Cytol. and Gen. 8:2^.
Seiber, J. W. , P. F. Landrum, S. C. Madden, K. D. Nugent and W. L. Winterlin.
1975 • Isolation and Gas Chromatographic Characterization of Some
Toxaphene Components. J. Chromatogr. 11^:361.
Stahl, E. , Ed. 1969. "Thin-Layer Chromatography" . P. 86. 2nd Ed.
Springer -Ver lag, New York, N.Y.
Turner, W. V., S. Khalifa and J. E. Casida. 1975. Toxaphene Component A.
Mixture of 2,2, g-endo ,6-exo, 8, 8, 9 , 10-Octachlorobornane and 2,2,5-
endo,6-exo,8,9,9,10-cctachlorobornane. J. Agr. Food Chem. 23:991.
Wade, R. S. and C. E, Castro. 1973. Oxidation of Iron ( II ) Porphyrins by
Alkyl Halides. J. Am. Chem. Soc. 95:226.
Walling, C. and M. F. Mayahi. 1959- Some Solvent and Structural Effects
in Free Radical Chlorination. J. Am. Chem. Soc. 8l:lM35.
Williamson, K. L. 1963. Substituent Effects on Nuclear Magnetic Resonance
Coupling Constants and Chemical Shifts in a Saturated System:
Hexcahlorobicyclo[2.2.l]heptenes. J. Am. Chem. Soc. 85:516.
Wong, R. Y., K. J. Palmer, M. A. Saleh and J. E. Casida. 1978. Unpublished
Results .
Zweig, G. and J. Sherma. 1972. Chapter 56. Toxaphene. Anal. Methods
Pestle., Plant Growth Regul.
51
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TECHNICAL REPORT DATA
read Instructions on the reverse before completing)
REPORT NO
EPA-600/1-78-060
TITLE AND SUBTITLE
5. REPORT DATE
September 1978
Toxaphene Composition and Toxicology
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSIOf^NO.
AUTHOR(S)
John E. Casida and Mahmoud Abbas Saleh
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Pesticide Chemistry and Toxicology Laboratory
Department of Entomological Sciences
University of California
Berkeley, CA 94720
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R-803913
2. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTF, NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The composition and metabolism of Toxaphene have been examined to aid in understanding
the conditions under which this insecticide can be most effectively and safely used. Each
of 8 Toxaphene samples manufactured by Hercules Chemical Co. from 1949 to 1975 shows the same
29 major peaks and in almost identical ratios. About 85% of the total peak area is accounted
for by these 29 peaks which individually vary from 1 to R% of the total. The 8 Toxaphene
samples were easily differentiated from 12 samples of chlorinated terpenes from other manufac-
turers in the United States and abroad. There is surprisingly little variation in the acute
toxicity of any sample.
Fivp major Toxaphene components (2,2,5-endo,6-exo,8,9,10-heptachloiobornane (I) and its
3-exo-chloro-, 8-chloro-, 9-chloro- and 10-chloro-derivatives) collectively account for up to
23* of the technical grade Toxaphene and up to 34% of those of chlorinated 2-exo,10-dichloro-
bornane. Chlorination of 2-exo,10-dichlorobornane provides a convenient source of I and other
chlorinated bornanes. The toxicity to mice, houseflies and goldfish of the octachlorobornanes
formed by introducing chlorine substituents into 1, relative to I itself, generally decreases
in the order: 9-chloro > 8-chloro > no added chlorine (i.e. I) > 3-exo-chloro, 5-exg-cnloro or
10-chloro.
Fat from chickens and mammals treated orally with Toxaphene contains products similar in
GLC characteristics to Toxaphene itself whereas liver and feces contain Toxaphene-derived
products of greatly altered GLC properties. Toxaphene preparations and related chlorinated
terpenes are mutagens in the histidine-requiring Salmonella typhimurium assay. The most
potent mutagenic components, which are not identified, reside in the polar fractions on
crystallization or solumn chromatography.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
insecticides
metabolism
composition(property)
toxicity
Toxaphene
07 C
06 A, T
IS. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21
NO. OF PAGES
65
20. SECURITY CLASS (Thispage/
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
52
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