EPA-600/1-77-012
February 1977
METABOLISM OF CARBAMATE INSECTICIDES
by
H. Wyman Dorough
Department of Entomology
University of Kentucky
Lexington, Kentucky 40506
Grant No. R-802005
Project Officer
Merrill Jackson
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
I1BB APY
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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.
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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 develops and revises 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 preparing 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.
This project was conducted to meet program needs on the fate of
carbamate insecticides. These results provide information on the rates
of metabolite degradation, sites and levels of storage of the metabolites
and their degradation products in animals.
John H. Knelson, M.D.,
Director
Health Effects Research Laboratory
111
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ABSTRACT
Studies were conducted to determine the metabolic fate of carbamate in-
secticides and to evaluate factors which influence metabolite formation
and their toxicological significance. Methomyl metabolism in rats was
investigated in detail while Croneton was studied in the rat, cow, pig
and chicken. Several insecticides, including carbaryl and carbofuran,
were administered to rats endotracheally either as aerosols or as com-
ponents of tobacco smoke and their fate determined. In the smoke exper-
iments, the quantity and nature of the residues inhaled were established
by analysis of mainstream smoke from cigarettes fortified with radioac-
tive insecticides. Carbaryl and nitrosocarbaryl were among a series of
pesticides assayed for mutagenic/carcinogenic activity using the Ames
assay system. In addition, a study was conducted to determine if the
in vitro metabolism of carbaryl under conditions of the Ames assay was
representative of that which occurs in vivo. The production of respira-
tory ^C-carbon dioxide from carbonyl-labeled carbamates was shown to be
an excellent quantitative method for evaluating the effects of various
exogenous and endogenous factors on the metabolism of carbamate insecti-
cides. Carbaryl and certain of its analogs were used in studies designed
to define the role of glutathione conjugation in carbamate metabolism in
rats. Conjugate and bound residues of carbaryl, carbofuran, Croneton
and aldicarb formed by plants were administered orally to rats and their
bioavailability ascertained. Biliary excretion and enterohepatic circu-
lation were considered in addition to urinary and fecal elimination.
This report was submitted in fulfillment of Grant Number R-802005 by the
University of Kentucky under the sponsorship of the Environmental Protec-
tion Agency. Work was completed as of October 1976.
IV
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CONTENTS
Paqe
List of Tables vi
Acknowledgments xii
Sections
I Conclusions 1
II Recommendations 2
III Experimental 3
Introduction 3
Metabolism of Methomyl in Rats 5
Fate of Croneton in Rats 27
Fate of Croneton in Large Animals 43
Fate of Insecticides Administered Endotracheally to Rats 62
Insecticide Residues in Smoke: Transfer and Fate in Rats 67
Screening Pesticides for Mutagenic Potential 100
Metabolism of Carbaryl in Ames Mutagenic Screening System 114
Synthesis of 1^C-Carbonyl Methylcarbamates 123
Influence of Dose on Carbamate Hydrolysis 129
Fate of 1-Naphthyl N-Hydroxymethylcarbamate in Rats 144
Bioavailability of Bound and Conjugated Metabolites 157
Plant Metabolism of Carbaryl 186
Glutathione Metabolism Studies 197
IV References 226
V Publications 234
VI Summary 236
v
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TABLES
No. Paqe
1. Radiolabeled materials used in the study of methorny1
metabolism in rats 7
2. Efficiencies of various trapping systems for collection of
radioactive carbon dioxide and acetonitrile in the respiratory
gases of rats treated with [ C] methomyl 1-2
3. Fate of radiocarbon from rats treated orally with syn
[1(+C=0] methomyl 14
4. Radiocarbon in respiratory gases of rats treated orally with
11+C=N labeled acetonitrile 16
5. [14C]-Carbon dioxide and [^C]-acetonitrile released in the
respiratory gases of female rats treated with [C=N]-labeled
methomyl or methomyl oxime 17
6. [14C]-Acetonitrile and carbon dioxide in the respiratory gases
of rats treated by IP injection with [14C=N]-labeled anti and
syn methomyl oxime 19
7. Silica gel thin layer chromatography of Croneton and its
metabolites 29
8. Nature of radiocarbon eliminated in the urine of rats treated
with 11+C-Croneton 35
9. Nature of radiocarbon eliminated in the urine of rats given
a single oral dose of 1^C-Croneton sulfoxide and Croneton
sulfone 38
10. Toxicities of Croneton and its oxidized sulfur analogs to rats
and mice 42
11. Nature of radiocarbon in 0-12 hour urine of cow and pig
treated with a single oral dose, 0.5 mg/kg, [11+C] ring-
treated 49
12. Nature of [^C]-Croneton equivalents in milk of cow treated
with a single oral dose, 0.5 mg/kg, of [11+C]-ring-Croneton 52
]3. [1I+C]-Ring Croneton equivalents in tissues of hens
administered the insecticide as a single oral dose or as
twice daily treatments for 7 days (0.5 mg/kg per dose) 53
14. Nature of radiocarbon in excreta of hens treated with [14C]-
ring labeled Croneton 55
vi
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TABLES - cont.
No. Page
15. Nature of [1LfC]-Croneton equivalents in eggs of hens dosed
every 12 hr with 0.5 mg/kg [^Cj-ring Croneton for 7 days 57
16. Comparative nature of residues in the urine of animals
treated with a single oral dose of [11+C]-ring labeled Croneton 59
17. [ C]-Croneton equivaletns in milk and eggs 61
18. Residues in tissues of rats 1 hour after inhalation of
11+C-insecticides 65
19. Radioactive compounds used in the study of the transfer of
insecticide residues to rats in cigarette smoke 69
20. Effect of smoke exposure method on retention of carbaryl-
naphthyl-*^C equivalents in the body of rats immediately
after the smoking process 79
21. Distribution of radiocarbon following smoking of cigarettes
with 5-ml and 35-ml puffs. Percent added to cigarette 81
22. Nature of radiocarbon in mainstream smoke of cigarettes
impregnated with ring- C-insecticides 86
23. Nature of radiocarbon in sidestream smoke of cigarettes
impregnated with ring-14C-insecticides and smoked using
eight 5-ml puffs 89
24. Effect of filters on the transfer and nature of carbaryl-
naphthyl-li+C equivalents in the mainstream smoke of
cigarettes smoked with eight 5-ml puffs 91
25. Radioactive residues in the inhaled and exhaled smoke of
cigarettes fortified with C-ring labeled insecticides 92
26. Fate of 14C-ring labeled insecticide residues inhaled by
rats in cigarette smoke 94
27. Spontaneous reversion frequency in TA1535 series of S.
typhimurium with and without rat liver homogenate 104
28. Mutagenic activity of known mutagens, carcinogens, and
carcingen analogs in S. typhimurium - TA1535 series 105
29. Mutagenicity in S. typhimurium - TA1535 series 107
Vll
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TABLES - cont.
No. Page
30. Mutagenicity screening of carbaryl bean plant water
soluble metabolites in S. typhimurium - TA1535 series 111
31. Distribution of radiocarbon after incubating carbaryl-
1-naphthyl- C with various components of the Ames
assay system 117
32. Carbaryl-l-naphthyl-14C remaining after incubation with
various components of the Ames assay system 118
33. Nature of the radiocarbon in the organo-soluble fraction
resulting from the incubation of carbaryl-l-naphthyl-ll|C
with all components of the Ames system except the micro-
organisms, treatment III 120
34. Nature of the radiocarbon in the organo-soluble fraction
resulting from the incubation of carbaryl-l-naphthyl-ll+C
with all components of the Ames system, treatment IV 122
35. R values of starting materials and products isolated by
silica gel tic systems 125
36. Major ions in the mass spectra of the synthesized
methylcarbamates 127
37. Relative symptoms apparent in rats given various doses
of t1^C-carbonyl]-Croneton 133
38. Mean elimination from rats of lt+CO2 per time interval
as a function of dose of [C-carbonyl]-Croneton 134
39. Mean cumulative elimination of CC>2 from rats
administered varying doses of [ C-carbonyl]-Croneton 134
40. Half-times of hydrolysis (1LtCO2 excretion) from rats
administered varying oral doses of [1^C-carbonyl]-
Croneton 137
41. Elimination of radiocarbon from rats administered
varying doses of C-carbonyl-Croneton 139
42. Cumulative elimination of radiocarbon from rats
administered [14C-carbonyl]-Croneton after receiving
50 mg/kg atropine sulfate i.p. 141
43. Partitioning characteristics of urinary radiocarbon from rats
as a function of time and dose of [ltf C-carbonyl]-Croneton 142
viii
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TABLES - cont.
No. Page
44. Radioactive components resulting from the metabolism
of l-naphthyl-l-^C carbaryl by rat liver microsomes 148
45. The chemical stability of ring-^C-N-hydroxymethylcarbaryl
stored under different conditions for 24 hr at 37°C 150
46. Urine and fecal elimination of radiocarbon by rats treated
with C-ring-labeled carbaryl or N-hydroxymethylcarbaryl 151
47. Recovery of 14CO2 from rats treated orally (20 mg/kg) with
carbonyl-lt+C-carbaryl or carbonyl-* V-N-hydroxymethylcarbaryl 153
48. Excretion of ll4C in the urine of rats treated orally
(20 mg/kg) with carbonyl-^ ^C carbaryl or carbonyl-ltfC
N-hydroxymethylcarbaryl 154
49. Elimination of radioactivity from rats treated orally
(20 mg/kg) with carbonyl-ltfc carbaryl or carbonyl-lkC
N-hydroxymethylcarbaryl 154
50. Attempted cleavage of water soluble metabolites in urine
of rats treated orally with 11+C-ring- or 1 ^C-carbonyl-
labeled N-hydroxymethylcarbaryl 155
51. Radioactive insecticides used in the treatment of bean
plants 159
52. Nature of radiocarbon in bean plants treated with
[^C]-labeled carbamate insecticides 169
53. Elimination of radiocarbon from rats treated with carbaryl,
carbofuran, and Croneton unextractable metabolites from
plants 170
54. Elimination of radiocarbon from rats treated with carbaryl,
aldicarb and carbofuran water-soluble metabolites from bean
plants 173
55. Elimination of radiocarbon from rats treated with Croneton
water-soluble metabolites from bean plants 175
56. Thin-layer chromatography of radiocarbon released by acid
and enzyme hydrolysis of the bound and water-soluble residue
from bean plants treated with carbamate insecticides 177
IX
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TABLES - cont.
No. Paqe
57. Thin-layer chromatography of radiocarbon released by acid
and enzyme hydrolysis of urine from rats treated with the
water soluble residue of carbamate insecticides from bean
plants 180
58. Extraction and partitioning characteristics of carbaryl and
metabolites after injection into bean plants 190
59. Extraction characteristics of radiocarbon in excised bean
plants following uptake via the stem of carbaryl water-
soluble metabolites (bean plant-generated) and aglycones
resulting from acid hydrolysis 192
60. In vitro metabolism of carbaryl and related compounds
by bean plant homogenates 194
61. Conjugation of naphthalene-l-11+C and carbaryl by
different subcellular fractions of rat liver homogenate 203
62. Nature of radiocarbon following incubation of naphthalene-
1-14C and l-naphthyl-l-llfC carbaryl with 15,000 g super-
natant of a rat liver homogenate 205
63. Radioactive components of the ether fraction from in vitro
metabolism of naphthalene-l-11+C and l-naphthyl-l-11+C carbaryl 207
64. Conversion of water soluble and ether extractable materials
by enzyme and acid treatments 208
65. Products fromed upon treatment of 2-hydroxy 1,2-dihydro-S-
(naphthyl-l-luC)glutathione (III) with 6 N HCl at 110°C
for 24 hr 210
66. Ether extractable radioactive metabolites in urine excreted
during a 72-hr period after rats were treated with naphtha-
lene-l-luC and l-naphthyl-l-11+C carbaryl 214
67. Water-soluble radioactive metabolites in the urine excreted
during a 72-hr period after rats were treated with naphtha-
lene-l-^C 215
68. Water-soluble radioactive metabolites in urine excreted
during a 72-hr period after rats were treated with 1-
naphthyl-l-11+C carbaryl 217
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TABLES - cont.
No.
69. Conversion of water-soluble metabolite of naphthalene-1-
C formed in vivo by rats to ether extractable materials
by enzyme and acid treatments 218
70. Conversion of water-soluble metabolites of 1-naphthyl-l-
14C carbaryl formed in vivo by rats to ether extractable
metabolites by enzyme and acid treatments 219
71. The major ions in mass spectra of N-acetyl-S-(1,2-dihydro-
2-hydroxy-naphthyl)cysteine and its analogs 221
72. Radioactive components in the ethyl acetate of urine
(adjusted to pH 2) excreted during a 72-hr period after
rats treated with naphthalene-1-11+C and l-naphthyl-l-li+C
carbaryl 224
XI
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ACKNOWLEDGMENTS
The principal investigator wishes to acknowledge the efforts and dedica-
tion of those students, post-doctoral fellows, and technical assistants
who have contributed so much to this research program on carbamate in-
secticides. Sincere thanks are extended to those individuals listed
below for their hard work and cooperation during the course of these
investigations.
Graduate Research Assistants
D. R. Roongsook
H. E. Hurst
T. C. Marshall
K. C. Chen
R. W. Rickard
M. S. Tanious
Post-Doctoral Fellows
L. D. Rodriguez
Y. H. Atallah
L. E. Phillips
J. H. Thorstenson
D. E. Nye
K. L. Huhtanen
R. C. Couch
Technical Assistants
H. E. Bryant
D. M. Jarrell
C. N. Thomas
S. C. Lolmaugh
N. E. Wilson
B. W. Quick
S. H. Pan
XI1
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I CONCLUSIONS
Hydrolysis, oxidation and conjugation are biochemical reactions common
to all insecticidal carbamates. Ester hydrolysis almost invariably re-
duces toxicity, while oxidation may result in compounds retaining a high
degree of toxicity. Croneton, for example, undergoes sulfur oxidation
to the sulfoxide which is more acutely toxic to rats and mice. The sul-
fone derivative is of similar toxicity as the parent compound. Methomyl
metabolism in rats involves isomerization and Beckmann rearrangement in
addition to the other reactions common to the carbamates. Carbamate in-
secticides entering the body via inhalation are metabolized similarly to
when ingested. When present as residues in tobacco, much of the intact
carbamate is transferred to the smoker by volatilization before being
degraded during the actual burning process.
Nitrosocarbaryl is a potent direct mutagen when assayed in the Ames
bacterial system. Carbaryl is inactive either with or without the rat
liver enzymes. In the presence of the liver enzymes, carbaryl is almost
completely metabolized within one hour to products commonly formed in
the in vivo mammalian system. Thus, the metabolites as well as the
parent compound were assayed for their mutagenic potential.
Water soluble metabolites of carbamates formed in bean plants are rapid-
ly absorbed from the gut of rats and are excreted almost quantitatively
in the urine even though biliary excretion is extensive. The bound re-
sidues in the plant are not bioavailable as evidenced by complete fecal
excretion without detection of residues in the bile. Reports that
glutathione conjugation plays a major role in carbaryl metabolism in
rats were not substantiated. It appears that the proposed mercapturic
acid derivatives of carbaryl were artifacts resulting from strong acid
treatment of the dihydrodiol metabolites of carbaryl.
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II RECOMMENDATIONS
Each carbamate insecticide must be subjected to thorough metabolic
studies with an awareness on the part of the investigator that metabolic
pathways are not always predictable. Moreover, reviewers of metabolism
studies, whether for publication or regulatory purposes, should look
specifically for evidence suggesting the existence of metabolites not
reported, and/or of intermediates which obviously must exist in order to
yield the products identified. Such scrutiny of the data would lessen
considerably the likelihood of a compound, as was the case with methomyl,
being evaluated on the basis of proposed metabolic pathways inconsistent
with the experimental evidence.
Efforts should be made to more fully define the toxicological signifi-
cance of the conjugated carbamate metabolites. Unlike the bound resi-
dues, the conjugates are bioavailable in animals and may be converted
in vivo to toxic carbamate materials. Chronic toxicological evaluations
should be conducted on individual conjugate metabolites if they consti-
tute a significant portion of the terminal residues in products destined
for human consumption.
Carbamate metabolites in animal and plant tissues should be classified
as bound residues only if demonstrated to be nonbioavailable when ad-
ministered orally to appropriate experimental animals. Such residues
occurring in substrates such as feed of dairy animals and crops returned
to the soil should be investigated to determine if the bound residues
are converted by microbial action, etc., to a bioavailable form and per-
sist as such in products consumed by man.
While there are many problems yet to be resolved relative to carbamate
metabolism, this group of chemicals remains a viable source of new pest
control agents. Their many desirable features justify continued efforts
to expand the utility of this very important family of insecticide.
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Ill EXPERIMENTAL
INTRODUCTION
Carbamate insecticides as we know them today had their start in the late
1950's with the introduction of carbaryl (1-naphthyl N-methylcarbamate,
Trade Name Sevin). The compound was rapidly accepted on the world mar-
ket because of its efficacy, moderate mammalian toxicity, and relatively
low persistence in the environment. From the metabolic point, carbaryl
was assumed to undergo simple ester hydrolysis to yield innocuous prod-
ucts.
Studies in the 1960's and early 1970's demonstrated that carbaryl metab-
olism was far more complex than originally reported and, in fact, was
among the most complex ever encountered with a pesticidal compound.
Even today, carbaryl metabolism is the topic of many investigations,
either as a model compound or in attempts to fully define its metabolic
fate. In the interim, other carbamates having vastly different chemical
and toxicological properties than carbaryl have been introduced. One
commonality persisted, however, and that was the complexity of metabo-
lism involving predominately hydrolytic, oxidative and conjugative reac-
tions (Kuhr and Dorough 1976).
Over the past 15 years a major portion of the principal investigator's
research program has been devoted to carbamate metabolism and its signi-
ficance relative to the safe use of these insecticides. Among those
compounds studied most extensively are carbaryl, aldicarb, carbofuran
and methomyl, each of which is currently widely used for insect control.
Since 1970, much of the work on carbamate metabolism has been funded
from a grant from the Environmental Protection Agency (Grant Number
R-802005). A progress report (identified as EPA-650/1-74-002, September
1973) was submitted covering the period of September 1, 1970 through
August 31, 1973. A renewal of the grant was requested and approved for
the period of February 1, 1974 through January 31, 1977. Progress made
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during the latter project period is contained in the present report.
The principal objective of the proposed research was to define the meta-
bolic fate of carbamate insecticides in various biological systems so
that this information could be used by regulatory officials in assessing
the safety of the compounds involved. During the process, studies were
performed which would contribute to ascertaining the significance of
metabolites encountered with the carbamates. Studies wherein physiolog-
ical and biochemical processes responsible for variations in carbamate
metabolism were also explored.
Those studies performed during the past three years towards attaining
the objectives of the project are presented below. Studies essentially
completed in 1973 and the results included in Report Number EPA-650/1-
74-002 have not been repeated herein. However, those conducted since
1973, even if published elsewhere, are made a part of the present report
to provide a single source of reference for those investigations sup-
ported by EPA Grant Number R-802005.
Research supported by this EPA grant has contributed enormously to
papers by the principal investigator and his colleagues which are not
experimental in nature. A book on carbamate insecticides published in
1976 (Kuhr and Dorough) resulted from an invitation by CRC Press to the
principal investigator based on his experience with the carbamates, much
of which was obtained while funded by EPA. A similar situation resulted
in a presentation to the Pesticide Division of ACS on the biological ac-
tivity of pesticide conjugates (Dorough 1976), and to the 1976 Gordon
Conference on Drug Metabolism on the topic of bioavailability of cova-
lently bound pesticide residues. Another such occurrence was the prep-
aration of a review article dealing with the state-of-the-art of carba-
mate residue analysis (Dorough and Thorstenson 1975). While the afore-
mentioned papers are not in the present report, they deal largely with
carbamate insecticides and are based on the data which are reported.
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METABOLISM OF METHOMYL IN RATS
Since the preliminary report of methomyl {s-methyl-N-[(methylcarbamoyl)
oxy]thioacetimidate} metabolism by Baron in 1971, and the detailed re-
ports in 1973 (Harvey and Pease, 1973; Harvey and Reiser, 1973; Harvey
et al., 1973), we have been intrigued by the metabolic pathway proposed
for this oxime carbamate in animals, plants, and soils. Using methomyl
radiolabeled in the ll*C=N position, the radioactive metabolic products
observed were simply carbon dioxide and acetonitrile. Wile some diffi-
culties were apparent in the quantitative collection of the volatile
metabolites, the qualitative evidence was sound in each of the afore-
mentioned studies.
A particularly interesting point concerning these results was the chem-
ical route leading to the radioactive carbon dioxide and acetonitrile.
This point was not raised by any of the authors and may have been con-
sidered self-evident. Nonetheless, their mechanism of bioformation
was not readily obvious to us, possibly because of our experience with
aldicarb metabolism (Dorough, 1970).
With aldicarb, the compound is metabolized similarly by most organisms.
Hydrolysis of the carbamate ester is usually very rapid, especially in
animals, and has been documented by the release of [1I+C] carbon dioxide
from f1 C] carbonyl-labeled aldicarb. The studies with methomyl did
not include a carbonyl radiocarbon so it could not be determined if the
carbon dioxide and acetonitrile formed from [ll*C=N] methomyl came from
intermediates containing the intact ester linkage or were formed sub-
sequent to hydrolysis.
Sulfur oxidation to form the sulfoxide (SO) and sulfone (SOa) of aldi-
carb and/or its oxime constituted the major metabolic reactions in all
systems tested. With methomyl, there was no indication that sulfur
oxidation was involved at all in its metabolism. Thus, the real dif-
ference in metabolism appears to be in the fate of the oxime hydrolysis
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products of aldicarb and methomyl. This, of course, requires the
assumption that methomyl is hydrolyzed rapidly as is aldicarb and other
N-methylcarbamate insecticides.
Considering the chemical differences which might account for the dif-
ferent metabolic pathways of aldicarb and methomyl, the most obvious
was the manner in which the thioalkyl groups are bonded to the imide
carbon. With aldicarb, the thioalkyl moiety is attached to the carbon
alpha to the imide carbon and is designated chemically as an alkylthio-
ether. The thioalkyl group of methomyl is bonded directly to the imide
carbon and is classified as a thiohydroximate ester. That these dif-
ferences could account for the metabolic differences for aldicarb and
methomyl was supported by reports on the metabolism of DS-15647
(Whitten and Bull, 1974) and thiocarboxime (Hutson, 1971). DS-15647,
an alkylthioether, was metabolized by hydrolysis and/or sulfur oxida-
tion in the same manner as aldicarb, whereas thiocarboxime, a thio-
hydroximate ester, was metabolized to carbon dioxide and acetonitrile
as was methomyl. Thus, it might be speculated that the metabolic path-
ways of alkylthioether and thiohydroximate oxime carbamates follow
different metabolic schemes. This being the case, the mechanism of
methomyl metabolite formation, if defined, would likely apply also to
other insecticidal thiohydroximate esters.
Materials and Methods
Chemicals - Radioactive materials used in this study are shown in Table
1 along with the position of the radiolabel and the abbreviated de-
signations used throughout this paper. [1'*C=0] Methomyl was prepared
from syn methomyl oxime and methyl [^C] isocyanate and purified by
tic. Its specific activity was 5.8 mCi/mmol. syn [1'*C=N] Methomyl
oxime and syn [ C=N] methomyl were synthesized from [ C] acetonitrile
using the method of Harvey et al. to give a specific activity of 1.4
mCi/mmol. The syn [1₯C=N] oxime was converted to the anti [1'*C=N]
isomer by ultraviolet irradiation and the anti [1'*C=N] methomyl formed
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Table 1. RADIOLABELED MATERIALS USED IN THE STUDY
OF METHOMYL METABOLISM IN RATS
Chemical Name
Abbreviations
syn S-Methyl N[(methylcarbamoyl-
carbonyl- C)oxy]thioacetimidate
syn S-Methyl [1-l^ClN-t(methyl-
carbamoyl)oxy]thioacetimidate
anti S-Methyl[1-lkC]N-[(methyl-
carbamoyl)oxy]thioacetimidate
syn S-Methyl[I-1"cjN-hydroxythio-
acetimidate
aiti S-Methyl[I-1^ClN-hydroxythio-
acetimidate
Ethyl di-n-propylthiocarbamate-
1 C carbonyl
syn[ C=0]methomyl
14
syn[ C=N]methomyl
anti [ C=N]methorny1
syn [1'*C=N]methomyl oxime
anti [1 ** C=N] me thorny 1 oxime
[ 1'*C=O] carbonyl Eptam
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by reacting it with methyl isocyanate.
Confirmation of structures was accomplished by comparing infrared
and 'H nmr spectra of the compounds with those previously reported
(Davies et al., 1968). Radioactive purity of the syn preparations,
as determined by tic and autoradiography, was 95% or greater. anti
[14C] Methomyl contained some of the syn isomer, up to 20%, when admin-
istered to test animals because of the ease with which it was converted
to the syn form, anti Oxime could be resolved by tic from the other
radioactive components in the crude preparation mixture, but rechroma-
tography after extraction from the gel showed varying amounts of syn
oxime and three to four unknown products. This instability prevented
direct structural confirmation by spectral analysis and its conversion
to the anti methomyl when reacted with methyl isocyanate was accepted
as proof of structure.
[llfC] Carbonyl-labeled Eptam (ethyl di-n-propylthio [llfC] carbamate) ,
specific activity 6 mCi/mmol,- was prepared from radioactive phosgene
following the method of Tilles (1959). Nonradioactive methomyl,
methomyl oxime and Eptam used as standards were analytical grade mate-
rials from our residue laboratory.
Thin Layer Chromatography - Silica gel chromatoplates (Analtech, 250y)
were used to resolve the syn and anti isomers of methomyl and its oxime
derivatives. The carbamate forms were separated by developing the
chromatoplates in ethyl acetate (syn-methomyl R 0.56, anti-methomyl
R 0.67). Effective separation of the two oxime forms was not accomp-
lished in this solvent system but the compounds were resolved by
developing the plates in a 5 to 1 dichloromethane and ether mixture
(syn-oxime R 0.71, anti-oxime R 0.88). Methomyl (R 0.16) was sepa-
rated from the oxime (R 0.67) using a 9 to 1 mixture of ether and
hexane as the solvent system. Isomeric configuration of the respective
compounds was then determined by tic analysis in the appropriate solvent
system just described. Radioactive materials on the chromatoplates
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detected by autoradiography using Kodak no-screen medical X-ray
film. Nonradioactive standards were viewed under short wave ultra-
violet light.
rreatment - Albino rats (Cox-SD, Laboratory Supply Co.) weighing
approximately 250 g each were treated with from one to two million
ipm's of the radioactive compound used in each experiment. For oral
3osing, the chemical was dissolved in 0.5 ml of corn oil and adminis-
tered to the rats via a feeding needle. All treatments were by the
oral route except in one experiment where syn- and anti-oxime were
injected intraperitoneally into the animals. Here, only one animal
was used for each compound, whereas all oral treatments consisted of a
minimum of two rats from which each set of data was obtained. Numerous
animals were treated for the sole purpose of developing and evaluating
methods of collecting radioactivity in the expired gases. Quantitative
data in this report do not contain the results obtained in the method-
ology experiments, but it is noteworthy that they invariably substan-
tiated the results which are presented.
Radioassay - Quantitation of radioactivity was accomplished by liquid
iscintillation counting (Packard Tri-Carb 3385/544) using a premixed
scintillation fluid (3a70B, Research Products International, Elk Grove
Village, IL) . Aliquots from traps used for the collection of respira-
tory [llfC] gases, and samples of rat urine were radioassayed directly.
Peces samples, approximately 0.5 g, were combusted using a Packard
Model 306 oxidizer, and the liberated [ C] carbon dioxide trapped in
a 2 to 1 mixture of 2-methoxyethanol and 2-aminoethanol for radioassay.
Collection of Samples - Treated animals were placed in glass metabolism
cages which allowed separate collection of the urine and feces. To
collect radioactive materials in the respiratory gases, air (about
100 ml/min) was passed through the cage into one or more solutions
designed for the trapping of carbon dioxide and/or acetonitrile. When
monitoring only for carbon dioxide and for relatively short periods,
-------
6-12 hrs, the trap consisted of 20 ml of a 2 to 1 mixture of 2-
methoxyethanol and 2-aminoethanol (System A).
Separate collecting of respiratory carbon dioxide and acetonitrile
was accomplished by passing the air from the animal cage through two
cold acetone (dry ice-acetone) traps to collect the acetonitrile and
then through 100 ml of 4 N sodium hydroxide for the collection of
carbon dioxide (System B). While this system effectively trapped the
compounds in question, there remained the possibility that other radio-
active materials could be present in the respiratory gases of [ C=N]
methomyl-treated animals. This possibility was evaluated by using a
third trapping system (System C) whereby the air was directed from the
cage into 4 N sodium hydroxide, and from there it was mixed with oxygen,
200 ml/min, and passed through a Beckman Biological Materials Oxidizer
(BMO). The purpose of the BMO was to combust any radioactive components
escaping the hydroxide trap, including acetonitrile, to carbon dioxide
which was then collected in a second hydroxide trap. To account for
the [ C] acetonitrile collected in the trap placed before the BMO,
barium hydroxide was added to the 4 N sodium hydroxide to precipitate
the radioactive carbonate. The trap solution was centrifuged, the
pellet radioassayed as an indication of [1'*C] carbon dioxide in the
trap, and the supernatant radioassayed to determine the [1'*C] acetoni-
trile content of the trap solution.
Acetonitrile Confirmation - Radioactivity collected in the cold acetone
traps from the respiratory gases of rats treated with [1!*C=N] methomyl
was assumed to be CHs l kC 3SJ (Harvey et al., 1973). Confirmation of its
identity was attempted by determining if the radioactivity could be
converted to acetophenone [CeH5-ll+C (O)CHs] . The latter has been pre-
pared from authentic acetonitrile (Shriner and Turner, 1930) and, there-
fore, the procedure was used for the attempted synthesis of the radio-
active form from the methomyl metabolite suspected of being CHs^CH" N.
The starting material consisted of 3 x 10s dpm of the metabolite in
about 50 ml of acetone and 10 ml of nonradioactive acetonitrile.
10
-------
Following distillation to a remaining volume of 6 ml for the complete
removal of acetone, the semicarbazone derivative of actophenone
[C6H5-C(CH3)NNHC(0)NH2] was prepared and recrystallized from ethanol to
a constant melting point and until a constant specific activity was, or
was not, evident.
Results
Trapping of [^C] Volatiles - Trapping of [11+C] carbon dioxide in the
respiratory gases of animals treated with [11+C = 0] methomyl was essen-
tially quantitative using a mixture of 2-methoxyethanol and 2-amino-
sthanol (Table 2, System A). It did not effectively trap [14C] aceto-
nitrile and thus could be used to selectively monitor carbon dioxide
if so desired. In studies using syn [lf*C = N] methomyl, which yields
ooth radiolabeled compounds, the amount of [ltfC] carbon dioxide
collected in System A was the same as that in systems allowing sepa-
rate quantitation of carbon dioxide and acetonitrile.
With trapping System B, the acetonitrile collected in the cold acetone,
primarily in trap I, while only small quantities of carbon dioxide,
3% or less of that generated, were in trap III, a 4 N sodium hydroxide
solution.
The incorporation of the BMO combustion apparatus into the trapping
system (System C, Table 2 ) was an attempt to quantitate total radio-
activity in the respiratory gases. Without trap I, the carbon dioxide
ds well as other components converted to carbon dioxide in the BMO
would collect in trap II, giving a single value for respiratory [J1*C]
components. Of course, the carbon dioxide was removed by the sodium
hydroxide trap I, but the solution also contained almost one-half of
i:he acetonitrile passing through the system. The most important point
relative to System C was that radioactive acetonitrile entering the
BMO was combusted and collected as carbon dioxide without any appre-
ciable loss. This suggested that other radioactive components of the
11
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Table 2. EFFICIENCIES OF VARIOUS TRAPPING SYSTEMS FOR COLLECTION OF
RADIOACTIVE CARBON DIOXIDE AND ACETONITRILE IN THE RESPIRATORY GASES OF
RATS TREATED WITH [lkC] METHOMYL.
% of total 1[*C passed through traps
Trapping system Carbon dioxide Acetonitrile
A. Trap I, 2:1 2-methoxyethanol- 100 1
2-aminoethano1
B. Trap I, Acetone, -10 to 0°C 2 92
Trap II, Acetone, -10 to 0°C 1 8
Trap III, 4 N Sodium Hydroxide 97 0
C. Trap I, 4 N Sodium Hydroxide 100 45
Combustion apparatus, then
Trap II, 4 N Sodium Hydroxide 0 55
Air from animal was passed through traps in order listed under each
system.
For recovery experiment, ll*CO2 was generated by treating animals with
[llfC=O] methomyl; CH31I+C=N was volatilized from a water solution by
bubbling air through the water and then into the indicated traps.
c
Air from first NaOH trap was mixed with oxygen, directed through a
Beckman Biological Materials Oxidizer, and then into the second
NaOH trap.
12
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expired air, if any, would be similarly quantitatible. The fact is,
however, that respiratory [ C] gases resulting from treatment of rats
with [ C=N] methomyl were of the same magnitude whether trapped in
System B or System C. These results demonstrated that there was no
radioactivity in the expired air which escaped System B and was trapped
in System C, and provided strong evidence that System B effectively
trapped the total respiratory radiocarbon liberated by the animals.
Nature of [^C] Volatiles - That radioactivity which could be trapped
by 2-methoxyethanol:2 aminoethanol and by 4 N sodium hydroxide, and
which could be precipitated from the latter with barium hydroxide was
characterized as carbon dioxide. The remainder of the radiocarbon in
the expired air of rats was identified as acetonitrile based upon its
synthesis to acetophenone semicarbazone. The final product, when re-
crystallized to a constant melting point and specific activity,
contained 83% of the radioactivity added to the initial reaction mix-
ture from traps I and II of trapping system B (Table 2 ).
Fate of syn [^0=0] Methomyl - Orally administered syn [14C=0] methomyl
to rats was rapidly hydrolyzed as indicated by the evolution of [14C]
carbon dioxide in the expired air (Table 3 ). While the rate of ester
hydrolysis appeared somewhat faster in females than in males during
the early stages of the experiment, there was little difference in
the total hydrolysis after 24 hr (87.7% of dose in females and 83% in
males). After the same period following treatment, the urinary radio-
carbon accounted for approximately 5% of the administered dose while
only 0.2% was voided in the feces. The average total recovery consid-
ering all treated animals was 90.4% of the dose.
Tic analysis of concentrated urine applied directly to the chromato-
plates and developed in ethyl acetate showed that none of the radio-
activity moved from the origin. Thus, the radiocarbon was not in the
form of the parent carbamate or its sulfoxide and sulfone analogs.
These results agreed with those earlier reported for methomyl and for
13
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Table 3. FATE OF RADIOCARBON FROM RATS TREATED ORALLY
lit
WITH SYN [ OO] METHOMYL
Nature of^C and hrs after dose
Carbon dioxide
1
2
4
6
10
24
Urinary- lkC, 24
Fecal- ^C, 24
Total Recovery, 24
Cumulative 3
Female
6.0
33.9
69.5
84.0
87.4
87.7
4.9
0.2
92.8
a
b of dose
Male
13.5
27.0
55.0
79.0
81.0
83.0
4.8
0.2
88.0
Each value is the average from two test animals.
14
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thiocarboxirae. Because of the very low level of urinary [ C]
metabolites, no attempt was made to further characterize the material.
Fate of [ _C=N] Methomyl and Oxime - Having established in the method-
ology experiments that [ ^C] acetonitrile was expired in the air of
rats treated with syn [ C=N] methomyl, it was considered essential to
determine if orally administered [llfC] acetonitrile would similarly be
expired. Data in Table 4 show that this indeed was the case and that
it was detectable in the expired air after only 1 hr. By 6 hr, almost
one-half of the administered dose had been expelled by this route. A
small amount of the dose, 9%, was trapped as [ C] carbon dioxide but
its formation was restricted to the first 2 hr following treatment.
The importance of these data, as will become more evident in the
results presented below, was the documentation that [^C] acetonitrile
rapidly and efficiently voided from the rats in the respiratory
gases; therefore, a delay in the detection of acetonitrile from syn
[1'*C=N] methomyl-treated rats should be indicative of its rate of
formation and not of its rate of elimination.
\s just suggested, there was a considerable delay in the formation of
acetonitrile in rats treated with syn [lkC] methomyl (Table 5). The
compound first occurred in the trap solution between 4 and 6 hr after
treatment, but then its rate of formation increased so that after 24
hr, respiratory [llfC] acetonitrile accounted for 11% of the dose.
[ C] Carbon dioxide, on the other hand, was detected in the first
assay of the trap, 2 hr after treatment, in quantities equivalent to
2.5% of the dose. Its continued liberation over the 24 hr test period
resulted in 19.5% of the dose being expired as carbon dioxide. Col-
lection of respiratory radiocarbon from one of the animals for 72 hr
showed that the carbon dioxide increased to 24.4% of the dose, while
acetonitrile increased to 14.9%. The pattern of elimination of these
products from syn [1 **(
Harvey et al. (1973).
products from syn [ 1*C=N] methomyl was the same as that reported by
15
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Table 4. RADIOCARBON IN RESPIRATORY GASES OF RATS TREATED ORALLY
WITH * "*C E N LABELED ACETONITRILE
Cumulative % of dose
Hours after treatment
Carbon dioxide
Acetonitrile
1
2
4
6
7.1
9.1
9.1
9.1
0.6
10.3
27.7
48.7
16
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Table 5. [ll*C] CARBON DIOXIDE AND [^C] ACETONITRILE RELEASED IN THE
RESPIRATORY GASES OF FEMALE RATS TREATED WITH [J "*C=N]-LABELED METHOMYL
OR METHOMYL OXIME
Cumulative % of dose
Hours after
treatment
2
4
6
10
14
22
24
anti- [llfC=N]
Me thorny 1
CO2 CH3CN
1.3 3.0
3.9 5.8
5.0 8.4
6.3 14.0
7.1 18.4
7.4 25.1
7.6 27.9
syn-[ll+C=N]
Methomyl
C02 CH3CN
2.5 0
6.0 0
8.5 0.7
12.0 3.0
15.5 5.5
19.0 10.5
19.5 11.3
syn-[1'*C=N]
Oxime
CO2 CH3CN
2.5 0
7.0 0.3
10.5 0.4
14.0 0.8
17.0 1.0
21.0 2.2
21.8 2.4
17
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Radiocarbon in the expired air of rats treated with anti [14C=N]
methomyl was predominately acetonitrile, 27.9% of the dose after 24 hr,
and it was readily apparent after only 2 hr (Table 5). [1'*C] Carbon
dioxide was eliminated in only small amounts, 7.6% of the dose after
24 hr, with the majority generated in the first 6 hr. Because the
anti methomyl contained up to 20% of the syn isomer, it is likely that
most of the COa was derived directly from this source. In any event,
the data do show that anti methomyl was metabolized differently than
syn methomyl, and pointed up the possibility that [1I+C] acetonitrile
from the syn methomyl treatment was generated from the anti form fol-
lowing in vivo isomerization.
syn [1'*C=N] Oxime, unlike its carbamate derivative, was not metabolized
appreciably to acetonitrile (Table 5). However, the amount of 11+CC>2
formed from the oxime was about the same as for syn methomyl. This
same situation was observed in studies of the metabolism of thiocarbox-
ime and its oxime in rats (Hutson et al., 1971). Apparently, the syn
oxime is more resistant to reactions resulting in acetonitrile than is
the syn carbamate and, thus, follows the metabolic route which yields
carbon dioxide. Because the anti [14G=N] oxime was even more unstable
than anti methomyl, it was not included in the oral dose studies.
The anti [1'*C=N] oxime preparation was included in a cursory examina-
tion of the fate of the oximes when injected intraperitoneally into
rats. Results of these experiments showed that the anti [ C=N] oxime,
while impure, formed 20 times more [ C] acetonitrile than did the syn
isomer (Table 6). Carbon dioxide formation from the injected syn
[1'*C=N] oxime was less than that resulting from oral administration of
the compound (Table 5). However, the quantities of [14C] acetonitrile
were similar for both routes of treatments. Even with the difference
in carbon dioxide expired, the data were sufficiently similar to show
that the degradation observed was not a function of reactions unique to
the gut. The relatively large amount of [1'*C] carbon dioxide formed
from anti [ll*C=N] oxime, 17.4% of the dose after 24 hrs, was incon-
18
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Table 6. [14C] ACETONITRILE AND CARBON DIOXIDE IN THE RESPIRATORY
GASES OF RATS TREATED BY IP INJECTION WITH [1 '*C=N] -LABELED ANT I AND
SYN METHOMYL OXIME
Cumulative % of dose
an t i- [ 1 ** C=N ] Oxime syn- [ l lfC=N] Oxime
Hours after treatment _ CO 2 _ CH3CN _ CO 2
6 10.2 4.9 6.2 0.3
14 13.8 12.1 11.6 0.7
24 17.4 20.5 16.5 1.7
19
-------
sistent with the amount, 7.6%, resulting from treatment of animals
with anti [ 1*C=N] methomyl (Table 5). Presumably, this resulted from
the several impurities, including syn oxime, contained in the anti
methomyl preparation.
Urine of rats treated orally with [1'*C=N] methomyl and with syn [14C=N]
oxime contained from 25 to 35% of the administered radioactivity after
24 hr. The chemical nature of the urinary [llfC] metabolites was not
critically evaluated. However, it was obvious that not more than 5%
of the methomyl dose was excreted in a form containing the carbonyl
carbon (Table 3) and, therefore, this represented the maximum amount
possible in the carbamate form. Concentration by freeze-drying of
urine from rats treated with syn [14C=N] methomyl resulted in 20% loss
of the radiocarbon, indicating that a portion of the [14C] components,
possibly acetonitrile, was very volatile. That radioactivity in the
urine concentrate was not moved from the tic origin when developed in
ethyl acetate. With thiocarboxime, a compound of similar structure to
methomyl, the urinary metabolites were primarily glucoside and sulfate
conjugates of the oxime.
Proposed Route of ^COa and CHa llfCEN Production from syn [1'tC=N]
Methomyl - Prior to the experiments with anti [ C=N] methomyl, the
possibility that [ll+C] acetonitrile and [llfC] carbon dioxide were de-
rived from an intermediate metabolite of syn [ C=4] methomyl capable
of undergoing two separate routes of degradation was considered. This
was based on reports that both methomyl and methomyl oxime are more
stable in the syn form and the absence of any evidence for the
biological isomerization of the compounds, or of the metabolic products
of the anti isomers if formed. The metabolic data obtained with the
anti form of this carbamate showed, however, that such a metabolite
probably did not exist. Production of both acetonitrile and carbon
dioxide could be explained if syn methomyl or its oxime was partially
converted, _in vivo, to the anti isomer. That is, [I1+C] acetonitrile
could be formed from the anti isomer and [lkC] carbon dioxide from the
20
-------
syn isomer.
Because of the rapid hydrolysis rate of methomyl (Table 3) , it was
evident that acetonitrile and carbon dioxide were not derived directly
from intermediates containing the intact carbamate ester. The majority
of these volatile metabolites were formed after 80% or more hydrolysis
of the carbamate had occurred. Studies with the syn and anti oximes
(Table 5 and 6) demonstrated that these metabolites of methomyl hydro-
lysis could serve as primary intermediates to carbon dioxide and aceto-
nitrile, respectively. The study with syn oxime also showed that this
isomer was relatively stable to isomerization in the rat; only 2.4% of
the dose was expired as acetonitrile. Thus, the major source of anti
oxime in the syn methomyl-treated rats was apparently from hydrolysis
of anti methomyl formed by isomerization of the administered carbamate.
The conversion of syn methomyl to the anti form obviously was not an
instantaneous reaction in the rat. If that had occurred, the presence
of acetonitrile would have been evident in the expired air immediately
after treatment and this was not the case (Table 5) . This is based
on the findings that acetonitrile, per se, was expired very rapidly
(Table 4) and that anti methomyl yielded acetonitrile at a rate almost
identical to the rate of carbon dioxide formation from syn methomyl.
Therefore, the delay in acetonitrile formation from syn methomyl treat-
ments probably resulted from the time required for its precursors, anti
nethomyl and anti oxime, to be generated in sufficient quantities to
allow biosynthesis of acetonitrile in detectable quantities.
the anti oxime preparation gave considerable quantities of carbon
dioxide (Table 6) , indicating isomerization to the syn form, its
formation was probably from impurities in the preparation and not from
.Ln_ vivo formation of syn oxime. A better estimate of the in vivo
stability of the anti oxime may be gained through analysis of results
obtained in the anti [1'*C=N] methomyl rat study (Table 5). While
hydrolysis to the anti oxime and subsequent metabolism to acetonitrile
21
-------
were evident after just 2 hr, and continued progressively for 24 hr,
where it equalled 27.9% of the dose, there was very little [14C] carbon
dioxide given off by the animals. Possibly, the carbon dioxide was
formed largely from syn methomyl, up to 20%, present in the anti
methomyl rather than resulting entirely from the isomerization of anti
methomyl or its oxime. Support for this was indicated by treating a
female rat with anti [ll*C=N] methomyl containing only 5% of the syn
form. After 24 hr, 34% of the dose was expired as [1'*C] acetonitrile
and only 5% as [ C] carbon dioxide. The conclusion is, therefore,
that little in vivo isomerization takes place with anti methomyl or
with anti oxime.
Having concluded that syn [14C=N] methomyl was isomerized, in part,
to the anti isomer in the rat, and that the carbamates were then hydro-
lyzed to their respective oximes which, in turn, were degraded to
carbon dioxide and acetonitrile, possible mechanisms for the latter
reactions were considered. The nature of the reactants and end prod-
ucts suggested that Beckmann type rearrangements were involved in the
metabolic degradation of the oximes. Such chemical reactions have
been reported for syn and anti methomyl oxime (Davies et al., 1968)
and provide the basis for the proposed in vivo mechanism for the form-
ation of carbon dioxide and acetonitrile. Under acid conditions, the
anti isomer underwent rearrangement to yield acetonitrile, a product
of its metabolism in the rat. The syn isomer underwent rearrangement
to form S-methyl N-methylthiocarbamate. Quite clearly, the S-methyl
N-methylthiocarbamate formed from syn oxime by Beckmann rearrangement
was not the respiratory product of syn oxime metabolism in the rat.
It was, however, a possible precursor to that end product, carbon
dioxide. Evidence that a [ll*C] carbonyl-labeled thiocarbamate is
metabolized to [ C] carbon dioxide in rats was obtained with Eptam.
This herbicide was metabolized very rapidly to [14C] carbon dioxide
and after 8 hr accounted for 35% of the administered radiocarbon. The
metabolic conversion of the carbonyl carbon of thiocarbamates to carbon
dioxide was previously proposed (Ong and Fang, 1970), but had not been
22
-------
demonstrated experimentally. With this evidence, the final step in
the metabolism of syn oxime appeared to be the bioalteration of the
thiocarbamate, CHsS-lf*C (O)NHCHs, to [ll*C] carbon dioxide.
Discussion
Known routes of pesticide metabolite formation in biological systems
are important in attempting to assess the potential hazards associated
with the use of a particular compound. With methomyl, formation of
carbon dioxide and acetonitrile from the imide carbon indicates that
this carbamate yields possibly the most innocuous terminal residues of
any pesticide currently in use. Terminal residues of such simple
structure must, however, have an intermediate source vastly different
than the parent carbamate or its oxime hydrolysis product. Data are
presented in the current study which indicate that intermediates of
unknown toxicological significance are formed in rats by Beckmann
rearrangement of syn and anti methomyl oxime. These products, while
in part predictable based on previous work with the compound, have not
heretofore been postulated as metabolites of methomyl.
While liberation of carbon dioxide and acetonitrile from the imide
carbon was the primary indication that unreported metabolites were
involved in methomyl metabolism, additional factors relative to its
fate in rats were also instrumental. Of utmost importance was the slow
rate of urinary elimination of total radiocarbon from syn [1I+C=N] meth-
omyl-treated animals. Twenty-four hr after treatment, only 34% of the
administered radioactivity was voided by this route. An additional 30%
was eliminated in the expired air, but these products, especially
acetonitrile, continued to be exhaled for 3 days, demonstrating that
their precursors were still in the body. Because 80% of the adminis-
tered carbamate was hydrolyzed by 6 hr (Table 3), it was apparent
that the [ C=N]-labeled residues remaining in the rat were predominan-
tly oximes or their metabolites.
23
-------
Since [11+C] urinary excretion of most carbamates radiolabeled on the
alcohol moiety equal that in the urine plus the expired air when the
carbamates are radiolabeled on the carbonyl carbon, the low quantity
of [ 4C=N]-labeled metabolites of methomyl in the urine was definitely
considered unusual for a member of this group of insecticides. With
other carbamates, including aldicarb, the hydrolysis products are
conjugated and almost completely excreted from the body within 24 hr.
It was assumed, therefore, that a portion of residues in rats treated
with [1<*C=N] methomyl were in a form other than the oxime. Otherwise,
far more than 34% of the radiocarbon, presumably oxime conjugates,
would have appeared in the urine by 24 hr.
The nature of the persistent precursor to acetonitrile and carbon
dioxide was not elucidated, but the evidence suggests that a rather
stable complex is formed between a normal body component(s) and the
methomyl oximes or their Beckmann rearrangement products. Because the
rate of [ C] acetonitrile liberation from anti [ C=N] methomyl was
almost the same as for [lkC] carbon dioxide from syn [1'*C=N] methomyl
and its oxime (Table 5), the stabilities of the syn and anti complexes
appear to be very similar. Therefore, the production of acetonitrile
beyond that of carbon dioxide from syn [ll*C=N] methomyl-treated rats is
probably a simple matter of the syn complex being formed before the
anti complex.
Complex formation of the type proposed for methomyl metabolism is
evident also for thiocarboxime, another thiohydroximic N-methylcarba-
mate insecticide (Hutson et al. , 1971). With syn [llfCH311*C=N]-labeled
material, rats exhaled 5.2% of the dose as [J1|C] carbon dioxide during
the first day, 2.4% on the second day and 0.5% on the third day follow-
ing oral treatment. Much greater quantities of [1I+C] acetonitrile
(20.9, 10.7, and 4.1% of the dose on each of the first 3 days, respec-
tively) were exhaled, indicating the degree of isomerization to the
anti isomer which had occurred.
24
-------
It should be noted, however, that Beckmann rearrangement of the anti
oxime would yield acetonitrile containing both radiocarbons from the
parent pesticide, whereas the carbon dioxide from the syn form would
contain only one of the radiocarbons. Therefore, the actual amount
of [1!*C] carbon dioxide produced would be double the amounts indicated
by the radioactivity collected in the traps. The other radiocarbon
would be converted to [14C] methylamine by degradation of the thio-
carbamate and excreted via the urine. Considering this stereospecific
aspect of acetonitrile and carbon dioxide formation from syn and anti
oximes, the amounts of these products formed from thiocarboxime (35.7
and 16.2% of dose after 3 days) were similar to those from methomyl
treatments (24.4 and 14.9% of dose after 3 days).
Like methomyl, isomerization of thiocarboxime to the anti form prior
to hydrolysis was indicated by treatment of rats with the syn
t1 CHa lfC=N] oxime. In this case, the [llfC] carbon dioxide expired was
about the same amount as when the carbamate was administered. But,
the amount of [ C] acetonitrile was reduced to only 7.3% of the dose
and this was all collected in the first 24 hr after treatment. It
seems that the route of the metabolism of the oximes is dependent
almost entirely upon its stereoconfiguration when entering the animal
body.
The absence of sulfur oxidation metabolites of methomyl was another
facet which gave an indication that previously unreported metabolic
products might exist. However, there remains the possibility that the
sulfoxide and sulfone derivatives are formed but are unstable. A com-
pound having some structural similarities to the thiohydroximic acids,
methylthiotriazine, reportedly was oxidized to the highly unstable
sulfoxide form in beans (Knuesli, 1964). Based on a possible similar
occurrence with methomyl, the effect of sulfur oxidation on the geomet-
ric configurations of thiohydroximic acids was reviewed. It was found
that S-methyl benzothiohydroximic acid took on the syn form, while the
sulfoxide and sulfone had the anti configuration (Benn et al., 1968).
25
-------
Applying this to methomyl, it seemed possible that syn oxime could be
formed from syn methomyl, and then converted to the anti isomer when
oxidation of the sulfur occurred. This way, isomerization after meth-
omyl hydrolysis could occur and subsequently yield both carbon dioxide
and methomyl. Because the administration of the syn oxime to rats
gave so little acetonitrile, this pathway was not considered adequate
to explain the observed results.
Assuming that the thiohydroximate esters, methomyl and thiocarboxime,
do not undergo sulfur oxidation as do the alkylthioethers, aldicarb
and DS-15647, the explanation may be in the basic chemical nature of
the two groups. For example, the alkylthio group of the thiohydroxi-
mate esters is bonded to a Sp hybridized carbon which is attached to
nitrogen. The effective electronegativity of this carbon atom should
be greater than a carbon that is Sp3 hybridized and bonded to another
carbon as is the case with the alkylthioethers. Consequently, the
oxidation of the sulfur in the thiohydroximate esters should be more
difficult.
In any event, it is now clear that all oxime carbamate insecticides
do not undergo the same general type of metabolism. Evidence has been
presented showing that in vivo isomerization and Beckmann rearrangement
reactions are important in the metabolism of methomyl and are probably
applicable to other thiohydroximate esters. These reactions, plus
carbamate ester hydrolysis and conjugation of the oximes, result in
far more complex routes of bioalteration than previously reported.
Moreover, the complexity of their metabolism appears to exceed that of
the alkylthioethers which are known to involve primarily defined routes
of hydrolysis, sulfur oxidation, and conjugation.
26
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FATE OF CRONETON IN RATS
Croneton, 2-ethylthiomethylphenyl N-methylcarbamate (Bay Hox 1901),
is an experimental plant systemic insecticide with excellent aphici-
dal properties. One of its principal projected uses is the control
of these insects on vegetable, fruit and cereal crops. Croneton is
highly toxic to aphids, LDsg of 5 mg/kg, while the mammalian oral
toxicity is rather low, with an LDso of 411 mg/kg to rats (Bayer In-
formation Bull. E.1-715/20 359, 1974).
The potential use of Croneton on food and feed crops necessitates
studies of its fate in both plants and animals. This paper reports
the results of investigations regarding the insecticide's metabolism
in the rat. The fate of Croneton and its sulfoxide (SO) and sulfone
(SO2) analogs was determined in rats following a single oral exposure.
Also, animals were exposed to the insecticide as a dietary supplement
to determine the degree of residue accumulation which might occur
following the more natural avenue of exposure.
Materials and Methods
Chemicals - Croneton was radiolabeled with ^C uniformly on the aroma-
tic ring (7.05 mCi/mmol) and on the carbonyl moiety (5.88 mCi/mmol).
The C-ring label was supplied by The Chemagro Agricultural Division
Df Mobay Chemical Corporation and was greater than 99% radiochemical-
ly pure. The C-carbonyl label was synthesized by the reaction of a
1.5 to 1 ratio of 2-ethylthiomethylphenol and ^ C-methylisocyanate in
dry benzene. Diacetoxydibutyltin was used as a catalyst.
?or use as metabolite standards, 2-ethylsulfinylmethylphenyl N-methyl-
carbamate (sulfoxide), 2-ethylsulfonylmethylphenyl N-methylcarbamate
(sulfone), 2-ethylsulfinylmethylphenol (phenol sulfoxide), and 2-ethyl-
sulfonylmethylphenol (phenol sulfone) were synthesized by the method
of Harvey et al. (1973), which utilized peracetic acid as the oxidiz-
27
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ing agent. Structural confirmation was performed by nuclear magnetic
resonance and mass spectrometry. For the NMR spectra, samples were
dissolved in deuterochloroform containing 1% tetramethylsilane and
measured with a model T-60 spectrometer (Varian Associates). Mass
spectra were taken at 60 eV using a Finnegan Model 1015 C quadropole
mass spectrometer.
2-Ethylsulfinylmethyl N-methylcarbamate - NMR spectra: 61.29 (t3H,
ethyl CH3), 62.60 (q2H, ethyl-CH2), 62.87 (d3H, N-CH3), 63.90 (dlH,
PhCH), 64.15 (dlH, PhCH), 65.52 (blH, NH), and 67.05-7.55 (m4H, Ph).
Mass spectra: m/e 184 (1.9%), 107 (100%), 79 (18.1%), 78 (17.5%), 77
(30.6%), 58 (2.5%), 57 (20.6%), 51 (10.0%).
2-Ethylsulfonylmethyl N-methylcarbamate - NMR spectra: 61.32 (t3H,
ethyl CH3), 62.68-3.10 (m5H, ethyl-CH2 and NCH3), 64.28 (s2H, PhCH2),
65.27 (blH, NH), and 67.08-7.68 (m4H, PH). Mass spectra: m/e 200
(5.5%), 107 (100%), 79 (10.0%), 78 (13.5%), 77 (16.5%, 58 (4.5%), 57
(9.0%), 51 (5.0%).
Croneton phenol, the phenol sulfoxide, and the phenol sulfone were al-
so confirmed by NMR and MS. Because a molecular ion (m/e 225) was
found for Croneton, the MS of Croneton phenol was easily distin-
guished. This was not the case for the oxidized material which could
only be differentiated from respective phenols by the presence of m/e
58 and 57 ions arising from the carbamate moiety. More positive
structural confirmation was obtained by NMR in these cases. In addi-
tion, the metabolite standards also were found to be identical to
materials supplied by Chemagro.
The purity of the synthesized compounds was determined by thin-layer
chromatographic means using the solvent systems given in Table 7. In
addition, purity was checked using high pressure liquid chromatography
under the following condition: Instrument, two M-6000 pumps plus a
model 660 solvent programer (Waters Associates); detector, a model 240
28
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Table 7. SILICA GEL THIN LAYER CHROMATOGRAPHY OF CRONETON
AND ITS METABOLITES
Compound
C:roneton sulf oxide
Croneton sulf one
Phenol sulfoxide
Phenol sulf one
Droneton phenol
Croneton
Rf values
A
.15
.85
.39
.93
.98
.97
in solvent systems
B
.29
.89
.63
.94
.97
.94
C
.15
.43
.38
.50
.71
.64
A - Ethyl Acetate
3-8:2 Acetonitrile-benzene
C - 6:4 Hexane-acetone
29
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UV spectrophotometer detector set at 270 run (Gilform Instrument Corpo-
ration) ; column - 1/4 in by 30 cm y Porasil; gradient elution, non-
linear 1% methanol in chloroform to 15% methanol in chloroform for 20
min at 1.0 ml/min. Under these conditions the six Croneton standards
were completely resolved.
Treatment - Albino rats (Cox-SD, Laboratory Supply Company) weighing
approximately 200 g each were used in all metabolism experiments. In
the single oral dose studies, male and female animals were treated
with 14C-ring and llfC-carbonyl-labeled Croneton at a dosage level of
0.5 mg/kg. The specific activity of the compounds was adjusted to 2.0
mCi/mmol and delivered to the animals in 0.5 ml of water via a feeding
needle. To determine the fate of the oxidation products of Croneton,
female rats were similarly treated with the ll+C-ring and li+C-carbonyl
sulfoxide and sulfone. Sufficient rats were treated so that all data
collected represented the results obtained with a minimum of 2 animals.
Croneton-carbonyl-11+C and -ring-11+C were administered as a dietary
supplement to female rats for up to 10 days. The animals were main-
tained on standard laboratory chow fortified with 6.6 ppm of the in-
secticide. At this concentration, animals consuming 15 g of feed per
day received 0.5 mg/kg/day of the insecticide. Feeding of the Crone-
ton-fortified diet was followed by a normal dietary regime for up to
1 wk. Three rats were used for each predetermined sacrifice time.
All animals were acclimated to their surroundings for 1 wk prior to
initiating treatment.
Sample Collection and Radioassay - Following treatment with either
ll+C-carbonyl- or 1LfC-ring-Croneton, the animals were placed in metab-
olism cages allowing for separation of urine and feces. Those treated
with the 14C-carbonyl material were placed in cages modified for col-
lection of exhaled gases. Urine samples were radioassayed by direct
liquid scintillation counting. Feces samples were collected at 24-hr
intervals over the 72-hr experimental period and radioassayed by com-
30
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bustion of 0.5 g samples using a Packard Model 306 sample oxidizer.
The ltfCO2 in the exhaled gasses of rats was collected by drawing air
from the cages through 300 ml of 1.0 N potassium hydroxide. Samples
were then taken for radioassay at hourly intervals for 6 hr post-
treatment, then at 3-hr intervals for the next 6 hr and at 12-hr in-
tervals for the remainder of the 72-hr experiment. Radioassay was
accomplished by direct scintillation counting of 1.0 ml of the trap
solution.
To obtain a measure of the rates of dissipation of 1^C-Croneton resi-
dues from selected tissues following single oral treatments, 2 animals
were sacrificed after 8, 24, 48, and 72 hr. Immediately after sacri-
fice, a blood sample was taken and the brain, fat, heart, kidney,
liver, and a sample of skeletal muscle and skin were excised for ra-
dioassay. Radioassay of 0.2-0.5 g tissue samples, with the exception
of fat where 50 mg samples were used, was accomplished in the manner
as the feces.
Following treatment of female rats with lltC-ring- and 11+C-carbonyl-
Croneton sulfoxide, or ^C-ring-Croneton-sulfone, the animals were
placed in metabolism cages as described above. Urine, feces, and ex-
haled gases were monitored for radiocarbon for 3 days after treatment.
Tissue samples were not analyzed in this series of experiments.
Upon initiation of feeding *^C-Croneton as a dietary supplement, sepa-
rate collections of urine and feces were made on a daily basis, but
no attempt was made to collect the exhaled gases. Sampling of excreta
and tissues, and quantitation of the radiocarbon therein, were accom-
plished using the same techniques as employed in the single dose
studies.
Samples collected during the course of the above experiments were
stored at -20°C after preliminary radioassay. Periodic analysis of
urine and liver over a 6-mo. period showed that the residues were
31
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stable at this temperature.
Analysis of Urinary Radiocarbon - Urine was lyophilized to dryness and
the solid residue washed with methanol repeatedly until no radioacti-
vity was detected in the wash. This process was quantitative in the
recovery of the urinary radiocarbon while leaving much of the inter-
fering urinary components behind. The methanol wash was then concen-
trated to a volume which allowed direct application to silica gel thin-
layer plates. Several solvents systems were used to resolve the radio-
active components of the urine (Table 7). In all cases, cochromato-
graphy with standards was used to indicate the nature of the metabo-
lites detected.
Analysis of the polar Croneton metabolites in the urine was carried out
by reconstituting an appropriate amount of the methanolic fraction
with distilled water. Extraction of this aqueous portion with chloro-
form removed the free carbamates and phenols and the aqueous layer was
then adjusted to 0.5 N hydrochloric acid and incubated in a water bath
at 37°C. The incubations were conducted in a sealed tube containing
an equal volume of chloroform for a period of 6 days. Periodic shak-
ing of the tubes extracted the hydrolyzed polar metabolites into the
chloroform layer to prevent further decomposition. The chloroform ex-
tracts were combined and analyzed by tic.
Toxicity Determinations - Oral LD5Q values were determined for Crone-
ton and its oxygen analogs in both rats and mice. In the case of rats,
the techniques of Deichmann and LeBlanc (1943) were used to obtain an
approximate LD50 only. The animals, 200 g female Cox (SD) albino
rats (Laboratory Supply Company), were treated with one of a series
of doses which increased by a factor of 1.5 on a mg/kg basis. The
compounds were administered in aqueous solution via a feeding needle.
For Croneton and its sulfone, 2% Tween-80 was used as the carrier.
The approximate LD50 values were then calculated on a 24-hr mortality
basis. In the case of the mice, LD5Q values were determined according
32
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to techniques of Litchfield and Wilcoxon (1949), using log-dosage/
probit analyses. Swiss female albino mice (Laboratory Supply Compa-
ny) weighing 20 g were treated in groups of 5 with one of six dosage
levels which varied over the range of the approximate LD5Q calculated
as above. Again, treatment was by feeding needle and in aqueous so-
lution or Tween-80 as descirbed above. Mortality values were calcu-
lated on a 24 hr basis.
Results and Discussion
Excretion - Administration of both 11+C-carbonyl- and llfC-ring labeled
Croneton to male and female rats as a single oral dose resulted in
rapid elimination of the radiocarbon. The pattern of elimination of
14C-carbonyl-labeled Croneton was found to be virtually the same as
the elimination pattern of ltfC-carbonyl-Croneton sulfoxide. An aver-
age of 47% of the dose was exhaled as 11*C02 and this occurred predomi-
nantly within the first 8 hr after treatment. An additional 41% of
the dose was recovered in the urine over a 3-day period while only 7%
of the dose was voided in the feces.
Approximately 96% of the 1^C-ring-Croneton dose was eliminated from
the rats in the urine and about 2% in the feces. Almost identical
patterns of excretion were exhibited by ^C-ring-labeled Croneton sul-
f oxide and sulf one. Like the 11+C-carbonyl label, most of the excre-
tion occurred during the first 24 hr following administration, and
there was no difference in the rates or quantities of radiocarbon
voided by males and females.
Excretion rates of radiocarbon from rats administered 1^C-carbonyl-
and 1^C-ring-Croneton as a dietary supplement were very similar to
those observed from the single oral treatments. Approximately 37% of
the average daily dose of ^C-carbonyl-Croneton was eliminated in the
urine and 7% in the feces. After return to untreated feed, radiocar-
bon in the urine and feces declined to below 1% of the daily dose in
33
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3 days. The rate of decline was the same for animals fed the treated
diet for 7 and 10 days.
Elimination of ^C-ring-Croneton equivalents in the urine averaged
89% of the average daily dose while only 5% was voided in the feces
during the treatment period. Like the carbonyl-^C continuous feeding
study, a plateau was reached after 3 days on the treated diet. Excre-
tion of radioactive residues declined to below 1% of the average daily
dose 3 days after the 1[|C-Croneton was removed from the diet. Cumu-
lative excretion of the total radioactivity consumed during the 7-day
feeding period was 86% at the time the animals were returned to an
untreated diet, and 90% 7 days later.
Nature of Urinary Radiocarbon - Results of the analysis of urine from
rats which received 1LfC-Croneton as a single oral dose or in the diet
are presented in Table 8. These data represent the results obtained
with the ring-11+C and carbonyl-ltfC Croneton. All metabolites identi-
fied as carbamates were detected in the urine of rats treated with
both ring-11+C- and carbonyl-^C-labeled Croneton. Those identified
as phenolic hydrolysis products were detected only in the urine of
rats treated with Croneton- ring-1L*C. These data, plus extensive
tic of the metabolites with authentic standards, provided the basis
upon which identification of the metabolic products was made.
Qualitatively, the urinary metabolites were sufficiently similar fol-
lowing treatment of the rats with single and multiple doses of the
carbamate to suggest that the metabolic pathway for Croneton in rats
was not altered by continuous exposure of the animals to the insecti-
cide in the diet. The parent carbamate was not voided in the urine,
except possibly in trace amounts which appeared in extracts of acid-
treated polar metabolites. While hydrolysis of the carbamate ester
was evident, the parent phenol constituted only a small portion of the
excreted radiocarbon, and this was almost entirely in the conjugated
form.
34
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Table 8. NATURE OF RADIOCARBON ELIMINATED IN THE URINE OF RATS
TREATED WITH 14C-CRONETON
Percent of dose/administered as:
Metabolite
Apolar
Croneton sulfoxide
Croneton sulfone
Parent phenol
Sulfoxide phenol
Sulfone phenol
Unknown A
c
Polar
Croneton
Croneton sulfone
Parent phenol
Sulfoxide phenol
Sulfone phenol
Unknown B
r?otal
Single oral dose
36.1
23.0
3.4
0.0
4.5
2.9
2.3
60.6
1.0
0.0
0.9
18.5
22.0
18.2
96.7
Dietary supplement
51.4
28.2
6.9
0.4
6.1
4.5
5.3
34.5
0.9
4.5
7.7
14.4
3.6
3.4
85.9
b
Combined urine voided over 3-day period following treatment.
Combine urine voided during 7-day feeding period.
Polar metabolites treated with acid to release indicated materials.
Radiocarbon not released by acid treatment.
35
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The remaining metabolites which were identified resulted, at least in
part, from sulfur oxidation (Table 8). Croneton sulfoxide was the
predominant carbamate metabolite in the urine while the sulfoxide and
sulfone phenols were the predominant non-carbamate. Further oxidation
of the sulfur resulted in the formation of the sulfone analog which
existed as the carbamate and phenol, but in lesser amounts than the sul-
foxide derivatives. The majority of the phenolic metabolites were pres-
ent as conjugates while the carbamate sulfoxide and sulfone were large-
ly in the free form.
In the continuous feeding study, Croneton sulfone was recovered from the
acid-treated polar metabolites in quantities equal to 4.5% of the total
radiocarbon consumed by the animals. The majority of this metabolite
was released immediately upon acidification. Its concentration was sim-
ilar in urine of rats fed both radioactive Croneton feed preparations,
and consistent results were obtained with replicate samples. It is
therefore, quite likely that Croneton sulfone was weakly conjugated in
rats and was excreted in this form. Croneton sulfoxide appeared in some
extracts of the acid-treated polar metabolites, but the quantities were
less than the sulfone and its presence was not consistent. It was con-
cluded that the sulfoxide was not conjugated but was in the polar frac-
tion in the free form as a result of incomplete extraction into the
apolar phase.
The metabolite designated as unknown A (Table 8) was tentatively iden-
tified as 2-ethylsulfonylmethylphenyl N-hydroxymethylcarbamate, or
hydroxymethyl Croneton sulfone. Since the metabolite was produced in
equal quantities with both the ring-14C and carbonyl-lt+C Croneton, the
ester linkage was assumed to be intact. This was further substantiated
by treating the ring-1I+C-labeled metabolites with 0.5 N sodium hydroxide
to yield a product having tic characteristics identical to sulfone phe-
nol. Reaction of this product with methyl isocyanate gave a compound
indistinguishable from Croneton sulfone. These data established that
the ring moiety of unknown A was the sulfone phenol and that some modi-
36
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fication of the carbamate group also had occurred. N-Methylhydroxyla-
tion was proposed since a positive color reaction was evident when the
.netabolite was treated with chromotropic acid. This test, which indi-
cates the formation of formaldehyde from N-hydroxymethyl compounds, has
seen used to denote the presence or absence of N-hydroxymethyl metabo-
lites of several carbamate insecticides (Dorough and Casida 1964, Met-
oalf et al. 1968, Black et al. 1973).
Unknown A was consistently present in the free form (Table 8) and was
sometimes detected in very small amounts in extracts of the acid-
created polar materials. However, its low level and sporatic presence
prevented confirmation of its identity as a conjugate.
Treatment of rats with 1^C-Croneton sulfoxide as a single oral dose re-
sulted in the same metabolic pattern (Table 9) as with Croneton, per
se. The same free metabolites were detected in the urine, and the po-
Lar products were of a similar magnitude. There is little doubt that
"he parent carbamate is very rapidly converted to the sulfoxide form
upon entering the animal body by ingestion. Data in Table 9 also con-
firm that the oxidation of the sulfoxide to the sulfone proceeds at a
nuch slower rate than the initial oxidation. When the sulfone itself
was given to the rats, the nature of the metabolites in the urine was
as one might predict. All of the free metabolites were sulfone deriv-
atives, and the amounts of polar metabolites were of the same general
Levels as observed with Croneton and Croneton sulfoxide treatments.
tissue - Selected tissues were radioassayed for ll*C-residues following
single oral administration of * C-carbonyl- and ^C-ring-Croneton. The
highest levels of residues were found in tissues of animals treated
with the 11+C-carbonyl-labeled material. As compared to the ring-1<+C
residues where maximum residues did not exceed 0.05 ppm, they were
higher by a factor of 1.4 in the muscle, and by factors of 2.3, 5.8,
cJid 9.1 for the kidney, blood and liver, respectively, when averaged
over the 72-hr experiment. Obviously, these increased residues with
37
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Table 9. NATURE OF RADIOCARBON ELIMINATED IN THE URINE OF
RATS GIVEN A SINGLE ORAL DOSE OF lkC-CRONETON SULFOXIDE
AND CRONETON SULFONE
Percent of dose
Metabolites Crone ton sulf oxide Croneton sulfone
Apolar 35.1 23.3
Croneton sulfoxide 23.8 0.0
Croneton sulfone 4.9 7.8
Sulfone phenol 1.2 10.9
Unknown A 5.2 4.6
Polar 58.3 65.4
Total 83.4 88.7
Combined urine voided over 3-day period following treatment.
38
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;he carbonyl-^C Croneton treatment were derived only from the carbamate
portion of the molecule. This would suggest that the radioactive car-
bon liberated upon hydrolysis was incorporated in natural tissue compo-
nents.
Because of the very rapid metabolism and excretion of Croneton, and the
.Low level of ring-14C residues in rat tissue 8 hr after treatment, an
experiment was conducted to determine if peak residue levels occurred
prior to the 8-hr sampling period. Female rats were treated orally
with 5 x 105 dpm (7.3 yg) of the ^C-ring-labeled Croneton and then an
cinimal sacrificed at each 30 min interval for 8 hr. Samples of the
blood, liver, kidney, and muscle were collected and radioassayed. The
point at which maximum 14C-ring-Croneton equivalents occurred in the
t.issues ranged between 2 and 2.5 hr after administration of the single
oral dose, with the blood and muscle showing the earlier peak. These
cata clearly demonstrated the extreme rapidity with which Croneton and/
cr its metabolites are absorbed from the gut, distributed throughout
the body and then eliminated. With such efficient metabolism and excre-
tion, there appears to be little potential for residue accumulation.
Analysis of tissue samples collected from rats maintained on a diet con-
taining 6.6 ppm of either C-carbonyl- or C-ring-Croneton substanti-
ated the results seen in the single oral dose studies. In those rats
fed the C-carbonyl-label material, the majority of the tissues in-
creased in Croneton equivalents as the time of exposure increased. How-
ever, the rate of increase was very slow after the second day of expo-
sure of the animals to the treated diet. At the end of 7 days the lev-
els were 1.18, 0.29, 0.24, 0.10, 0.18, and 0.06 ppm for the liver, kid-
nsy, blood, muscle, brain, and fat, respectively. Those animals fed the
C-ring-labeled compound showed much lower C-residue levels in the
same tissues and they increased very little after the second day of
feeding.
When the animals were returned to an untreated diet, C-ring-residues
39
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in the tissues declined at a much faster rate than the ^C-carbonyl-
labeled residues. This pattern of dissipation would be expected if the
latter residues resulted in part from the incorporation of the radioac-
tive carbon into normal tissue constituents. ^C-Carbonyl-Croneton
equivalents were between 0.04 and 0.11 ppm 7 days after removal from
treatment, while the lt+C-ring-labeled residues had decreased below the
0.01 level after only 2 days.
Because of the low levels of Croneton-ring-^^C residues in the tissues,
no attempt was made to characterize the radiocarbon therein. However,
livers of rats fed Croneton-carbonyl-ll|C in the diet were evaluated as
to extraction characteristics, but their levels were too low for evi-
dence required for identification to be obtained. Homogenization of
livers of rats fed 1, 2, 4, and 7 days with a 3:1 acetonitrile-water
mixture removed approximately 30% of the ^C-residues in each of the
samples. Treatment of the tissue solids with 1.0 N hydrochloric acid
at 80°C for 1 hr released an additional 44% of the residues; these could
not be extracted from the aqueous phase with chloroform. The acetoni-
trile-water extract of the livers also was extracted with chloroform
and, in this case, about two-thirds of the radiocarbon partitioned into
the organo-solvent layer. Treatment of the aqueous layer with 1.0 N
hydrochloric acid for 1 hr at 80°C did not convert any of the radioac-
tivity into chloroform-extractable materials.
Thin-layer chromatography of the chloroform extract of the liver homoge-
nates was unsuccessful because of the small amount of radioactivity and
the large quantities of oils and other interfering materials present.
Treatment of the chloroform-extractables with a coagulation solution
(1.25 g ammonium chloride and 35 ml of phosphoric acid diluted to 1
liter) resulted in all of the radioactivity being removed from the solu-
tion along with the precipitate. Thus, none of the ^C-residues in the
liver of rats fed 1<4C-carbonyl-Croneton exhibited extraction and/or par-
titioning characteristics typical of free or acid-released Croneton-type
metabolites.
40
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Toxicity - An an indicator of the biological significance of Croneton
,md its metabolites, the toxicities of the parent carbamate and its
oxidized sulfur analogs were determined in both rats and mice (Table
LO). The mice were much more sensitive to the carbamates than the rats.
Cn mice, the toxicity of Croneton and its sulfoxide was not statistical-
ly different (71 and 59 mg/kg, p<.05) while Croneton sulfone was only
.ibout one-fourth as toxic. While less toxic to rats, the relative tox-
.Lcities were approximately the same in rats as in mice.
41
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Table 10. TOXICITIES OF CRONETON AND ITS OXIDIZED SULFUR
ANALOGS TO RATS AND MICEa
Compound
Croneton
Croneton sulfoxide
Croneton sulfone
APPROXIMATE
Rats
338
150
506
LD50(mg/kg)
Mice
150
67
225
LD50 (mg/kg)
Mice
71
59
282
a
Twenty-four hr LD5Q following single oral dose.
Determined by the approximation method of Deichmann and LeBlanc (1943)
Q
Determined by log-dosage/probit analyses of Litchfield and Wilcoxon
(1949).
42
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FATE OF CRONETON IN LARGE ANIMALS
Croneton (2-ethylthiomethylphenyl-N-methylcarbamate) is an experimental
insecticide which may be used on crops constituting a portion of the
diets of livestock. Therefore, it is important to determine the fate of
the insecticide in these animals so that the potential for the transfer
Df residues to the diet of man can be estimated. In the present study,
results of the fate of a single oral dose of radioactive Croneton to a
lactating Holstein cow, a male Yorkshire pig, and to laying White Leg-
norn hens are presented. Experiments with the hens were extended to in-
clude daily treatments with Croneton for 7 days so that the effect of
multiple exposure on metabolism and disposition of the carbamate could
be evaluated. The fate of Croneton in rats following single and multi-
ple treatments of the insecticide has been determined in our laboratory
and the results previously reported (Nye et al. 1976).
Materials and Methods
Chemicals - To determine the fate of Croneton in the animals, [ll|C]-
ring-labeled insecticide was used (specific activity, 2.0 mCi/mmol).
This was supplied by the Chemagro Corporation and had a radiochemical
purity of greater than 99% as determined by thin-layer chromatographic
c.nd autoradiographic techniques. To aid in metabolite identification
by cochromatography, the following compounds were synthesized by methods
previously described (Nye et al. 1976); 2-ethylsulfinylmethylphenyl N-
methylcarbamate (Croneton sulfoxide), 2-ethylsulfonylmethylphenyl N-
methylcarbamate (Croneton sulfone), 2-ethylthiomethylphenol (Croneton
phenol), 2-ethylsulfinylmethylphenol (phenol sulfoxide), and 2-ethyl-
sulfonylmethylphenol (phenol sulfone).
Treatment and Sampling - Cow and pig - Both the lactating Holstein cow
(weighing approximately 500 kg with milk production of 22 kg/day) and
the male Yorkshire pig (approximate weight of 50 kg) were acclimated to
the experimental ambience for 3 days prior to treatment. Croneton was
43
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administered at a level of 0.5 mg/kg in a gelatin capsule containing
feed grain. Following treatment, the cow was placed in a metabolism
stall and separation of urine from feces was accomplished by an intra-
urethral catheter implanted prior to treatment. The swine was also
placed in a metabolism stall which allowed for separate collection of
urine and feces.
Urine eliminated by the cow was sampled on an hourly basis for the first
6 hr after treatment, at 2 hr intervals for the next 6 hr, and at 4 hr
intervals for the next 12 hr. Urine eliminated by the pig was sampled
periodically during the 24 hr experiment and collection was dependent
on the intervals of urination. Radioassay of the urine samples was by
direct liquid scintillation counting of 0.5 ml samples. Feces samples
were collected from both animals periodically and radioassayed by com-
bustion of 1.0 g samples using a Packard Model 306 Sample Oxidizer.
In addition to the urine and feces samples, blood (5.0 ml) and milk from
each quarter (5-10 ml) were taken from the cow at the same time inter-
vals as the urine samples. At 6 and 21 hr after treatment, the animal
was milked in a normal manner using a mechanical milker. The blood sam-
ples, taken from the abdominal vein, were immediately heparinized and
later radioassayed by combustion of 0.5 ml samples as described above.
At 24 hr post-treatment, the cow and pig were slaughtered and represen-
tative tissue samples immediately frozen for later analysis. Radioassay
of 0.5 g samples (0.05 g in the case of adipose tissue) was accomplished
by combustion in the same manner as for the blood and feces samples.
Hens - In the first study, eight hens were treated with a single oral
dose of Croneton at a rate of 0.5 mg/kg (sp. act., 2.0 mCi/mmol). The
dose was prepared by adding the ring labeled insecticide to gelatin cap-
sules containing a small amount of laying mash. Following treatment,
the birds were placed in individual cages and eggs and excreta were col-
lected at 12-hr intervals for 3 days. No attempt was made to separate
44
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urine and feces. At 8 hr, and at 1, 2, and 3-day intervals after
treatment, two birds were sacrificed and tissue samples collected.
In the second study, 10 birds were given the capsules containing Crone-
ton on a twice-daily basis at about 12-hr intervals for 7 days. The
specific activity of the ring [11+C] material was 4.0 mCi/mmol to facil-
itate tissue radiocarbon analysis which was difficult at the dosage
level used in the first study. Eggs and excreta were collected prior to
treatment and on a daily basis after treatment was initiated. On the
4th day of treatment and prior to administration of the second daily
dose, one bird was sacrificed and tissues taken for radioassay. At 4 hr
following the final treatment, four birds were sacrificed in an attempt
to achieve maximum tissue [11+C]-ring Croneton equivalents. Two birds
were sacrificed 1 day after the final treatment and one bird sacrificed
at 2, 4, and 7 days after the last capsule was given to establish rates
sf dissipation of [1XtC]-ring Croneton residues.
In all experiments, the birds were maintained on a 12-hr light and 12-hr
iark photo period and were given laying mash and water ad libitum. All
samples were stored in the freezer until analyzed.
Tissue samples and the excreta were radioassayed by combustion as pre-
viously described. The eggs also were analyzed by combustion, but they
were first divided into shell, yolk and whites, and analyses were per-
.formed using 0.2-g samples.
Metabolite Identification - Excreta - Aliquots of urine from the cow and
sswine, or excreta from the hens were lyophilized prior to the determina-
tion of the nature of the radiocarbon content. The dry solid residues
were then repeatedly washed with methanol which removed greater than 95%
of the [ C]-ring Croneton equivalents while leaving a large portion of
the interfering components behind. The methanolic fraction was concen-
trated to a volume suitable for direct application to silica gel thin
layer chromatographic plates. Two basic solvent systems were used to
45
-------
develop the tic plates; a 6:5 hexane-acetone mixture for one-dimensional
chromatography, and a two-dimensional system using 6:5 hexane-acetone in
one direction and 10:1 ethyl acetate-hexane in the second direction.
The first system was used as a preparative method for analysis of free
metabolites, while the two-dimensional system was used for attempted co-
chromatography of metabolites with known standards. In addition, a
benzene-acetonitrile solvents system (of varying composition) was used
to confirm the results of the two-dimensional system.
The polar metabolites remaining at the origin in the above systems were
subjected to analysis by incubation with 0.5 N hydrochloric acid. The
acidified samples were incubated for 6 days at 45°C and the acid-released
metabolites were removed by extraction with chloroform at 24-hr intervals.
In addition to acid hydrolysis, the polar metabolites were incubated
with 3-glucuronidase (type V-A, Sigma Chemical Company) and aryl sulfa-
tase (type III, Sigma Chemical Company). Fifty units of B-glucuronidase
or 10 units of aryl sulfatase were added to the extracted metabolites in
acetic acid-sodium acetate buffer (0.1 M, pH 5.6) and the mixture incu-
bated for 24 hr at 37°C. The preparations were then extracted with
three equal volumes of chloroform, and the chloroform concentrated and
applied to tic.
Milk - To determine the nature of the radiocarbon residues in milk, 100
ml of the 6-hr sample were mixed with 200 ml of acetone to precipitate
the solids. The solids were separated by filtration and the filtrate
extracted three times (100 ml, 75 ml, and 75 ml) with chloroform. The
combined chloroform extracts were concentrated and analyzed by tic us-
ing the 6:5 hexane-acetone solvents system.
The aqueous phase of the milk was acidified to 1.0 N with hydrochloric
acid and heated for 1 hr at 80°C. Radiocarbon released by acid treat-
ment was extracted into chloroform and analyzed by tic.
46
-------
2ggs - Eggs collected during the continuous feeding study were analyzed
Dy homogenizing the combined whites and yolks in acetonitrile for 3 min
in a Lourdes homogenizer. The solids were removed by filtration and
bwice again homogenized with acetonitrile. The acetonitrile filtrates
were combined and 100 ml of 0.5 N hydrochloric acid were added. This
mixture was extracted with four 60-ml aliquots of chloroform, the chlo-
roform dried over anhydrous sodium sulfate, and then evaporated to dry-
less. Several washings of the evaporating flask with a precipitation
solution (1.25g of ammonium chloride and 25 ml of phosphoric acid in
sufficient water to make a final volume of one liter) were used to trans-
fer the residue to a separatory funnel. The solution was extracted as
oefore with chloroform, the solvent dried with sodium sulfate and then
concentrated for application to tic plates.
The water resulting from the first chloroform extract was acidified to
L.O N with hydrochloric acid and heated at 80°C for 1 hr. This solution
was extracted with chloroform. Solids remaining after homogenization
were similarly treated with a 1.0 N hydrochloric acid solution for 1
ir at 80°C and the solution filtered. The filtrate was extracted with
chloroform and both phases were radioassayed.
Results
Cow and Pig - Uptake and excretion - Maximum concentrations of [ C]-
ring Croneton equivalents in cow blood, 0.32 ppm, occurred 3 hr after
treatment. The appearance of radiocarbon residues in the milk was
.somewhat slower with the maximum concentration of 0.15 ppm observed 4
ir after administration of the capsule. These results are similar to
;hose observed in rats where blood and tissue [^C]-Croneton equiva-
lents were highest after 2.0 to 2.5 hr (Nye et al. 1976). [11+C]-Cro-
neton equivalents in the urine reached a maximum concentration 3 hr af-
:er administration. Cumulative excretion of [lltC] Croneton equivalents
via the urine was greater than 90% of the dose by 12 hr. After 24 hr,
97.8% of the dose had been voided in the urine while less than 1% was in
47
-------
the feces. In the pig, elimination was also principally via the urine,
with 80% of the dose excreted within the first 12 hr. After 24 hr, 90%
of the dose had been eliminated in the urine and 5.1% was present in
the feces.
Tissues - Following slaughter of the swine 24 hr after treatment, com-
bustion analysis of various tissues revealed no detectable radioactive
residues. Essentially the same findings were obtained with the bovine
tissues. Of 18 different tissues analyzed, only the kidney (0.016 ppm),
liver (0.017 ppm), and skin (0.05 ppm) contained detectable levels of
radiocarbon.
Urinary metabolites - There were several quantitative differences in the
nature of the Croneton metabolites in the cow and pig urine (Table 11).
The cow, for example, excreted Croneton principally as water soluble
metabolites, 86.9% of the dose, with the phenol sulfoxide and phenol
sulfone being the only significant free metabolites. The swine, on the
other hand, eliminated the [ll*C]-ring-Croneton equivalents to a lesser
extent as water soluble metabolites (53.3% of the dose). Of the total
radiocarbon in the pig urine, 26% was as free Croneton phenol, 9% as
free phenol sulfoxide and 6% as free phenol sulfone.
Analysis of the polar urinary [11+C]-components, those remaining at the
tic origin, was attempted by acid hydrolysis. In the case of urine col-
lected from the cow, incubation at 45°C for 6 days in 0.5 N HC1 or at
80°C for 2 hr at 4.0 N HC1, gave identical results. Only 25% of the
polar metabolites was recovered by chloroform extraction. Nearly one-
half of the radiocarbon released with acid was identified as the phenol
sulfoxide. The remainder consisted of Croneton sulfoxide, phenol sul-
fone, Croneton, phenol, and unknown II. The latter metabolite had the
same characteristics as a metabolite formed in rats (unknown A, Nye et
al. 1976) and in chickens (unknown II). Incubation of the polar metabo-
lites with 3~glucuronidase was more successful at releasing the [ C]-
Croneton equivalents from the polar form than treatment with acid
48
-------
Table 11. NATURE OF RADIOCARBON IN 0-12 HOUR URINE OF COW AND
PIG TREATED WITH A SINGLE ORAL DOSE, 0.5 MG/KG,
[lkC] RING-CRONETON.
Metabolites
F - Free
\ - Acid-released
% of total [llfC] in sample
J tJ-LU^UXUU-LLlClJit: J-ti-Ltici&tJU
3 - Sulfatase-released
Croneton
Croneton sulfoxide
Croneton phenol
:?henol sulfoxide
Phenol sulfone
Unknown II
b
Polar unknown
- A
- F
- A
- G
- S
- F
- F
- A
- G
- S
- F
- A
- G
- S
- A
- A
- G
- S
Cow
0.0
0.5
0.9
0.0
0.0
0.0
8.7
12.5
37.9
4.8
2.4
1.0
6.3
1.8
7.5
66.5
44.2
81.8
Pig
9.0
0.0
0.0
2.1
2.0
25.5
9.1
12.2
12.1
18.0
6.0
1.1
4.3
4.3
0.0
33.6
40.9
35.1
After removing the free metabolites, the polar radiocarbon was sepa-
rated into 3 portions and individually treated with acid, glucuroni-
dase or sulfatase. Therefore, the released metabolites are not cumu-
lative.
Radiocarbon remaining in water phase after indicated treatment.
49
-------
(Table 11). Tic analysis revealed that conjugated phenol sulfoxide con-
stituted 37.9% of the radiocarbon in the urine and the phenol sulfone,
6.3%. Aryl sulfatase treatment released only 4.7% and 1.7% as the phe-
nol sulfoxide and phenol sulfone, respectively. In neither case did
the enzyme treatment release unknown II.
Only 33.6% of the Croneton equivalents in the swine urine remained as
water soluble components after treatment with acid (Table 11). About 9%
of the total [1JtC] in the urine was released as phenol sulfoxide, while
only 1% was released as the phenol sulfone. In addition, 9% was released
as a product with an R,. identical to Croneton. Unknown II was not ob-
served in the acid released fraction of the swine urine.
Enzymatic hydrolysis of the polar origin Croneton equivalents was only
slightly more effective than treatment with acid, and the radiocarbon
released was nearly equal for glucuronidase and sulfatase. As was the
case for the cow urine, the major component released by enzymatic hydro-
lysis was the phenol sulfoxide. After glucuronidase treatment, 41% of
the radioactivity in the urine remained in the water phase while 35% re-
mained after treatment with sulfatase.
It appears that the differences that occurred between the two animals
with regard to the fate of Croneton was their ability to oxidize the
parent carbamate as well as to conjugate the phenolic metabolites of
Croneton. The cow had the greater ability to perform these functions,
while the swine had a somewhat restrictive ability to form conjugates
of the products resulting from hydrolysis of the carbamate. It is im-
portant, however, that this did not limit the swine's ability to elimi-
nate the ingested compound.
Milk metabolites - While no appreciable radiocarbon was detectable in
the milk 21 hr following administration of the bolus, analysis of the
metabolites in the 6-hr sample (0.3% of the dose) gave a good indication
of the nature of the products excreted in milk. Of the total radiocar-
50
-------
bon in the milk, 67.3% was organosoluble and identified primarily as the
isulfoxide and sulfone of the carbamate (Table 12). Small amounts of the
phenol sulfoxide and sulfone were also present in the free form.
Acid hydrolysis of the water soluble fraction yielded organo extract-
-------
Table 12. NATURE OF [14C] CRONETON EQUIVALENTS IN MILK OF COW
TREATED WITH A SINGLE ORAL DOSE, 0.5 MG/KG,
OF [14C] RING-CRONETON.
PPB and % distribution/6-hr milk sample
Metabolites or fraction
Free
Croneton sulfoxide
Crone ton sulfone
Phenol sulfoxide
Phenol sulfone
Water soluble
Acid-released
Croneton sulfoxide
Croneton sulfone
Croneton phenol
Phenol sulfoxide
Phenol sulfone
Unknown II
Remaining in water
Milk Solids
Total
PPB
86.1
50.6
24.7
7.2
3.6
37.8
i-LZ
7.2
0.4
0.6
4.8
1.5
1.2
H-i
4.0
127.9
%
67.3
39.6
19.3
5.6
2.8
29.6
12._3
5.6
0.3
0.5
3.8
1.2
0.9
17;3
3.1
100.0
52
-------
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53
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Accumulation of insecticide equivalents in the tissues was minimal as
indicated by the low levels found in isolated tissues 4 days after ini-
tiation of the daily treatments. Although [11+C]-residues were higher in
the tissues at 7 days, this was due largely to the early sampling time
after the last dose was administered (4 hr) rather than from accumula-
tion. With the 4-day samples, the hens were sacrificed 8 hr after the
prior treatment. [14C]-Residues in all tissues dissipated very rapidly
and most were below the level of sensitivity (.005 ppm) within 2 days
after treatment was terminated.
Eggs collected following the single oral treatment contained no [ C]-
ring Croneton equivalents in either shell, white, or yolk. [1I+C]-Resi-
dues in eggs collected during the continuous feeding study were evident
2 days after initiation of treatment. The shell showed the lowest level
of insecticide residues, reaching a high of 16.1 ppb on the third day.
Residues in the white and yolk were nearly equal for the first 4 days of
treatments and averaged 38 ppb. While residues in the white increased
very little thereafter, those in the yolk continued to rise and were on
the order of 70 ppb on the last day of treatment.
A large variation existed between the eggs sampled at any given time
during the continuous feeding experiment. The location of residues
within the eggs appeared to be dependent on the stage of egg development
at the time the dose was administered and certain eggs within each sam-
pling time were found to contain the entire residue within either the
white or the yolk.
Nature of metabolites - The nature of the [ll+C]-ring-Croneton equivalents
found in the excreta is given in Table 14. The differences in metabolism
that existed between application by single oral dose or continuous feed-
ing occurred in two areas. First, the quantity of polar materials in
the excreta of the single-dosed hens was lower than that observed in the
continuous feeding experiment. This difference was minimized after acid
hydrolysis, as similar amounts of [14C]-ring Croneton equivalents remained
54
-------
Table 14. NATURE OF RADIOCARBON IN EXCRETA OF HENS
TREATED WITH [1UC]-RING LABELED CRONETON
Metabolites
F = Free
A = Acid-released
Croneton sulfoxide
C::oneton sulfone
C::oneton phenol
Phenol sulfoxide
Phenol sulfone
Unknowns
Unknown polar
_ T?
- A
- F
F1
- F
- A
- F
- A
- F
- A
c
',
Single
oral
dose
0
0
0
2.9
23.9
1.0
36.5
1.6
0
7.1
27.0
6 of total [1UC]
Continuous
1
2.7
0
0.9
1.3
2.6
9.8
13.3
15.1
12.1
7.2
35.0
in sample
treatments/days
4
1.3
1.2
0.9
2.6
5.1
2.6
29.3
12.3
9.6
9.4
25.7
7
1.7
0
0.7
2.5
6.0
3.4
20.2
14.1
10.6
13.3
27.4
Radiocarbon remaining in water phase after acid treatment.
55
-------
as unknown polar products in both studies. A second difference was in
the nature of the radiocarbon which existed as free metabolites, and
which could be released with acid. Like the single oral dose experi-
ment, the major metabolites in the excreta of the multiple-dosed hens
were the phenol sulfoxide and phenol sulfone. However, conjugation of
these products was much greater in the continuous feeding study. Some
small amounts of the sulfoxide and sulfone of the carbamate were also
found in the continuous feeding study, but not in the single dose ex-
periment.
Major differences in the quantity of unknown metabolites were found in
the two studies (Table 14). Unknown II (10.6% of the dose) has the same
R as an unknown isolated and tentatively identified as the N-hydroxy-
methyl Croneton sulfone in previous rat studies (Nye et al. 1976). As
found in the earlier study, unknown II in the chicken excreta was un-
stable, and was converted to the sulfone phenol under acid conditions.
Several other unknowns were also released by acid hydrolysis but were
not identified because of their low individual quantity. Treatment of
the water soluble metabolites with 3-glucuronidase and aryl sulfatase
did not release any t^Cl-ring Croneton equivalents into the chloroform
extract.
The nature of [ C]- residues in the tissues was not evaluated. Total
radiocarbon was either too low in tissues of bulk, or the sample too
small to yield sufficient [ll*C] to follow through the various fractions
resulting from extraction and partitioning. This was not the case with
eggs (Table 15).
A majority of the [ll*C]-residues in the eggs was organosoluble and, as
was the case for metabolites in the excreta, those in the eggs were
principally sulfoxidation products of Croneton phenol. Combined, the
sulfoxide and sulfone of the phenol accounted for 71.4, 75.5, and 78.3%
of the total residues in the eggs collected on day 3, 6, and 7. Small
amounts of the oxidized carbamates and unknown products were also de-
56
-------
Table 15. NATURE OF [lkC] CRONETON EQUIVALENTS IN EGGS OF HENS
DOSED EVERY 12 HR WITH 0.5 MG/KG [lkC] RING CRONETON FOR 7 DAYS.
Metabolites
or fractions
Free
Croneton sulfoxide
Crone ton sulfone
?henol sulfoxide
.Phenol sulfone
Unknowns
Water Soluble
Acid- released
Remaining in water
Unextractables
Acid-released
Remaining in solids
^otal
PPB and %
3
PPB
41.82
2.01
3.68
9.79
24.00
2.34
2.25
0.57
1.68
3.27
0.50
2.77
47.34
distribution
6
PPB
48.99
2.78
3.14
11.05
30.89
1.13
2.70
0.69
2.01
3.92
0.63
3.29
55.61
in eggs laid on day
7
PPB
63.34
2.77
2.89
14.04
42.04
2.60
3.35
0.85
2.50
4.86
.79
4.07
71.55
%
88.5
3.9
4.0
19.6
58.7
2.3
4.7
1.2
3.5
6.8
1.1
5.7
100.0
57
-------
tected. [11+C]-Residues in the water phase and solid fraction contained
a combined total of 5 to 10% of the total radiocarbon in the eggs.
Discussion
The carbamate insecticides are generally characterized as being effi-
ciently metabolized and rapidly excreted by animals (Kuhr and Dorough,
1976). This very desirable feature has been of paramount importance in
keeping alive the search for carbamates of commercial value during the
past ten years. While progress was slow in the 1960's, several carba-
mates have more recently achieved commercial success in the United
States and in other countries, and this success has stimulated greater
research efforts on this group of insecticides.
Croneton, a Bayer product designated by the company initially as BAY HOX
1901, is one of the newer carbamate insecticides and one whose metabo-
lism in animals has been studied extensively in our laboratory. Results
obtained in the present study with the cow, pig, and hen are similar to
those found with the rat (Nye et al. 1976). However, species differ-
ences do exist which are both of interest and of possible practical sig-
nificance. Differences, as well as similarities, can best be demonstrated
by considering the radiocarbon in the excreta of animals treated with a
single oral dose of [11+C]-ring Croneton (Table 16) .
It is evident that the efficiency of excretion of [14C] Croneton equiv-
alents over a 24-hr period following treatment varied little among the
species tested. In every case, 90% or more of the dose had been excret-
ed. A major difference in the nature of the excreted radiocarbon was
that the rat urine contained rather large quantities, 24% of the dose,
of free carbamates, mostly Croneton sulfoxide. None of the large ani-
mals eliminated any free metabolite with the carbamate ester linkage in-
tact. The pig and hen, however, did excrete much of the dose as free
phenolic analogs of Croneton. They were minor constituents in cow and
rat urine. Excretion of the free metabolites by the pig and hen, and
58
-------
Table 16. COMPARATIVE NATURE OF RESIDUES IN THE URINE OF ANIMALS
TREATED WITH A SINGLE ORAL DOSE OF [14C]-RING LABELED CRONETON.
Metabolites
Free
Croneton sulfoxide
Croneton sulfone
Croneton phenol
Ehenol sulfoxide
Phenol sulfone
Water solubles
Total
5
Rat
22
2
0
3
3
60
90
& of dose as
Cow
0
0
0
9
2
87
98
indicated metabolite
Pig
0
0
26
8
6
50
90
a
Hen
0
0
3
23
34
31
91
Total excreta collected.
59
-------
the corresponding low levels of water soluble products demonstrated that
conjugation is not always necessary for efficient elimination of pheno-
lic metabolites of the carbamates.
Because of the very efficient excretion of Croneton by the animals, resi-
dues in the tissues were either absent or too low in quantity for char-
acterization. Examination of the milk and eggs (from hens treated daily
for 7 days) showed that these products contained [11+C] residues quite
different in nature (Table 17). While a majority of the residues in
each substrate was of an organosoluble nature, these free metabolites in
milk were comprised largely of Croneton sulfoxide and Croneton sulfone,
but were almost entirely Croneton phenol in eggs. Levels of bound, or
unextractable, residues were exceedingly small in both milk and eggs.
One could continue to dissect the data generated in the animal studies
with Croneton, and continue to reveal interesting similarities and dif-
ferences among species. However, the most germane points have been em-
phasized and further comparisons can best be made individually by those
with specific points of interest. Generally, it may be concluded that
the metabolism of Croneton in animals is quite typical of that expected
of an N-methylcarbamate containing an ethylthiomethyl substituent on a
phenyl ring. While different animal species exhibited metabolic differ-
ences which could be toxicologically significant, the rapid rate of ex-
cretion of free as well as conjugated Croneton metabolites lessened
their potential significance considerably. Thus, the production of free
metabolites of sufficient polarity to effect urinary elimination without
further metabolism (for example, Croneton sulfoxide in rats) is one
characteristic which distinguishes Croneton from most other carbamate
insecticides.
60
-------
Table 17. [ll*C] CRONETON EQUIVALENTS IN MILK AND EGGS
Metabolites
Free
Croneton sulf oxide
Croneton sulfone
Phenols
Unknowns
Water solubles
Unextractables
% of f1^
Milk3
67
39
19
9
0
30
C] in sample,
Eggs
88
4
4
79
1
5
7
Milk sample collected 6 hr after single oral dose, 0.5 mg/kg, of
[14C] ring Croneton.
Eggs samples on 7th day of treatment of hens; 0.5 mg/kg twice daily.
61
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FATE OF INSECTICIDES ADMINISTERED ENDOTRACHEALLY TO RATS
The evaluation of the fate of Insecticides in animals has been classi-
cally determined after oral, dermal, and/or intraperitoneal administra-
tion of the compounds. Little information is available on the metabolic
fate of these chemicals when inhaled, although inhalation toxicity stud-
ies are commonly conducted on products being developed for commercial
use. The chemical and biological fate of inhaled insecticides could
greatly influence their toxicity to animals, and a knowledge of their
fate would be useful in estimating the potential hazards of these toxi-
cants following exposure by inhalation.
In the present study, insecticides were quantitatively administered as
aerosols to the lungs of rats via the trachea. The relative rates of
absorption by the blood and the fate of the insecticides in the animals
were determined.
Materials and Methods
Insecticides - Four 1LtC-labeled insecticides were selected for this
study: carbaryl [l-naphthyl-ll+C N-methylcarbamate (6,600 dpm/yg) and
the carbonyl-11+C form (4,400 dpm/yg)], leptophos [0-methyl 0-4-bromo-
2,5-dichlorophenyl-1'*C phenylphosphonothioate (7,800 dpm/yg) and 0-
methyl 0-4-bromo-2,5-dichlorophenyl phenyl-11+C-phosphonothioate (8,000
dpm/yg)], parathion [0,0-diethyl 0-p-nitrophenyl-2,5-lltC phosphorothio-
ate (13,600 dpm/yg) and the diethyl-^C form (13,000 dpm/yg)] and
chlordane-^C (11,500 dpm/yg).
Treatment - Female Sprague-Dawley rats weighing approximately 200 g each
were surgically prepared for treatment using techniques previously des-
cribed (Atallah and Dorough 1975, Burton and Shanker 1974). Each animal
was anesthetized with 13 mg of sodium pentobarbital, IP, which immobi-
lized them for the duration of the experiment. A cannula was inserted
between the fourth and fifth tracheal ring below the thyroid cartilage
62
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through which the insecticide solution was administered.
The radiolabeled insecticide in 20 yl of ethanol was delivered to the
lungs as an aerosol by depressing the plungers of the 50-yl and 5-ml
syringe simultaneously. Thus, the insecticidal aerosol was delivered to
the lungs in a total air volume of 5 ml, held for 15 sec to achieve maxi-
mum retention, and then the cannula leading from the trachea connected
to a carbon dioxide trap (2:1 solution of 2-methoxyethanol and 2-amino-
ethanol). Blood samples were taken from the tail periodically for one
hour and the animals then sacrificed and tissue samples collected for
analysis.
Blood and Tissue Analysis - The blood, 0.2-0.4 ml, and tissue samples,
400-800 mg, were placed in quartz vessels and combusted in a Beckman
Biological Materials Oxidizer. The radiocarbon generated as 14CO2 dur-
ing combustion was trapped and an aliquot of the solution radioassayed
by liquid scintillation counting (Packard Model 3380/544).
Excretion Studies - Carbaryl-ring-^C, leptophos-phenoxy-ll*C, parathion-
phenyl-11+C, and chlordane- * ^C were administered to female rats in the
manner described above. In this case, however, ether was used as the
anesthetic and the incision closed immediately after administration of
the insecticides. The animals were placed in plexaglass metabolism
cages and the excreta monitored periodically for radiocarbon until ex-
cretion of radioactivity had ceased.
Results and Discussion
Administration of the dose to the rats via the trachea resulted in quan-
titative retention of the inhaled insecticides. None of the intact in-
secticides was detected in the exhaled air. In fact, the only radiocar-
oon exhaled at all was C-carbon dioxide, 2.5% of the applied dose,
from animals treated with 14C-carbonyl-labeled carbaryl.
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Carbaryl-ltlC and chlordane-11+C residues appeared rapidly in the blood
with maximum concentrations, 10 and 4% of dose, occurring after only 2
to 5 minutes. Then, the residues immediately began to dissipate from
the blood. Leptophos and parathion treatments resulted in a somewhat
slower accumulation of ll*C-residues in the blood but the maximum con-
centrations were similar to that observed with carbaryl. The organo-
phosphorus residues did not dissipate after reaching peak concentrations
as did those of carbaryl and chlordane. Rapid ester hydrolysis of car-
baryl, leptophos and parathion was evidenced by the different levels of
radiocarbon in the blood following treatment with the same chemical, but
with the radioactive carbon on the acid or alcohol moieties. In each
case, the levels of residues in the blood were almost doubled when the
radiolabel was on the alcohol portion of the molecule.
Levels of C-residues in selected tissues of the rats one hour after
treatment are given in Table 18. Concentrations in the lungs were
highest with leptophos and chlordane, 30 and 24% of the inhaled dose,
while carbaryl and parathion residues were only one-third these levels.
Results obtained with those compounds radiolabeled on the acid and alco-
hol moieties were very similar. Indicating that the ester linkages were
intact.
With carbaryl, parathion, and leptophos, residues in the kidney and
bladder at the end of 60 minutes were higher for the C-alcohol-labeled
insecticide than for the corresponding 14C-acid-labeled compound. The
liver, on the other hand, contained higher residues with the C-acid-
labeled materials, demonstrating that the acid moieties released upon
hydrolysis were not removed from the liver as rapidly as the alcohol
moieties. That carbaryl showed higher ll|C-equivalents in the liver sug-
gests that there was a delay between the formation of methyl carbamic
acid and its further deqradation to carbon dioxide. Chlordane- C resi-
dues in the liver were equivalent to 20% of the dose but only trace
levels were detected in the kidney or bladder (Table 18).
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Table 18. RESIDUES IN TISSUES OF RATS I HOUR AFTER INHALATION OF
14C-INSECTICIDES.
% of administered radiocarbon/tissue
Insecticide Liver Lung Kidney Bladder
Carbaryl-ring- * ^C
Carbaryl-carbonyl- * ^C
Leptophos-phenoxy- * ^C
Leptophos-phenyl- 11*C
Parathion-ring- 1 ^C
Parathion-ethyl- 1 ^C
4.8
9.2
8.9
15.3
4.0
8.5
10.5
9.2
31.8
29.4
8.1
7.3
3.7
2.9
3.9
3.3
10.7
2.5
5.9
2.7
4.8
2.3
18.0
10.8
Chlordane 19.6 23.9 0.3 0.1
Includes urine contained therein.
65
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While there was rapid absorption of the inhaled compounds into the body
following exhalation, the excretion rates also were quite rapid. Car-
baryl, leptophos, and parathion equivalents were eliminated in the urine
in amounts greater than 90% of the inhaled doses by 3 days. The feces
contained an additional 2 5o 5% of the doses. For chlordane, excretion
was primarily via the feces and was slower than the other compounds.
About 52% of the dose was eliminated in the feces after 6 days, while the
urine contained an additional 12%. The rates of excretion following in-
halation are in good agreement with those reported for orally adminis-
tered carbaryl (Knaak et al. 1965), chlordane (Barnett and Dorough 1974),
parathion (Nakatsugawa et al. 1969), and leptophos (Holmstead et al.
1973). It would appear, therefore, that the ultimate fate of these in-
secticides when inhaled is not appreciably different than when the com-
pounds enter the body by ingestion.
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INSECTICIDE RESIDUES IN CIGARETTE SMOKE: TRANSFER AND FATE IN RATS
A degree of human exposure to insecticides and other types of pestici -
dal chemical residues is unavoidable because of their ubiquitous nature
in the environment and particularly on food commodities. This source
of contamination has long been recognized and stringent use limitations
are established for each pesticide in an effort to assure that levels
of exposure are kept well below those believed to be hazardous. Tobac-
co smoke is another possible source of human exposure to insecticides
but their actual significance to the health of the smoker is largely
unknown (Wynder and Hoffman, 1967). However, it is known that various
insecticide residues occur in commercial tobacco products (Dorough
and Gibson, 1972) and that insecticides and their pyrolysis products
may be transferred to the mainstream smoke of cigarettes (Tso, 1972).
Even so, tobacco smoke has not been generally considered as an
important source of human exposure to insecticides and, not being a
food crop, tobacco is not included among those crops requiring official
pesticide tolerances. Nevertheless, regulatory officials do carefully
consider the levels and nature of residues in tobacco and in smoke
condensates as part of the evaluation of pesticides whose proposed use
includes the control of tobacco pests. If hazardous conditions appear
likely, the use may be denied or proper limitations imposed to circum-
vent the potential problems involved. This type of consideration is
relatively new and there is a critical need for information which will
assist in determining the significance of pesticide residues in tobacco
and tobacco smoke. A most obvious void in this regard is information
relating to the fate of insecticide residues, their metabolites and/or
pyrolysis products after being inhaled. Studies of this nature should
be conducted with animals and, as is the case with pesticide residues
on food crops, the data extrapolated to humans.
In the current study, a simple but quantitative smoking device was
constructed whereby smoke containing radioactive insecticide residues
67
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was transferred directly from the cigarette to the lungs of rats via
the trachea. Rats were selected because they are commonly used in
metabolic fate studies of pesticides, and the existing information
would be useful in interpreting the significance of data obtained in
the smoke experiments. While it is desirable to administer the smoke
to the animal in a more natural manner, an effective quantitative
system that would allow the "smoking" of animals similar to the process
in humans has yet to be developed. Enclosing animals in cylinders or
chambers (Moore and Bock, 1956; Holland et al., 1958) may be sufficient
for studying the pathological effects of chronic smoke exposure, but
the quantitative aspects necessary for pesticide fate studies and for
determining exposure potential of pesticides to the smoker are not
provided by such methods. Forced smoking via the trachea (Wynder and
Hoffman, 1967; Armitage et al., 1969) allows an excellent means of
administering quantitative doses of smoke containing insecticide
residues to the lungs of animals, and the subsequent determination of
the fate of the inhaled residues.
Materials and Methods
Treatment of Cigarettes - Radioactive insecticides were used in all
experiments. The identity of the compounds and the position of the
1I+C-label on the molecule are shown in Table 19. These insecticides
were selected to represent different chemical types and not as examples
of compounds used for the control of tobacco pests. In fact, of those
insecticides listed in Table 19, only carbaryl and carbofuran are
currently approved for this use. DDT was registered for use on
tobacco until 1970 and residues do exist in commercial cigarettes
(Dorough and Gibson, 1972).
Reference research cigarettes 1R1 were provided by the Tobacco and
Health Research Institute (University of Kentucky, Lexington, Kentucky)
The desired quantity of insecticide was dissolved in 0.1 ml of acetone
and applied to a 17 mm segment, extending from a point 25 mm from the
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Table 19. RADIOACTIVE COMPOUNDS USED IN THE STUDY OF THE TRANSFER OF
INSECTICIDE RESIDUES TO RATS IN CIGARETTE SMOKE.
Chemical identity and position
of carbon-14
Designation
Specific
Activity
mC/mM
l-Naphthol-l-^C
1-Naphthyl-l-1 4C methylcarbamate
1-Naphthyl-methylcarbamate
(carbonyl-ll*C)
1-Naphthyl methyl-1^C carbamate
2,3,-dihydro-2,2-dimethyl-7-
1 4
benzo- C furanyl-N-methyl-
carbamate
2,3,-dihydro-2,2-dimethyl-7-
benzofuranyl-N-methylcarbamate
(carbonyl-^C)
0-(2,5-dichloro-4-bromophenyl-
^C) 0-methyl phenylthiophos-
phonate
0-(2,5-dichloro-4-bromophenyl)
i 4
0-methyl phenyl- C-thiophos-
phonate
0,0-Diethyl-O-p-nitrophenyl-
llfC phosphorothioate
1,3,4-methenododecachloroocta-
hydro-2H-cyclobuta [c,d]
pentalene (^C UL)
l,l,l-Trichloro-2,2-bis (p-
14
chlorophenyl- C) ethane
l-Naphthol-^C 15.2
Carbaryl-naphthyl-1'*C 0.6
Carbaryl-carbonyl-llfC 26.4
Carbaryl-methyl-^C 1.1
Carbofuran-ring-^C 2.85
Carbofuran-carbonyl-^C 3.83
i k
Leptophos-phenoxy- C 7.34
Leptophos-phenyl- C
Parathion
Mirex-1IfC
DDT-^C
6.22
1.8
6.34
2.7
69
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butt end, of the cigarette using a micro syringe. The needle of the
syringe was inserted into the cigarette 10 mm from the treatment zone
towards the end to be lighted, passed through the center of the cigar-
ette until the point was at the center of the 17-ram band, and the
solution slowly injected into the cigarette. Each insecticide was
applied to the 200 mg of tobacco in the treated zone at a concentration
of 100 ppm. To evaporate the solvent, the cigarettes were left at
room temperature for 2 hr and then refrigerated overnight before use.
No degradation of the insecticides occurred during storage of the
cigarettes. Impregnation of hundreds of cigarettes in this manner,
and subsequent radioassay of subsections of the impregnated zone,
showed that the radioactivity was distributed in a bell shape fashion
with just trace amounts on either side of the marked zone.
Smoking Device - A smoking device was designed to collect, or transfer
to the lungs of rats, known volumes of smoke at designated intervals.
The mainstream smoke outlet was attached to a valve, (3-way Teflon
valve, Hamilton ML-3000) which allowed a puff to be drawn into a
syringe and then transferred to the lungs through a polyethylene tube,
(1.6 mm ID, 2.1 mm OD). Manual operation of the 3-way valve and
delivery of a 5-ml puff to the animals required 2 to 3 sec. A 50-ml
syringe was attached td the tracheal tube by a 27G needle for the
collection of the exhaled smoke. For mainstream smoke analysis, the
smoke was collected directly in an air tight syringe at the end of the
tracheal tube. For each test, a cigarette was placed in the holder,
lit and the cover of the smoking chamber was put into place. Air was
passed through the chamber at 500ml/min to remove the sidestream
smoke and to keep the cigarette burning. Sidestream smoke was passed
through traps of glass wool, cold ethanol, and 2 carbon dioxide trap
solutions consisting of a 2:1 mixture of 2-methoxyethanol and 2-amino-
ethanol. After the appropriate smoking sequence, the air flow through
the smoking chamber was stopped to extinguish the cigarette and then
operated again momentarily to clear the chamber of any smoke before it
was opened to collect the ash and butt.
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Inhalation Studies - Female white rats (Sprague-Dawley) weighing
180-200 g and 9 to 10 weeks old were used in the smoking studies.
For each inhalation experiment, an impregnated cigarette was placed
in the holder, lit, and the smoking chamber cover put into place.
Immediately, a rat was anesthetized with ether, and a midline longi-
tudinal incision made to expose the trachea. A thread was introduced
around the trachea and a small hole was clipped between the 4th and
5th tracheal rings caudal to the thyroid cartilage. Just as the
cigarette burned to the treatment zone, the tracheal tube of the
smoking system was introduced into the trachea and the thread was
tied over the trachea and the tube. A 5-ml puff was drawn into
syringe C and directed into the lung of the rat. The smoke was held
in the lungs for 12 sec, then 5 ml were withdrawl into syringe E. The
complete process required 15 sec. After repeating the process for a
total of 8 puffs, during which the entire treatment zone was burned,
the trachea was immediately clamped and the air flow through the
system was stopped to extinguish the cigarette.
A blood sample was removed from the right ventricle of the heart and
the lung and heart were removed and placed in a freezer. Blood was
taken about 2.5 min after the last exhalation and the heart and lungs
were excised about 1 min later.
Analysis - The blood was immediately radioassayed by combustion in a
Beckman Biological Materials Oxidizer and the radioactive carbon di-
oxide trapped in a 2 to 1 mixture of 2-methoxyethanol and 2-amino-
ethanol. Total blood volume of the animals was calculated to be 12.5
ml. Heart and lung tissues were similarly analyzed after 1 hr.
Radioactive residues in the exhaled air were removed by washing the
syringe with ethanol and carbon dioxide trap solution. First, the
ethanol was pulled into the syringe, the syringe shaken thoroughly,
and the ethanol forced from the syringe until only the needle was
filled with solvent. An air space was left between the syringe pumper
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and solvent to contain any gases and/or other volatile components not
soluble in ethanol. The ethanol wash was repeated, followed by 2
washings of the syringe with the carbon dioxide trap solution. This
same procedure was used in collecting the mainstream smoke radiocarbon
for direct analysis. The ethanol wash was concentrated-and applied to
thin layer plates (Merck, silica gel F-254). Solvent system for the
different compounds were: carbaryl-7:3 benzene:ether; carbofuran-
5:1 ether:hexane; letpophos-6:4 chloroform:benzene; mirex-n-heptane;
DDT-n-hexane. Individual components resolved by tic were quantitated
but no attempt was made to identify the pyrolysis products. The
radiocarbon suspected of being the original insecticide added to the
cigarettes was extracted from the gel and rechromatographed as a
mixture with the authentic compound. Cochromatography was achieved
with each of the insecticides evaluated.
To determine if the radiocarbon in the carbon dioxide trap solution
was actually llfC-carbon dioxide, a barium hydroxide trap was used in
place of the 2-aminoethanol and 2-methoxyethanol. Approximately the
same quantity of material was trapped in the barium hydroxide solution
and, upon acidification, about 90% of the radiocarbon dissipated.
These data indicated that the radiocarbon was predominately llfC-
carbon dioxide and not other volatile components of the insecticides
formed during the smoking process.
Radiocarbon in the ash, butt and smoking chamber was determined by
washing or extracting the residues with ethanol. Aliquots of the
liquid sidestream smoke traps were radioassayed directly while the
glass wool was washed with ethanol and then the wash assayed. Ethanol
solubles from the sidestream smoke, which included the smoking
chamber washes, were analyzed by tic as described for the mainstream
smoke.
All radioassays were performed using liquid scintillation counting
(Packard Tri-Carb Model 3380) and a commercial blend of scintillation
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counting fluid (3a70B, Research Products International Corp., Elk
Grove Village, 111.). All data reported herein were derived from a
minimum of 5 replicates for each experimental parameter investigated.
Inhalation Parameters - During the development of the smoking process
described above, various studies were performed to determine the
effects of puff volume and lung retention time on the amount of C-
residues retained in the animal body. The objective was to obtain the
maximum level of radiocarbon in the body following the smoking process
so that its fate in the rats could be determined.
Cigarettes were impregnated with carbaryl-naphthyl-1'*C as previously
described and then the entire treated zone was smoked using 2.5-ml
and 5-ml puff volumes. For each puff volume, the smoke was held in
the lungs for 6, 9 and 12 sec. The number of puffs required to smoke
the treated portion of the cigarettes decreased as the lung retention
time was increased, with the numbers being 22, 18 and 16, respectively,
when the 2.5-ml puffs were used. For the 5-ml puffs, the number
required was reduced by one-half. Radiocarbon remaining in the body
after the last smoke exhalation was determined by radioassay of the
lungs, blood and heart.
In another experiment, the smoking device was modified by replacing
the 3-way valve with an in-line flutter valve which opened as the
animal inhaled, thus drawing smoke into the lungs, and closed when
the animal exhaled, forcing the expired smoke into the collection
syringe. This was a totally automatic process since the plunger of the
syringe lubricated with ethanol, was pushed outward by exhalation but
maintained its position during inhalation. Using this system, anes-
thetized animals smoked the treated zone of the cigarettes in approx-
imately 180 puffs. The average single puff volume was 0.8 ml with the
inhalations per min being between 85 and 95. This situation represen-
ted the minimum puff volume and lung retention time attainable with
the smoking device used in these studies. Rats were allowed to smoke
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cigarettes impregnated with carbaryl-naphthyl-ll+C by the self-
inhalation method and the level of 1 "*C-residues remaining in the
body were compared with those obtained with the standard eight 5-ml
puff smoking process, and with a single inhalation of 5 ml of smoke.
The levels of carbaryl-naphthyl-1^C equivalents in the blood of animals
during the self-inhalation and eight 5-ml-puff smoking process, and
for 4 min after the last puff, were determined. Blood was drawn from
the subclavian vein at various intervals after the first inhalation
and the total radiocarbon in the samples determined by combustion
analyses. In this particular study, 10 animals were used for each
method of smoke exposure since considerable variation in the blood
levels was encountered.
Initially, flutter valves were used in the smoking apparatus to admin-
ister smoke to the lungs of rats rather than using the manually
operated 3-way valve. A flutter valve was inserted which allowed
the smoke to be drawn into the syringe and then transferred to the
lungs. Although this arrangement was superior to the 3-way valve in
regard to ease of operation and rapidity of smoke delivery, it was
discarded because 50 to 75% of the 1'*C-residues in the mainstream
smoke of 5-ml puffs was deposited directly on the valves. With the
3-way valve, which operates in a completely open or completely closed
manner, the amount of mainstream smoke radiocarbon on the valve was
generally less than 1%.
35-ml Puff Volumes - The use of rats in smoking studies for the purpose
of estimating the fate of inhaled insecticide residues in humans would
be valid only if the nature of the inhaled residues were the same,
and if the amounts were comparable based on lung capacity. These
factors were evaluated by determining the quantity arid nature of 1**(>
residues in the mainstream smoke of cigarettes impregnated with each
of the insecticides, but not all radioactive preparations, listed in
Table 19, and smoked using 35-ml puffs of 2 sec duration at 1 min
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intervals. This is the procedure normally used with commercial
smoking machines to represent human smoking (Wynder and Hoffmann,
1967). The impregnated zones of the cigarettes were burned completely
by 3 puffs. All other aspects of these studies (treatment of cigar-
ettes, collection of smoke, analyses, etc.) were the same as reported
for the 5-ml puffs.
Filters - The effect of filters on the quantity of carbaryl-naphthyl-
C equivalents transferred to the mainstream smoke of cigarettes was
evaluated using the eight 5-ml puff sequence. Pour commercial brands
of filter cigarettes were impregnated with the insecticide and the
radiocarbon in the ash, butt, sidestream smoke and mainstream smoke
was quantitated. Ethanol-soluble 1'*C-residues in the mainstream smoke
were analyzed by tic. The same procedure was followed using cigar-
ettes from the same package which had the filters removed. Reference
cigarettes, 1R1, were included in the study with the butt length
adjusted to be equivalent to the tobacco plus filter of the filter
cigarettes. When the filters were removed, an equivalent length of the
1R1 cigarettes was also removed. Therefore, it was possible to compare
the trapping effect of the filters with a comparable length of plain
tobacco.
Commercial cigarettes used in these experiments, designated as brands
A-D, each had a filter length of 20 mm except brand B which was 25 mm.
The nature of the filters was as follows: Brand A, 2 cellulose filters
separated by charcoal granules; Brand B, an inner cellulose filter
(towards the burning end) containing charcoal granules and an outer
cellulose filter; Brand C, an inner charcoal-impregnated cellulose
filter and an outer cellulose filter; Brand D, a single cellulose
filter. The treatment zone of the filter cigarettes, 17 mm, was 25 mm
from the point where the filter was joined to the cigarettes, making
the tobacco-containing butt the same length as for non-filter cigar-
ettes.
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Fate of Inhaled Residues - In one series of experiments, the tracheal
tube was disconnected from the smoking apparatus immediately after
administering the smoke to the rats and attached to a Y connector, one
end of which led to a carbon dioxide trap while the other end was open
to the atmosphere to provide air to the animal. The carbon dioxide
trap solution, 2:1 methylcellosolve-ethanolamine, was radioassayed at
5 min intervals for the first 15 min, then at 15 min intervals until
the experiment was terminated. Meanwhile, 0.25 ml blood samples were
collected from the subclavian vein at approximately 3 min intervals for
the first 10 min, then at 10 min intervals thereafter. At the end of
the experiment, 90 min, the urinary bladder was ligated and removed
intact. The other organs and tissues were immediately excised and
frozen.
In another series of experiments, the tracheal tube was quickly with-
drawn after the last of eight 5-ml smoke inhalations and exhalations.
The incision in the skin was then closed with a wound clip and the rat
immediately placed in a metabolism chamber (Nuclear Associates, Inc.,
N. Y.). Inhalation-quality compressed air was passed through the
chamber at the rate of 200 ml/min and then through a carbon dioxide
trap solution. The feces and urine were collected separately. At the
end of 6 hr, the animal was transferred to a larger metabolism cage for
4 days and then sacrificed. Various tissue samples were taken and
immediately frozen.
The urine samples were monitored for total radiocarbon by direct
scintillation counting of 0.5 ml in 10 ml of a commercial blend of
scintillation counting fluid (3a70B, Research Products International
Corp., Elk Grove Village, 111.). Urine samples having 500 dpm/ml or
more were pooled and lyophilized to approximately 1 ml, then applied to
thin layer plates (Merck, silica gel F-254). Radioactive residues in
the feces were quantitated by combustion followed by scintillation coun-
ting of the carbon dioxide trap solution.
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The deposition of the insecticide residues in the rat body were investi-
gated after 90 min and 4 days. Each sample was weighed and combusted
in a Beckman Biological Materials Oxidizer. For calculation purposes,
a 200 gm female rat was also dissected and different tissues and organs
separated and weighed. The percent of total body weight for the tissues
was as follows: lung, 1.2; heart, 0.5; skin, 14.2; hair, 2.1; muscles,
39.8; fat, 9.1; digestive system, 7.8 (3.4 without food); liver, 3.7;
urinary system, 1.0; reproductive system, 0.5; spleen, 0.2; brain, 0.9;
skeleton, 11.9 (bones, 7.9, and cartilage, 4.0); tongue, 0.4; total
weight 99.6%. The total radiocarbon in the body was based on these
actual weights and on previous reports in the literature.
Pyrolyses of Carbofuran - Controlled pyrolyses were conducted in an
apparatus recently designed by Smith and Patterson (1973) to more
closely correlate with the burning process in cigarettes. A 9 mm x
170 cm horizontal quartz tube in which the pesticide had been evenly
distributed over an 80-cm segment, was connected to 2 cold traps, dry
ice-chloroform, and swept with nitrogen at 40 ml/min. The oven consist-
ed of a nickel rod, 44 mm in diameter and 50 mm in length, supported in
an insulated aluminum cabinet (Chemical Data Systems, Oxford, Penn.).
The nickel rod was drilled to provide for three 150 watt 1/4" x 2"
Chromalox CIR cartridge heaters (Emerson Electric Company, Pittsburg,
Penn) and an integrated platinum temperature sensor-thermocouple
cartridge. A 13 mm hole was drilled through the center of the rod to
accomodate the combustion tube. Temperatures of 650° and 700°C were
maintained by a CDS-200 power-proportioning temperature controller
(Chemical Data Systems). The oven, attached to a moveable plate, was
driven at 2.7 cm/sec along the quartz tube containing the insecticide.
A vertically positioned Vycor combustion tube (2.5 cm x 94 cm) contain-
ing 20 cm of Berl Saddles positioned in the heated zone was used to
represent the more conventional pyrolysis conditions. The tube was
connected to cold traps and the system swept with nitrogen. The com-
bustion tube was heated to 783°C by a Lindberg furnace.
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Noncondensable gases were collected in a 10 cm IR gas cell (Wilks
Scientific, South Norwalk, Conn.) and the spectra recorded on a Beckman
IR 8. Precoated silica gel plates were used for tic analyses of the
components of the cold traps. After development in 7:3 benzene-methanol,
the spots were visualized under ultraviolet light or by spraying with
0.5% methanolic p-nitrobenzenediazonium fluoroborate. GLC analyses
were performed on a Varian 1400 instrument equipped with an FID. Neutral
pyrolysis products were examined on 4 mm x 180 cm glass column packed
with 5% OV-101 while the acidic ones were analyzed on a column packed
with 10% EGSSX on Gas Chrom Q. Injector and detector were maintained
at 210 and 22°C. Column temperatures were programmed from 100° to
230°C at l°/min with the OV-101 column and 128°C to 200°C at 4°/min for
the EGSSX support. The nitrogen carrier gas flow was 35 ml/min. GC/MS
data were obtained on the Finnigan 1015C at 70 eV, and the NMR data on a
Varian HA-60 spectrometer interfaced with a Varian 1024 computer.
Results and Discussion
Inhalation Parameters - Retention of carbaryl-naphthyl-14C equivalents
in the body of rats immediately after exposing the animal to cigarette
smoke using different puff volumes, numbers, and lung-contact times is
shown in Table 20. Of primary importance in this regard was the period
of time the smoke was held in the lungs. Maximum retention of the
radiocarbon, 76% of that inhaled, was obtained with a single 5-ml puff
held in the lungs for 12 sex. Of course, the actual amount of carbaryl-
ll*C equivalents in the body after the single puff was very small, only
0.9% of that radioactivity added to the cigarette. Repeating the 5-ml
puff 8 times in the same manner, during which the entire impregnated
zone of the cigarette was smoked, increased the total carbaryl- C
equivalents inhaled by a factor of 9 and the amount retained by a factor
of 8. This smoking sequence resulted in 7.5% of the radioactivity added
to the cigarette being retained in the lungs, blood and heart. Unlike
the single 5-ml puff smoking process where 78% of the retained residues
were in the lungs, the eight 5-ml puffs resulted in almost equal amounts
78
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of residues in the lungs and blood. Because the quantity of radio-
carbon remaining in the body using eight 5-ml puffs was greater than
with any other smoking procedure/ the process was selected as the
standard method of exposing rats to insecticide residues in cigarette
smoke.
In addition to establishing conditions for maximum body retention of
14C-insecticide residues inhaled in cigarette smoke, the data in Table
20 indicate some of the factors effecting the degree of transfer and
retention of the residues. For example, while the percentage transfer
of carbaryl- C equivalents to the mainstream smoke was obviously great-
er as the total volume of smoke increased, there was very little differ-
ence when considered on a per ml basis. This was true not only for
carbaryl, but for each of the insecticides evaluated, and for each puff
volume used (Table 21). Under conditions used in these experiments,
each ml of mainstream sm6ke contained an equivalent of between 0.2 to
0.3% of the radioactive insecticide in the tobacco burned during the
smoking process. Increasing the number of puffs required to burn the
same amount of tobacco did tend to reduce the percentage transfer or
residues to the mainstream smoke. However, this reduction was grossly
evident only when the number of puffs was 180, that required to burn
the impregnated zone using the normal respiration rate of anesthetized
rats (Table 20).
Retention of the inhaled 11+C-residues was primarily a function of the
period of time the smoke was held in the lungs, and to a lesser extent
to the volume of the puff. Increasing either of these parameters resul-
ted in increased retention of the inhaled radiocarbon. Of the retained
residues, less was located in the blood as the lung contact time of each
puff was increased from 6 to 12 sec; the percentage remaining in the
lung tissue showed a corresponding increase. Therefore, these data show
that, with repeated inhalation, the deposition of insecticide equiva-
lents in the lungs exceeded the rate of their removal by the blood.
80
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Following the smoke-exposure period, carbaryl- C equivalents continued
to increase in the blood. Maximum concentrations were approximately
28% of the total radiocarbon inhaled with both the eight 5-ml puff
smoking process and when the animals were exposed to the smoke at
normal respiration rates. Blood of rats exposed to smoke by normal
respiration rates of anesthetized animals contained maximum levels of
radioactivity 1.5 min after the last smoke exhalation, and then the
levels began to dissipate gradually. Peak concentrations of carbaryl-
^C equivalents in the blood of rats given the eight 5-ml puffs were
reached after 3.5 min, and remained unchanged after 4 min, or 6 min
after initiating the smoking process. In these experiments, the rats
were allowed to continue breathing after smoke exposure, but for all
other data reported herein, the trachea was clamped immediately after
the last smoke exhalation. Blood samples were then collected after
about 2.5 min. Levels in the blood when sampled in this manner were
approximately twice that at the end of the smoking sequence. That the
accumulation of carbaryl-^C equivalents in the blood was not apprecia-
bly altered by clamping the trachea was demonstrated by the fact that
1'*C-residue levels in the blood were very similar when the trachea was
clamped (Table 20) and when the animals were permitted to continue
breathing. This is in agreement with the observation that the heart
functioned for about 3 min after clamping the trachea.
Smoking Process and Transfer to Mainstream Smoke - The impregnated zone
of the cigarettes weighed 200 +_ 29 mg while the amount of tobacco burn-
ed per 5 ml puff, including the interval between puffs, was 25 + 3.8 mg
and contained 12.5 +_ 6.2% of the compound added to each cigarette.
Variation in the amount of 11+C-insecticide equivalents per puff was
caused largely by the bell-shaped distribution of the insecticide in
the spiked zone. Such variations probably did not effect retention of
the insecticide equivalents in the rat lung since Egle (1970) reported
no difference in acetaldhyde retention in humans with up to 50% varia-
tion in concentration, A similar lack of concentration effect was
noted in our studies using carbaryl at 25 and 100 ppm.
82
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One of the major objectives of this study was to perfect a simple,
reproducible system capable of comparing the potential of different
insecticides to be transferred in a toxic form to humans in tobacco
smoke. Toxic residues on the leaf at market or in smoke condensates
are significant only if they survive the smoking process and are
retained in the body at sufficient levels and periods to induce patho-
logical conditions. Based upon the excellent accountability of the
radiocarbon added to the cigarettes (Table 21), it appears that the
procedure described herein could serve as a general method for estimat-
ing, at least in part, the significance of pesticides and their meta-
bolites in tobacco products. With the exception of leptophos-phenyl-
1I+C where the recovery was only 67%, total recovery of the compounds
ranged from 85% for carbaryl-carbonyl-^C to 102% for DDT. The loss of
radioactivity with certain of the insecticides indicated that volatile
pyrolysis products were formed which were not collected in the traps
of the smoking apparatus.
There was no consistent difference in the recovery of the insecticides
using puff volumes of 5 and 35 ml (Table 21). Neither were there any
great differences in the percentage transfer of pesticide-llfC equivalents
to the mainstream smoke when considered on a per ml basis. With the
5-ml puffs, 40 ml total, from 7 to 14% transfer occurred, whereas with
the 35 ml puffs, 105 ml total, the transfer to the mainstream smoke was
21 to 32%, resulting in an overall transfer of 0.18 to 0.34% per ml of
smoke. Therefore, the quantities of residues inhaled by rats given 5-
ml puffs would be comparable in regard to lung capacity to that inhaled
in 35 ml puffs by man. With the 35-ml puffs, more of the 11(C-residues
were deposited in the butt and less given off in the sidestream smoke
than with the 5-ml puffs. Only minute residues were in the ash of the
cigarettes, regardless of the puff volume or compound involved.
The use of carbaryl, carbofuran and leptophos radiolabeled at different
positions on the molecule showed that the distribution of radiocarbon
following smoking of the cigarettes varied little with the position
83
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of the radioactive carbon. Carbaryl-naphthyl-11+C and carbaryl-methyl-
14C behaved similarly to l-naphthol-14C, but with carbaryl-carbonyl-
1'*C, the levels of residues in the butt were much less. This apparent-
ly resulted from the production of 1HC-carbon dioxide from the carbonyl
carbon which did not condense in the butt as did the other 1I+C-labeled
pyrolysis products. With the exception that radioactive residues in
the sidestream smoke were higher with carbofuran-carbonyl-1 "*C/ the
distribution of residues was about the same as for carbofuran-ring-
1I+C. In the case of leptophos, the phenyl-labeled material gave lower
residues in all fractions than did the phenoxy-^C material. Obviously,
some phenyl-14C pyrolysis product(s) was generated which escaped the
sidestream and/or mainstream smoke traps.
To compare the distribution and nature of residues in the mainstream
smoke using our smoking apparatus with that of a commercial smoking
machine, leptophos-phenoxy-lt*C was added to cigarettes and smoked with
both systems using 35-ml puffs. With the syringe method, a 17 mm
portion of the cigarette was impregnated with 100 ppm leptophos in the
manner already described. Cigarettes prepared for use on the commercial
smoking machine were impregnated throughout their entire length at the
same concentration. These latter cigarettes were smoked, 35-ml puffs
of 2 sec duration at 1 min intervals, to a butt length of approximately
25 mm and the mainstream smoke, butts and ash were analyzed. In both
cases, the distribution of radiocarbon was calculated on the basis of
the leptophos-lf*C content of the tobacco actually burned during smoking.
Results of these analyses showed that the distribution of radiocarbon
in the 3 fractions, and the nature of the 1'*C-residues in the mainstream
smoke, were virtually the same as obtained using our smoking apparatus
(Table 21).
As mentioned earlier, 35-ml puffs are used to represent that of man,
and have been used in previous studies of the transfer of insecticides
to the mainstream smoke of cigarettes. One such study with DDT
(Hoffmann and Rathkamp, 1968), showed that 12.4% of the parent compound
84
-------
was transferred to the mainstream smoke. Our 35-ml puff experiment
gave almost identical results, showing 12.1% transfer of the applied
material. Hengy and Thirion (1971) showed that the amount of the
chlorinated hydrocarbon, endosulfan, transferred intact to the main-
stream smoke was 15% of that contained in the burned tobacco. This is
in contrast to the 3% transfer reported in another study (Guthrie and
Bowery, 1962). Some of the difference in the 2 values may be explained
by the fact that in the latter study, calculations were based on radio-
activity added to the total cigarette and not restricted to that in
the burned tobacco. Endosulfan was not evaluated in the present study,
but with the chlorinated material mirex, 23% was as unaltered compound
in the mainstream smoke.
In our studies, 10% of the organophosphorus insecticide leptophos was
transferred intact to the mainstream smoke. With the carbamates
carbaryl and carbofuran, the transfer was 11 and 20%, respectively.
These values are far in excess of the 1% transfer reported for carbaryl
and guthion, an organophosphate, in the review article by Guthrie (1968)
However, they are similar to the 9% transfer of intact malathion, a
rapidly biodegradable organophosphate, obtained in the studies by
Hengy and Thirion (1970).
Mainstream Smoke - Smoking the ^C-ring-insecticide-impregnated cigar-
ettes with 35-ml puffs generally did not degrade the compounds as
extensively as did the 5-ml puffs (Table 22). Only with carbaryl and
DDT were the percentages of parent compound less with the higher puff
volume. Mirex was the most stable compound with approximately 70% of
the 1^C-components in the mainstream smoke as the applied compound.
Three pyrolysis products were detected, the major one constituting 52%
of their total. This product chromatographed similar to the monode-
chlorinated derivative of mirex formed by photolysis (Gibson et al.,
1972). As with all other compounds evaluated, the number and relative
magnitudes of the mirex pyrolysis products were similar with 5-ml and
35-ml puff volumes.
85
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86
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Carbofuran was almost as stable as mirex, but only 2 pyrolysis
products of carbofuran were detected by tic analysis. The products
existed in approximately equal concentrations and one chromatographed
the same as the phenolic derivative of carbofuran.
The percentage of the C-residues in the mainstream smoke as the
applied compound, 40 to 45%, was the same with carbaryl-naphthyl-l C
and DDT. Both compounds yielded 7 pyrolysis products, with 66% of the
pyrolysis products of carbaryl consisting of 1-naphthol. DDT has 2
major degradation products occurring in almost equal quantities which
together constituted 64% of the pyrolytic materials resolved by tic.
1-Naphthol was degraded during the smoking process to form all but 1
of the pyrolysis products observed with carbaryl-naphthyl-1£*C. How-
ever, more 1-naphthol survived the smoking process than did intact
carbaryl.
Unaltered leptophos-phenoxy-1 **C accounted for less of the total 1'*C-
residues in the mainstream smoke than with any of the other compounds.
Only 21% was as the parent compound with 5-ml puff volumes and 35%
with 35-ml puffs. Four pyrolysis products detected by tic analysis
made up about 50% of the mainstream smoke C-residues. Of these, 90%
was as a compound with the same chromatographic behavior as the phenolic
analog of leptophos.
Radioactive carbon dioxide accounted for 15 to 20% of the 1^C-residues
in the mainstream smoke when cigarettes were smoked with 5-ml puffs
(Table 22). with the 35-ml puffs, the amount of lkC-carbon dioxide
ranged from 5% to 12% of the total residues in the smoke. Since more
complete degradation, especially to carbon dioxide, of the insecticides
took place using eight 5-ml puffs than with three 35-ml puffs, it is
apparent that exposure of the compounds to the 850-900°C heat at the
burning point of the cigarette was greater with the smaller puff
volume. Actually, this is probably a function of puff numbers, or
frequency, and not to the volume of the puff. The amount of parent
87
-------
compound in the mainstream smoke clearly shows that much of the intact
insecticides were volatilized away from the intense heat, and suggests
that extensive pyrolysis occurred only to that material "trapped", or
not volatilized, from the burning zone- This trapping effect apparent-
ly takes place to a greater degree during the initial phase of each
puff, with increased volatility of the insecticides occurring as the
temperature rises in the area just behind the burning zone of the
cigarette. Therefore, less pyrolysis of the insecticide would be
expected when the same amount of tobacco was burned with 3 puffs than
with 8 puffs.
Sidestream Smoke - Insecticide residues in the sidestream smoke of
cigarettes may serve as a source of contamination to the non-smoker
as well as to the smoker. Analysis of the sidestream smoke of insect-
icide- 14C-impregnated cigarettes smoked with eight 5-ml puffs showed
that the smoke contained considerable quantities of intact pesticide
(Table 23). Intact leptophos constituted 21% of the sidestream smoke
^C-residues, the same as it did in the mainstream smoke. In fact,
the entire composition of leptophos-phenoxy-ll*C equivalents in the
sidestream smoke was almost the same as in the mainstream smoke.
With all other compounds, the percentage of the residues in the side-
stream smoke as the applied material was 2- to 4-fold less than that
in the mainstream smoke; conversion of the insecticides to 1'*C-carbon
dioxide was much greater in the sidestream smoke.
Pyrolysis products of the insecticides, as determined by tic analysis
of the components of the glass wool and ethanol traps, were identical
in number and similar in chromatographic behavior in both sidestream
and mainstream smoke. No attempt was made to establish that the
pyrolysis products were actually the same, but R values of correspond-
ing products were similar enough to suggest this possibility. Ratios
of the pyrolysis products of each insecticide were different in the
sidestream smoke than in the mainstream smoke as evidenced by the auto-
radiographs; the products in the sidestream smoke were not individually
88
-------
Table 23. NATURE OF RADIOCARBON IN SIDESTREAM SMOKE OF CIGARETTES
IMPREGNATED WITH RING-l**C INSECTICIDES AND SMOKED USING EIGHT 5-ML PUFFS
Percent of total ^C-residues in sidestream smoke
Phenoxy- 1
1 ^C-Material 1-Naphthol Carbaryl Carbof uran Leptophos Mirex DDT
Unaltered
compound 17.1 14.9 16.7 20.7 37.1 22.6
Carbon dioxide 62.1 61.4 45.9 28.5 36.4 35.5
Pyrolysis
products 15.1 18.2 10.0 45.4 17.0 28.6
Loss 5.7 5.5 27.4 5.4 9.5 13.3
89
-------
quantitated.
Loss of radioactivity during the analysis of the sidestream smoke was
generally of the same magnitude as observed in the mainstream smoke
analysis. The major exceptions were with carbofuran and DDT where
greater loss occurred during analysis of the sidestream smoke.
Effect of Filters - Different types of commercial filters reduced the
carbaryl-naphthyl-1'*C equivalents in the mainstream smoke by 36 to 49%
as compared to the same cigarettes without filters (Table 24). In-
creasing the butt length of unfiltered 1R1 cigarettes to that equivalent
to the filter lengths reduced the mainstream smoke "*C-residues by 28%.
Therefore, the reduction of 14C-residues as the result of filters was
only 8 to 21% more effective than an equivalent length of tobacco.
Two of the charcoal containing filters, A and C, were more efficient
in trapping the residues than filter D which had no charcoal. However,
the third charcoal-containing filter, B, was slightly less efficient
than filter D. Very little selectivity was demonstrated by any of the
filters. The reduction in the amount of radiocarbon in the mainstream
smoke resulted from partial trapping of the parent compound and of the
pyrolysis products except Il*C-carbon dioxide. Consequently, the 1I+C-
carbon dioxide accounted for a greater percentage of the mainstream
smoke radiocarbon with the filter cigarettes than with the non-filtered
ones.
Exhalation of Residues - During the smoking period, 26.2 to 41.9%
of the total inhaled radiocarbon contained in the mainstream smoke was
exhaled (Table 25). Of the individual ^C-residues, 20.7 to 46.7% of
the unaltered insecticides and 38 to 45% of gaseous pyrolysis products
(of which at least 85% was 1'tCO2) were expired. The extent of exhalation
of nongaseous ^C-pyrolytic products during smoking (about 20%) was
similar for carbaryl, leptophos, parathion and DDT. Non gaseous
residues of carbofuran exhibited nearly twice the clearance rate. Most
of the exhalation of the residues occurred within the first 12 min
90
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after smoking, but traces of radiocarbon were detected in the exhaled
air for up to 5 hr (Table 26). Elimination of ll+C-residues through
exhalation removed 64.7% of the inhaled carbofuran products and about
50% of the leptophos, carbaryl, and DDT residues. This route of
elimination of parathion residues was less, with only 35% being removed.
Residues in the Respiratory and Circulatory Systems - Immediately
following smoking, 22.8 to 39.5% of the inhaled radiocarbon was in the
lungs (Table 26). After 1.5 hr, more than 90% of the residues had
been removed by further exhalation and by absorption into the blood.
Four days later, only 0.6% of the radioactivity in the inhaled main-
stream smoke from DDT fortified cigarettes was detected, while lesser
amounts were observed from the inhalation of carbaryl and parathion
residues. Residues of leptophos and carbofuran were not detected.
Inhaled llfC-residues accumulated rapidly in the blood during the
smoking period and for 3 min thereafter. Peak levels of 39.8%, 36.0%
and 27.3% of total inhaled radiocarbon from leptophos, parathion, and
DDT residues respectively were observed. !4C-Carbofuran and carbaryl
pyrolysis products reached maximum blood concentrations of 18.1 and
32.5% of the inhaled residues during this time. Levels of llfC-carba-
mate residues, carbaryl and carbofuran, corresponding to 28 to 30%
of peak levels were detected in the blood after 90 min. Blood levels
of leptophos, parathion, and DDT *4C-residues exhibited a more rapid
decline with only 9 to 12% of the maximum values being observed after
90 min. After 4 days, parathion and leptophos residues were not
detected in the blood, whereas 0.2 to 0.3% of the inhaled radiocarbon
from carbaryl, carbofuran, and DDT fortified cigarettes still remained.
14C-Residues in the heart immediately following smoking ranged from
0.4 to 3.6% of the inhaled activity in the mainstream smoke. After
90 min, the amount had declined by approximately 75 to 85%. No
residues were detected in the heart after 4 days.
93
-------
Table 26. FATE OF 11
-------
Table 26 . continued
Carbaryl Carbofuran Parathion Leptophos DDT
Fat
90 minutes
4 days
Q
Other tissues
90 minutes
4 days
Total recovery
90 minutes
4 days
9.1
0.5
0.4
0.0
90.6
100.2
3.8
0.0
7.3
0.0
101.6
106.8
10.4
0.9
0.6
0.0
96.8
96.7
1.5
0.8
0.3
0.0
81.4
91.7
9.7
33.1
1.2
0.7
95.6
110.9
b
c
Each value represents the average obtained from 4 animals.
Collected during smoking period.
Includes spleen, brain, ovaries and uterus.
95
-------
Residues in Urine and Feces - Urinary excretion removed 4.0 to 58.5%
of the total inhaled ll*C-insecticide residues (Table 26). With
carbaryl, carbofuran, and parathion, this amounted to 93 to 98% of the
residues retained in the rat after the smoking process. In the case
of leptophos, 78% of the retained ^C-material was removed in the
urine, while 23% was found in the feces. Clearance of DDT residues
from the rat was much slower with only 7 and 12% of the residues
excreted in the urine and feces after 4 days.
Tic analysis of the urine revealed that 93% and 84% of the excreted
carbaryl and carbofuran residues were polar materials. The apolar
carbaryl metabolites, 7%, contained 4 products, none of which was
carbaryl. Four apolar metabolites and the intact insecticide composed
16% of the carbofuran residues excreted. The parent compound accounted
for 6.2% while the other residues, tentatively identified as 3-keto
carbofuran, 3-hydroxycarbofuran phenol, 3-hydroxycarbofuran and carbo-
furan phenol, were present in about equal amount. The nature of the
excreted residues from the other insecticides was not determined.
^C-Residue in Tissues - Ninety min after smoking, ll*C-residues from
all the compounds were detected in the liver, kidney, muscle, fat,
spleen, brain, ovary and uterus. The persistence of the residues, how-
ever, was quite variable. After 4 days, only the fat contained resi-
dues greater than 0.2% of the total inhaled ^C-pyrolysis products
derived from the organophosphates. At this time, the total body
burden amounted to 1.0% of the inhaled activity. 14C-Carbaryl residues
remaining after 4 days amounted to 1.6% of the inhaled radiocarbon, 75%
of which was in the fat, kidney, and liver. Carbofuran radiocarbon,
0.6%, was found only in the liver and blood.
While 1I+C-DDT residues declined in the liver, kidney, muscle and other
organs during the 4 days, concentrations in the fat increased to over
3 times the level observed at 90 min. The percentage of DDT pyrolysis
products remaining in the body 4 days after exposure was 48.7% of the
96
-------
inhaled radioactive residues.
Carbofuran Pyrolyses - The technique of mechanical pyrolysis, i.e. the
use of pyrolysis chambers, etc., has been extensively used in efforts
to determine the chemical processes which occur during tobacco combus-
tion. While the dangers of extrapolating the pyrolysis results to
processes actually occurring during smoking are well understood, dis-
crepancies may be even more apparent when considering the fate of
volatile materials on the burned tobacco. This issue was evaluated
in the present study by comparing the pyrolytic behavior of carbofuran
in a "conventional" pyrolysis apparatus, i.e. a vertical combustion
tube encased in a fixed position oven, to that using a horizontal pyro-
lysis apparatus recently developed. In the latter, the sample is
evenly distributed inside a horizontal quartz tube and heated by a
movable, variable temperature oven. In contrast to the "conventional"
apparatus, the slow movement of the oven along the tube results in a
burning process similar to that of a burning cone of a cigarette.
For pyrolysis using the horizontal quartz tube, 5.5 g of carbofuran
was evenly distributed along 80 cm of its length. After preheating to
the desired temperature, ca. 1 hr, the oven was set in motion, result-
ing in a 5.1 g of brown semisolids collected at the end of the tube
and in the first cold trap. Trituration of the pyrolyzate with cold
1:1 hexane-benzene yielded 1.6 g of crystalline carbofuran, confirmed
by melting point and tic. The filtrate was diluted with 100 ml of
benzene and quickly extracted 3 times with 20 ml of cold 5% sodium
hydroxide. The organic phase was dried with sodium sulfate and con-
centrated to yield an additional 380 mg of the intact insecticide.
The basic extract was acidified and repeatedly extracted with ether.
The organic layers were combined, dried and concentrated under vacuum
to yield 2.1 g of an oily residue, most of which was the phenol of
carbofuran. A minor component ( <1%) of the phenolic fraction, col-
lected by preparative glc, was tentatively identified as 2-methyl-2-
benzofuranol; ir(CHC!3) 2.90, 6.21 and 6.93 U; nmr (CDC13)6; 2.38
97
-------
(s, 3p), 6.35 (s, lp) and 6.6 to 7.1 (m, 4p); ms 70eV m/e, 148 (P),
131, 119, 91 and 65. The infra-red spectrum of the evolved gases
showed characteristic bands of a mixture of methyl isocyanate, methane,
and carbon dioxide.
With the vertical pyrolysis apparatus, the pyrolysis tube was heated
to 783°C, and 8.6 g of carbofuran introduced into the top of the
column over a 30 min period. At the completion of the experiment, the
apparatus was cooled to room temperature and 2 g of material which
had solidified before reaching the cold traps were triturated with
hexane-benzene to yield 1.2 g of parent compound. The contents of
the traps, 4.3 g, were dissolved in chloroform and quickly extracted 3
times with 50 ml of ice cold 5% sodium hydroxide. The resulting chloro-
form phase was dried with magnesium sulfate, concentrated under vacuum
and the solid residue washed with hexane-benzene and filtered to furn-
ish 2.2 g of carbofuran. The filtrate was concentrated to 680 mg of
oil that was composed of at least 46 compounds as indicated by glc.
GC/MS analysis showed the presence of naphthalene, benzofuran, biphenyl
and anthracene in the neutral fraction.
The aqueous layer was acidified and extracted with ether and the
extracts combined, dried, and concentrated under vacuum to give 1.7 g
of dark oil. Glc analysis on OV-101 and EGSSX columns showed that
about 90% of the material consisted of two major components whose
spectral and chromatographic characteristics were identical to the
phenol of carbofuran and to 2-methyl-7-benzofuranol. The balance of
the material was composed of at least three unknowns. Gaseous pyro-
lysis products consisted of methane, carbon monoxide, carbon dioxide
and methyl isocyanate.
Results of the carbofuran pyrolysis experiments clearly show that the
horizontal combustion apparatus more closely approximated the smoking
process than did the vertical unit. The latter system resulted in
numerous carbofuran derivatives which were not detected in the main-
98
-------
stream smoke of cigarettes, and would suggest a far more complicated
situation than actually exists. Since pyrolysis of pesticides in the
absence of tobacco greatly facilitates isolation and identification
of their pyrolysis products, such a process is very desirable. How-
ever, great care must be taken to assure that the nature of the
products is the same as that in tobacco smoke. It appears that this
can be largely accomplished by using the horizontal pyrolysis appara-
tus.
99
-------
SCREENING OF PESTICIDES FOR MUTAGENIC POTENTIAL USING THE AMES ASSAY
A wide variety of test systems with differing degrees of simplicity and
sensitivity have been developed to detect the mutagenic and carcinogenic
potential of chemicals, tobacco smoke fractions, and extracts of air-
borne pollutants. The methods involve use of cultured cells, micro-
organisms, plants, or intact animals, and have been recently reviewed
by Stolz et al. (1974) and Epstein and Legator (1971).
Ames et al. (1973 a,b) have introduced an assay for the detection of
mutagens using histidine-requiring mutants of Salmonella typhimurium.
The assay is quite sensitive to detect point mutations of both the frame-
shift and base-pair substitution type, is easy to perform, and includes
a capacity to approximate mammalian metabolism by the addition of a rat
liver homogenate fraction. In the present study, we have employed the
Ames assay procedure to evaluate the mutagenic potential of several
pesticides which have been used extensively for the control of crop and
animal pests.
Materials and Methods
Chemicals - Nitrosocarbaryl (N-nitroso-N-methyl-1-naphthylcarbamate) was
prepared according to procedures outlined by Elespuru et al. (1974).
The structure was confirmed by a mass spectral analysis (70 eV), which
showed a parent ion at m/e 230 and fragments at m/e 143 (naphthoxyl),
m/e 127 (naphthyl), and m/e 115, as previously reported. Purity was
confirmed by silica gel thin-layer chromatography in three solvent sys-
tems: 9:1 carbontetrachloride:ether; 1:1 Chloroform:ether; and 1:1
ethyl acetate:ether. Aroclor 1254, DDT, DDE, dieldrin/heptachlor, hep-
tachlor epoxide, carbaryl, diazinon, linuron, and captan were obtained
in a pure form from stock of standards used in residue analysis. N-methyl-
-N-nitro-N'-nitrosoguanidine (MNNG) and 2-acetylaminofluorene (AAF) were
purchased from Aldrich Chemical Company. Sodium phenobarbital was ob-
100
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tained from the W. A. Butler Company. Benz[a]anthracene, 9-aminoacri-
dine, 7-iodoitiethyl-12-methylbenz [a] anthracene, 7 ,12-dimethylbenz [a]an-
thracene (DMBA), DMBA dialdehyde, and 6-chloromethylbenzo[a]pyrene were
supplied by Dr. James Flesher, Department of Pharmacology, University
of Kentucky.
Bacterial System - The bacterial strains (TA1535, TA1536, TA1537,
TA1538) were acquired from Dr. Ames (Biochemistry Department, University
of California, Berkeley). These are histidine-requiring mutants which,
in addition, lack an excision repair system, and the lipopolysaccharide
barrier making them more sensitive and permeable to foreign compounds.
Strain TA1535 (Ames et al. 1973a) is a base-pair substitution mutant,
while the other three are frameshift mutants.
The agar medium was prepared according to Ames et al. (1973a) as was the
"S-9" liver preparation. Induction of the liver enzyme system was ac-
complished by providing 200 g Sprague-Dawley male rats with drinking
water containing 0.1% phenobarbital for seven days, or by injecting the
rats i.p. with Arochlor 1254 at 200 mg/kg 5 days before sacrifice.
The test methods used were exactly as described by Ames et al. (1973b).
All the compounds tested were dissolved in dimethylsulfoxide (DMSO) in
concentrations varying from 1 to 10 mg/ml. Up to 0.5 ml of DMSO solution
was added with the soft agar to each plate, as was 0.1 ml of bacterial
tester strain culture (10^ bacteria/ml estimated by measuring turbidity
on a Klett spectrophotometer). These plates were incubated for 48 hours
at 37°C and data quantitated by counting the number of colonies per
plate. Each colony represents a reverse mutation from the histidine-
dependent to the histidine-independent form. 2-Acetylaminofluorene and
N-methyl-N-nitro-N'-nitrosoguanidine were used as active standards or
positive controls in the prescribed manner (Ames et al. 1973b). Cellu-
lar toxicity was determined by mixing each compound with the tester
strain as described above, but with the top agar containing an excess of
histidine (3 ymoles/plate). The plates for toxicity determinations were
101
-------
incubated for 24 hours at 37 °C and evaluated for growth inhibition by
comparison with control plates.
The water soluble metabolites from bean plants each treated with 500 yg
of carbaryl were tested in the Ames assay. Since the injection to the
bean plants contained a small amount of carbaryl ring [1LfC] as a tracer,
the carbaryl equivalents in the water soluble metabolites were determined
by radioassay. The water soluble metabolites of carbaryl and the water
fraction from control plants were lyophilized to dryness. The carbaryl
water soluble metabolites were tested in a solution of 1 mg equivalents
of carbaryl to 1 ml DMSO at levels up to 500 yg equivalents of carbaryl
per plate. Equal volumes of the control plant residue were tested for
mutagenicity and toxicity. Samples of carbaryl ring [1I+C] were tested
at levels up to 100,000 dpm (twice the radioactive content of the high-
est level of carbaryl water soluble metabolites). Carbaryl itself was
tested up to 1000 yg per plate.
DDE, dieldrin, hpetachlor, heptachlor epoxide, and diazinon were tested
from 5 to 1000 yg per plate, and DDT was tested from 5 to 2500 yg per
plate. Linuron and captan were tested from 1 to 200 yg per plate.
Results and Discussion
The Ames assay is an in vitro testing system which uses histidine re-
quiring mutants of Salmonella typhimurium to screen chemicals for po-
tential in vivo mutagenicity. If the chemical being tested causes a
mutation in a cell of one of the strains (TA1535, TA1536, TA1537, or
TA1538) at a point in the gene responsible for histidine dependence,
such that the cell becomes histidine independent, then a reverse muta-
tion has taken place. This cell will grow into a revertant colony on
histidine deficient agar. The number of these colonies is a measurement
of the potential mutagenicity of the compound in vivo. The higher the
number is, the greater the potential.
102
-------
In order to obtain an appreciation for the amount of spontaneous rever-
tant colonies to expect, many plates were assayed with only the ingre-
dients of the nutrient agars and 0.5 ml dimethylsulfoxide, the solvent
used to dissolve the test compounds. The results of this undertaking
are illustrated in Table 27. The mean spontaneous reversion frequencies
were 10.7 with 1535, 0.6 with 1536, 10.2 for 1537, and 17.3 for 1538.
Another series of plates were assayed with the same ingredients plus a
rat liver homogenate (S-9 RLH). The mean spontaneous reversion frequen-
cies in this series were 10.3 for 1535, 2.7 for 1536, 15.0 for 1537, and
32.1 for 1538. Along with the mean spontaneous reversion frequencies
listed in Table 27 are the standard deviations of these means. The con-
siderable variation observed could have been narrowed by two procedures:
1) expressing the reversion frequency in terms of number per million
viable bacteria, which would require a dilution series plate count on
each date of testing; and 2) standardizing the stock cultures each time
they are grown (every seven days) to 10 bacteria per ml by a dilution
series plate count, instead of estimating this on a spectrophotometer.
However, these operations were considered unnecessary for the purpose of
screening large numbers of chemicals for mutagenic potential. Therefore,
it was decided that in order for a compound to be declared mutagenic in
this test system, it must demonstrate a reproducible dose response
curve with a peak reversion level well above several standard deviations
from the spontaneous reversion frequency.
Table 28 is a compilation of mutagens, carcinogens, and carcinogen
analogs, along with results of tests in the Ames assay. There was a
wide variety of activity demonstrated by these compounds, but the known
carcinogens, 2-acetylaminofluorene (AAF), N-methyl-N'-nitro-N-nitroso-
guanidine (MNNG), 7-iodomethyl-12-methyl-benz[a]anthracene, 7,12-dimethyl-
benz[a]anthracene, and 6-chloromethyl benzo[a]pyrene, were at least mild-
ly active. The carcinogen benz[a]anthracene was not active in the four
tester strains, and neither was the DMBA dialdehyde, a noncarcinogen.
These data illustrated certain characteristics of the Ames assay. For
example, with AAF and DMBA no mutagenic activity was manifested until
103
-------
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104
-------
Table 28. Mutagenic activity of known mutagens, carcinogens, and car-
cinogen analogs in S. typhimurium - TA1535 series.
Compound
2-acetylamino-
fluorene (AAF)
N-methyl-N1 -nitro-
N-nitrosoguanidine
(MNNG)
Ug/plate
±RLH*
50-
50+
2-
5-
Revertant
22(16)
18(21)
1812(14)
10560(20)
colonies
1(1)
0(1)
0(0)
per plate/strain
12(18)
88(29)
10(12)
7(17)
28(32)
5611(44)
18(29)
13(22)
9-aminoacridine
100-
11(10)
900(12)
11(18)
benz[a]anthracene
100+
6(14)
18(18)
21(38)
7-iodomethyl-12-
methylbenz [a] -
anthracene
DMB A- dial dehyde
7 , 12-dimethyl-benz-
[a] anthracene
(DMBA)
6-chloromethyl-
benzo [alpyrene
10-
10+
100-
100+
25-
25+
25-
25+
8(10)
8(14)
8(10)
15(14)
2(5)
6(8)
1(0)
3(1)
0(0)
14(7)
~
0(0)
1(2)
198(12)
268(18)
6(12)
12(18)
17(18)
63(18)
3(8)
10(15)
480(18)
456(38)
10(18)
20(38)
36(38)
90(38)
1280(16)
1600(32)
* with (+) or without (-) rat liver homogenate
( ) spontaneous reversion frequency for each test
105
-------
the S-9 rat liver homogenate fraction was added. The capacity of mam-
malian metabolism enables this test system to detect many carcinogens
as mutagens which, otherwise, would not be detected. On the other hand,
MNNG and 9-aminoacridine were quite active without the liver homogenate.
It is believed that these two chemicals require no in vivo biochemical
activation to express their mutagenic properties (Miller 1970). These
data supported the claim that the Ames assay was a good screening test
for potential mutagenicity and carcinogenicity (Ames et al. 1973b).
Table 29 contains the results of tests using the Ames assay on four
pesticides purported to be potential human carcinogens, DDT, dieldrin,
heptachlor, and captan. Also tested were DDE, heptachlor epoxide,
diazinon, carbaryl, nitrosocarbaryl, and linuron, all pesticides or
pesticide derivatives, which have demonstrated some mutagenic manifes-
tation in other assay systems (Vaarama 1947, Amer 1965, Markaryon 1967,
Wuu and Grant 1967, Epstein and Shafner 1968, Kaszubiak 1968, Legator
et al. 1969, Elespuru et al. 1974). The control values represent the
spontaneous reversion frequencies of the tester strains both with and
without rat liver homogenate. Where no activity was observed, only the
highest dose tested or the maximum tolerated concentration (the highest
concentration which did not appreciably inhibit growth) is listed. How-
ever, all the compounds were evaluated at concentrations as low as 5 yg/
plate with each successive level increased by a factor of two up to the
highest level. The toxicity data were useful in determining the con-
centrations that gave maximum reversion with a minimum of growth inhibi-
tion, an influence that would obviously affect the dose-response corre-
lation.
The results revealed two active compounds, captan and nitrosocarbaryl.
Both are active as base-pair substitution mutagens as evidenced by their
ability to back mutate the 1535 strain. According to Ames et al. (1973a),
the exact base-pair changes which revert this strain are not clear, but
it is suspected that most, if not all, of the six possible base-pair sub-
stitutions can be detected.
106
-------
Table 29. Mutagenicity in S. typhimurium - TA1535 series.
Compound
DDT
DDE
Dieldrin
Heptachlor
Heptachlor
epoxide
Diazinon
Carbaryl
Nitrosocarbaryl
Linuron
yg/plate
±RLH*
2500-
2500+
1000-
1000+
1000-
1000+
1000-
1000+
1000-
1000+
1000-
1000+
1000-
1000+
0.5-
5-
50-
100-
5+
50+
300+
500+
1000+
25-
200+
Revertant
1535
6( 8)
15(17)
4( 7)
18(17)
3( 8)
17(17)
4(10)
8(17)
10(13)
15(17)
12(10)
22(17)
19( 9)
9(17)
51( 8)
2976 ( 8)
2932 ( 8)
3( 8)
57(10)
870(17)
5252(17)
3892(17)
1268(17)
8( 8)
11(13)
colonies per plate/strain
1536
0(0)
3(8)
0(1)
3(8)
0(1)
18(8)
1(0)
8(8)
0(0)
7(8)
1(0)
7(2)
0(2)
10(8)
0(0)
0(0)
2(2)
0(2)
2(2)
2(0)
1(1)
1537
10(15)
45(43)
10(13)
22(43)
8(11)
37(43)
16(11)
41(43)
10(13)
55(43)
9(11)
3(18)
19(11)
33(43)
92(15)
115(15)
21 ( 8)
27( 8)
37 ( 8)
45(15)
4(22)
8(17)
1538
22(19)
69(56)
17(13)
69(56)
16(18)
65(56)
12(12)
53(56)
14(10)
65(56)
13(13)
13(33)
22(23)
51(56)
96(20)
52(20)
56(56)
61(56)
42(56)
38(56)
13(28)
40(46)
Toxic
level
ug
±RLH*
>2500-
>2500+
>2500-
>1000+
>2500-
>1000+
>2500-
>1000+
>2500-
>1000+
>1000-
>1000+
>2500-
>1000+
100-
>1000+
50-
^ 400+
107
-------
Table 29. Continued.
Compound
Captan
yg/plate
±RLH*
2.5-
10-
25-
50-
25+
50+
200+
Revertant
1535
100 ( 7)
248(10)
320(10)
596(10)
117(14)
231(14)
221(16)
colonies per plate/strain
1536
0(1)
2(1)
0(1)
2(1)
0(1)
1537
335 (
47 (
0(
148 (
288 (
78 (
7)
7)
7)
9)
9)
2)
1538
19(32)
11(32)
0(32)
32(32)
51(32)
34(32)
Toxic
level
ug
±RLH*
100-
MOO+
* KLH=S-9 rat liver homogenate
( ) Spontaneous reversion rate for each test
108
-------
Nitrosocarbaryl was extremely potent, demonstrating increased reversion
at 0.5 yg level in the base substitution revertible strain 1535. This
compound has demonstrated mutagenic characteristics in two other bac-
teria, Escherichia coli and Haemophilus influenzae (Elespuru et al.
1974). Beattie and Kimball (1974) reported that mutagenesis induced in
H. influenzae by nitrosocarbaryl consists of both replication-dependent
and replication-independent components. Our results with strain 1537
and 1538 indicate that nitrosocarbaryl may be slightly active as a
frameshift mutagen at higher concentrations. TA1538 is particularly
sensitive to the polycyclic aromatic hydrocarbons due to a specific
"hot spot" in the DNA (Ames et al. 1973a). Presence of the "S-9" liver
homogenate decreased the mutagenic action of nitrosocarbaryl, thus indi-
cating that activation was not required.
Four series of tests on nitrosocarbaryl in TA1535 representing four dif-
ferent stock cultures of that strain varying in age from two to seven
days were combined for a regression analysis. It was found that the age
of the culture had no significant contribution to the variability of the
system. Utilizing all the data generated in this study with nitrosocar-
baryl, a correlated T-test showed that the 1 yg level of nitrosocarbaryl
without rat liver homogenate and the 50 yg level with rat liver homoge-
nate were the concentration levels showing the least significant differ-
ence (p<.01) from the spontaneous reversion frequency.
Based on the results with nitrosocarbaryl and assuming that the variabil-
ity is inherent within the method regardless of the activity of the com-
pound, a hypothetical compound tested twice would need to demonstrate a
reversion mean at the peak response level of at least 135 without rat
liver homogenate, and at least 320 with rat liver homogenate in order to
conclude that the compound was mutagenic in strain 1535 (P=.05). This
conservative approach is necessary to eliminate the false positives which
could occur if one attempted to apply the standard T-test. However, the
strict adherence to numbers obtained from such a statistical analysis is
not recommended until a large number of active compounds are studied
109
-------
under similar conditions. Even then the results should be viewed only
as a. guideline by the researcher.
Captan was much less potent than nitrosocarbaryl, but also was active as
a frameshift mutagen in strain 1537. Similarly, it seemed to act direct-
ly since the liver homogenate decreased its activity. However, activa-
tion via the metabolic pathways of the bacteria cannot be ruled out com-
pletely for captan or nitrosocarbaryl. Several in vitro studies (Epstein
and Shafner 1968, Legator et al. 1969) have shown captan to be mutagenic,
while results of other in vivo studies fail to correlate with these
findings (Kennedy et al. 1975). This may be due to captan's short half
life in serum (Epstein and Legator 1971).
The other eight compounds were negative in this system. Unless toxicity
was noted, they were tested in concentrations from as low as 1 to 2500
yg/plate. Ideally, compounds should be tested up to levels of growth
inhibition as was done with linuron, but at the highest levels shown in
Table 29, precipitation was seen with the chlorinated hydrocarbons and
with carbaryl. The use of DDT, dieldrin and heptachlor has been banned
or severely limited by the Environmental Protection Agency because of
their carcinogenic activity in experimental mammals. A lack of activity
of these chemicals in the Ames assay may be indicative of its insensitiv-
ity to certain types of mutagens and/or carcinogens, or of the inconclu-
siveness of studies conducted in higher organisms.
The carbaryl water soluble metabolites from bean plants treated with 500
yg of carbaryl were tested in the Ames assay. The results are shown in
Table 30. These metabolites were not mutagenic at levels up to 500 yg
equivalents of carbaryl. Higher levels were not possible due to the
limited amount of test material. Carbaryl, itself, was not mutagenic at
levels up to 1000 yg/plate. It is of interest to note the inhibition of
growth seen at the 500 yg level (concentration = 1 mg equivalent of car-
baryl per ml) in the carbaryl water soluble metabolite plates, since
carbaryl itself was not toxic to the bacteria at 2500 ng/plate. However,
an equivalent amount of control plant water soluble material was also
110
-------
Table 30. Mutagenicity screening of carbaryl bean plant water soluble
metabolites in S. typhimurium - TA1535 series.
Treatment Revertant
Compound
Carbaryl
bean plant
water soluble
metabolites
Control
bean plant
water soluble
fraction
Naphthyl[ltfC]-
carbaryl
Carbaryl
per plate
±RLHS
10 ygC-
50 yg -
100 yg ~
500 yg -
10 yg +
50 yg +
100 yg +
500 yg +
d
10 yl -
50 yl -
100 yl -
500 yl -
10 yl +
50 yl +
100 yl +
500 yl +
50,000 dpm^-
100,000 dpm -
50,000 dpm +
100,000 dpm +
1000 yg -
1000 yg +
1535
4(11)
10
5
1
10(14)
9
3
0
6(10)
4
5
7
9(10)
10
14
15
5(4)
3
12(7)
7
19(9)
9(17)
colonies
1537
5(8)
10
8
33(22)
38
10
8(6)
6
8
9(6)
5
16
3(5)
7
10(5)
5
19(11)
33(43)
m b
per plate Toxic
level
1538 ±RLHS
9(10) 500 yg°-
8
10
0
15(14) 500 pg +
16
21
d
7(7) 500 yl -
9
9
5
26(21)
20
23
11(10)
10
15(16)
16
22(23) >2500 -
51(56) >1000 +
( ) Spontaneous reversion frequency for each test series.
a
b
c
d
e
f
With (+) and without (-) rat liver homogenate.
Inhibition of bacterial growth was used as an indication of toxicity.
yg equivalents of carbaryl as determined by radioactive content.
yl equivalents of the control plant water soluble/plate.
.23 yg carbaryl.
.46 yg carbaryl (up to twice the 11+C content of the carbaryl plant
water soluble plates).
Ill
-------
toxic at the 500 pi/plate level/ indicating some inherent toxicity of
this plant fraction not related to any carbaryl metabolites. The ring
[11+C] carbaryl (specific activity = 19.6 mCi/mM) was included in this
study as a check on any mutagenicity due to the low radioactivity pres-
ent in the carbaryl water soluble metabolites tested. This radioactive
preparation was tested at dpm levels up to twice the amount present in
the highest level of the carbaryl water soluble metabolites (50,000 dpm).
No mutagenicity was demonstrated.
The fact that carbaryl water soluble metabolites were negative in this
test system is noteworthy. As with many carbamate insecticides, a large
portion of carbaryl plant insecticide residues are of a water soluble
nature, thus resulting in long term, low level exposure to the human
population. Under these conditions carcinogenicity, mutagenicity, and
teratogenicity are toxic effects of greatest concern to regulatory
agencies. Although none of these effects have been implicated due to
the ingestion of any pesticide conjugate (which the water soluble metabo-
lites are comprised of), the possibility of chronic toxic effects does
exist (Dorough 1976). The sulfate ester of N-hydroxy-2-acetylamino-
fluorene is a good example (DeBaun et al. 1966). This metabolite is a
more potent carcinogen than the parent compound, 2-AAF.
Given the chemical structures and the theory on alkylating agents per-
taining to their mechanism of mutagenesis, the results obtained in these
experiments are somewhat predictable. Both nitrosocarbaryl and captan
have good "leaving groups", thus they could be expected to interact with
cellular nucleophiles such as DNA and proteins. Conversely, the other
compounds have no such easily identifiable group. Of course, this does
not rule out their being active by other mechanisms.
As part of the report prepared for the Mrak Commission (Secretary's Com-
mission on Pesticides and Their Relationship to Environmental Health) in
1970, Epstein and Legator (1971) recommended a program for mutagenesis
testing of pesticides. This included three mammalian test systems (the
112
-------
dominant lethal, host mediated, and in vivo cytogenetic), and several
ancillary microbial systems for detecting both single nucleotide changes
and effects involving more than one gene. Results reported herein in-
dicate that the Ames assay would be a valuable asset to such a battery
of tests, because of its sensitivity, ease of performance, and mammalian
metabolizing capacity. Not only would the assay aid in evaluating the
mutagenic potential of a pesticide, but also its carcinogenic hazard,
since a very high correlation exists among known carcinogens and their
ability to back mutate the TA1535 series of S. typhimurium (Ames et al.
1973b) .
Any conclusions based solely on the Ames assay, though, are not the
final answer. One need only scan the results in Table 29 relative to
the pesticides screened in the Ames assay to realize that problems exist
in detecting weak or potential carcinogens, such as DDT, heptachlor, and
dieldrin. Even the known carcinogen benz[a]anthracene was negative with
the four tester strains shown in Table 28. However, recently introduced
strains of S. typhimurium have shown this compound as an active mutagen
(McCann et al. 1975). A priority search is under way in the laboratory
of Ames to find S. tyhpimurium strains that are sensitive to the chlori-
nated insecticides suspected to be carcinogens. The possibility exists,
though, that these chemicals are not mutagens. On the other hand, cap-
tan has demonstrated no evidence of being a genetic hazard in mammalian
species, but it is active in several bacterial strains including those
of Ames (Kennedy et al. 1975, McCann et al. 1975). Obviously, caution
should be used in extrapolating effects observed in bacteria to higher
organisms.
113
-------
METABOLISM OF CARBARYL IN THE AMES MUTAGENIC SCREENING SYSTEM
The Ames mutagenic assay system has become a valuable tool to screen
chemicals for possible mutagenic/carcinogenic activity. Success of
this assay procedure can be partially attributed to the addition of S-9
rat liver homogenate (RLH) which is assumed to simulate mammalian metab-
olism. The metabolic activity of the S-9 RLH has not been fully demon-
strated and it is possible that the agar-bacteria-liver enzyme combina-
tion may either (a) produce none of the metabolites formed in animals,
or (b) produce active metabolites that are not common to the in vivo
animal system. The inability of the Ames system to detect 10% of the
175 carcinogens tested (McCann et al. 1975) may be attributed to the
failure of the system to metabolically activate the compounds. Such
possibilities have prompted this study which has analyzed the metabolic
capabilities of the Ames system on the test compound carbaryl.
Materials and Methods
Chemicals - Carbaryl-1-naphthy1-l4C (19.6 UCi/mmole), carbaryl-carbonyl-
11+C (5.38 yCi/mmole) , 4-hydroxy carbaryl, 5-hydroxy carbaryl, N-hydroxy-
methyl carbaryl, 5,6-dihydrodihydroxy carbaryl, 5,6-dihydrodihydroxy
naphthol, and a-naphthol were obtained from laboratory stocks. Purity
of each compound was confirmed by thin-layer chromatography (tic) .
Test System - Experimental conditions for the Ames test are described
by Marshall e_t al. (1976) . Because of interfering properties of dimeth-
yl sulfoxide with tic analysis, carbaryl was added as an acetone solu-
tion not to exceed 20 yl of acetone per plate. The experiment was de-
signed to evaluate the metabolic capabilities of each major constituent
of the Ames system. This necessitated the analysis of possible chemi-
cal degradation occurring in the complete top agar (Treatment I), mic-
robial metabolism in the inoculated top agar (Treatment II), "mammalian"
metabolism in the S-9 RLH-top agar complex (Treatment III) , and a combi-
nation of all of these (Treatment IV). Duplicate plates, representing
114
-------
each treatment and various incubation periods, were prepared and incu-
bated at 37°C.
After incubation the plates were immediately frozen at -20°C to stop
the metabolic reactions. Each plate contained approximately 112,000
dpm (0.52 yg) of carbaryl-l-naphthyl-ltfC. The test organism used
throughout this study was Salmonella typhimurium strain TA1535, a base-
pair substitution mutant. Since carbaryl could not promote growth of
the histidine-requiring mutants, excess histidine (0.63 yg/plate) was
added to additional plates from treatments II and IV to evaluate the
metabolism of carbaryl by actively growing organisms. Zero-hour, con-
trol plates from treatments III and IV received inactivated (boiled)
S-9 RLH.
The metabolism of carbaryl-carbonyl-^C was used as an aid in ascertain-
ing the identity of some of the carbaryl metabolites. Comparison of
metabolites from both labeling patterns indicated which metabolites re-
tained the carbonyl carbon.
Extraction Procedure - The contents of each petri plate were blended
with 25 ml of distilled water and 25 ml of ethyl acetate using a Lourdes
blender. The mixture was centrifuged for 30 min at 3300 x g, and the
liquid was decanted into a 125-ml separatory funnel. The organic phase
was collected and the aqueous and agar phases were re-extracted with an
additional 25 ml of ethyl acetate. Aliquots of each fraction were quan-
titated by liquid scintillation to determine the distribution of radio-
carbon. Each sample was stored at -10°C until further analysis.
Thin-Layer Chromatography - Organo-soluble metabolites were analyzed by
one-dimensional thin-layer chromatography using a methylene chloride:
ethyl acetate (1:1 v/v) solvent system. Radioautograms were prepared by
exposing tic plates to X-ray film for a minimum of seven days. Radio-
active bands were scraped and quantitatied by liquid scintillation
counting.
115
-------
Results and Discussion
The Ames test is routinely conducted both with and without the addition
of S-9 RLH. This allows the detection of direct-acting mutagens or ac-
tive metabolites of the test compound. Two basic assumptions must be
made with this type of procedure. The first assumption is that neither
the chemical environment nor the microorganisms have the ability to
detoxify the active product to prevent a positive response. Secondly,
it must be assumed that the S-9 RLH is representative of mammalian
metabolism. These assumptions must be questioned particularly in cases
where the Ames test has failed to detect known carcinogens.
Although carbaryl is not a mutagen/carcinogen, its susceptability to
microbial and mammalian metabolism makes it an excellent reference com-
pound to test the metabolic activity within the Ames system. Distribu-
tion data in Table 31 indicated the rate of carbaryl conjugation occur-
ring in the Ames system. The insignificant percentage of water soluble
products detected in plates from Treatment I suggests that the chemical
environment, consisting of agar, histidine, biotin, and glucose, had
little effect on carbaryl metabolism. Similar results were obtained
with inoculated plates from Treatment II, suggesting a lack of metabo-
lism by the Salmonella typhimurium mutants. Analysis of the organo-
soluble products revealed that 77% of the parent compound still remained
even after 48 hours of incubation (Table 32). Since no major metabo-
lites were detected, the 22% loss of carbaryl was believed to result
from a general loss of product during tic analysis. The addition of
excess histidine to enhance the microbial growth resulted in a slight
accumulation (<1%) of N-hydroxymethyl carbaryl, but this was considered
insignificant in relation to the total concentration of carbaryl.
These results indicate that the microorganisms had little effect upon
the metabolism of carbaryl.
The metabolic ability of the S-9 RLH is quite apparent from the distri-
bution data (Treatment III) presented in Table 31 Within one hour of
116
-------
Table 31. DISTRIBUTION OF RADIOCARBON AFTER INCUBATING
CARBARYL-l-NAPHTHYL-^C WITH VARIOUS COMPONENTS OF THE AMES
ASSAY SYSTEM
Treatment
and fraction
% per fraction/hr of incubation
12
24
48
I.
Organo-soluble
Water soluble
Bound (agar)
Total
II.
Organo-soluble
Water soluble
Bound (agar)
Total
III.
Organo-soluble
Water soluble
Bound (agar)
Total
IV.
Organo-soluble
Water soluble
Bound (agar
Total
100.0
0.1
0.1
100.0
97.8
0.1
0.1
98.0
95.3
0.1
0.1
95.5
95.9
0.0
0.0
95.9
100.0
0.1
0.1
100.0
96.7
0.1
0.1
96.9
49.7
24.3
15.4
89.4
50.8
24.5
14.5
89.8
100.0
0.1
0.1
100.0
100.0
0.1
0.1
100.0
47.6
27.0
17.8
92.4
45.4
24.7
17.3
87.4
83.3
0.3
0.2
83.8
82.2
0.3
0.2
82.7
44.1
28.0
16.9
89.0
37.6
26.7
17.7
82.0
87.3
0.8
0.5
88.6
94.1
1.7
0.5
96.3
41.8
26.9
19.1
87.8
38.4
26.3
20.9
85.6
80.4
2.6
2.3
85.3
89.2
2.1
1.9
93.2
45.9
27.1
20.1
93.1
39.6
30.1
20.6
90.3
80.7
7.2
5.8
93.7
86.6
5.3
4.8
96.7
45.9
17.0
18.0
80.9
42.7
27.9
16.9
87.5
Each treatment conducted under conditions of the Ames test. Petri
plates contained the solid bottom agar plus the:
( I) complete top agar consisting of soft agar, histidine, and bio-
tin;
( II) complete top agar and 108 mutants;
(III) complete top agar and the S-9 rat liver homogenate;
( IV) complete top agar, the mutants, and the S-9 rat liver homog-
enate .
117
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Table 32. CARBARYL-l-NAPHTHYL-llfC REMAINING AFTER INCUBATION
WITH VARIOUS COMPONENTS OF THE AMES ASSAY SYSTEM
% as carbaryl/hr of
a
Treatment
I
II
III
IV
0
99.
95.
93.
94.
0
9
3
0
1
NA
NA
8.7
10.9
3
NA
NA
2.4
2.8
6
NA
NA
0.8
0.8
incubation
12
87.
92.
2.
1.
3
0
5
5
24
NA
NA
0.9
0.4
48
77.9
82.7
4.0
1.1
Each treatment conducted under conditions of the Ames test. Petri
plates contained the solid bottom agar plus the:
( I) complete top agar consisting of soft agar, histidine, and
biotin;
( II) complete top agar and 108 mutants;
(III) complete top agar and the S-9 rat liver homogenate;
( IV) complete top agar, the mutants, and the S-9 rat liver
homogenate.
NA = not analyzed
118
-------
incubation, approximately 25% of carbaryl was converted to water solu-
ble products and 15% to agar-bound materials. The distribution of
radioactivity among the phases remained fairly constant after one hour.
This demonstrated that the enzymes had lost most of their activity
within the first hour of incubation at 37°C. However, analysis of the
organo-soluble products from plates of Treatment III suggested that the
S-9 fraction was slightly active for up to six hours in that the concen-
tration of carbaryl continued to decline during this period (Table 33).
This agrees with the contention of Ames that the solid agar tends to
prolong the activity of the liver enzymes, although it is clear that,
as with other in vitro RLH systems, the major activity is evidenced
shortly after the mixing of enzyme and substrate. The degree of activi-
ty of the S-9 RLH is indicated by the production of nine metabolites in
the organic phase.
Tentative identification of the metabolites was based upon cochromato-
graphy with known reference standards and comparisons of metabolites us-
ing two different labeling patterns. Five of the metabolites were iden-
tified as follows: 5,6-dihydrodihydroxy carbaryl, 5,6-dihydrodihydroxy
naphthol, N-hydroxymethyl carbaryl* 4-hydroxy carbaryl, and 5-hydroxy
carbaryl. The identity of the remaining products could not be confirmed
because of their low yield. Unknown 4 cochromatographed with a-naphthol
but the carbonyl labeled experiment revealed that this product retained
the carbonyl carbon as did the other unidentified metabolites. Further
characterization of the unknown metabolites has not been attempted since
it was not the intent of this study to completely characterize carbaryl
metabolism, but to evaluate the metabolic activity of the S-9 RLH.
Results of this study show that the homogenate was capable of rapidly
metabolizing carbaryl primarily via oxidative and conjugative routes.
The identified free products were all oxidative metabolites and were
probably metabolized further to glucuronides and sulfate conjugates as
in other mammalian systems. The identity of these conjugates was not
verified but their presence was indicated by the production of water
119
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Table 33. NATURE OF THE RADIOCARBON IN THE ORGANO-SOLUBLE
FRACTION RESULTING FROM THE INCUBATION OF CARBARYL-1-NAPHTHYL-11+C
WITH ALL COMPONENTS OF THE AMES SYSTEM EXCEPT THE MICROORGANISMS,
TREATMENT
Metabolites
resolved
by TLC
Unknown 1
Unknown 2
5,6-dihydro-
dihydroxy
carbaryl
5 , 6-dihydro
dihydroxy
naphthol
Unknown 3
N-hydroxymethyl
carbaryl
4-hydroxy
carbaryl
5-hydroxy
carbaryl
Carbaryl
Unknown 4
% of
Rf
0.00
0.04
0.14
0.30
0.39
0.50
0.68
0.79
0.90
0.98
14C in
0
0.2
0.0
0.0
0.0
0.0
0.0
0.0
0.0
99.0
0.8
organo-soluble
1
8.3
1.7
35.6
2.8
2.7
9.6
6.0
6.6
21.0
5.7
3
9.1
2.5
47.4
4.5
4.9
6.5
7.2
8.3
6.1
3.3
fraction/hr of incubation
6
7.1
2.5
50.7
7.2
6.1
3.7
9.8
8.1
2.2
2.6
12
10.6
2.8
45.7
8.9
4.1
4.1
6.4
5.9
7.1
4.6
24
10.5
2.9
55.0
15.3
3.9
1.3
2.8
2.5
2.3
3.6
48
14.1
3.9
32.3
19.5
3.5
2.1
3.5
3.6
10.6
6.9
Petri plates containing the bottom agar, the complete top atar, and
the S-9 rat liver homogenate of the Ames system, but without S.
typhimurium.
120
-------
soluble products. Evidence obtained thus far has demonstrated that the
S-9 RLH is an efficient in vitro metabolic system which appears repre-
sentative of mammalian metabolism. The rapid rate of metabolism indi-
dated that metabolites would be produced in sufficient time to elicit a
detectable response if indeed they were mutagenic in the Ames assay.
The concentration of an active metabolite required to produce a re-
sponce is unknown and likely varies with each compound. For this rea-
son, each chemical must be screened over a wide range of concentrations.
This poses the question of the effect of concentration on the rate of
metabolism. While the problem was not considered in the present study,
such an investigation is planned for the future.
Another question which deserved attention concerns the effects of the
microorganisms upon the metabolism of the S-9 RLH. It was demonstrated
that the microbes had a negligible effect upon the parent compound but
their effect upon RLH-produced metabolites has not been evaluated. The
addition of excess histidine, however, did increase the concentration
of 5-hydroxy carbaryl and greatly decreased the concentration of 5,6-
dihydrodihydroxy naphthol after 48 hours of incubation (Table 34). The
data indicate that the microorganisms may attack certain carbaryl me-
tabolites, but late in the testing procedure. The critical time in the
screening test is during the first few hours since the organisms must
have sufficient time to reproduce into visable colonies subsequent to
mutation.
The metabolism of carbaryl within the Ames assay system was shown to be
representative of in vivo mammalian metabolism. The microorganisms
were unable to significantly alter carbaryl and did not influence the
effects of the S-9 RLH metabolism. The majority of the metabolic activ-
ity within the Ames system was attributed to the S-9 RLH. This study
serves only to demonstrate that the Ames S-9 assay system is capable of
metabolyzing carbaryl. However, there is no apparent reason why the
system should not also metabolize other compounds of similar chemical
configuration. It remains to be seen if the system metabolizes vastly
different chemicals such as DDT the same as the intact organism.
121
-------
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122
-------
SYNTHESIS OF l4C-CARBONYL METHYLCARBAMATES
While [14C-carbonyl]-methyl isocyanate has been used widely for the
preparation of radioactive insecticidal methylcarbamates, yields are
often low, and in some cases reactions completely unsuccessful, because
of the sensitivity of the isocyanate to moisture. This paper describes
a process whereby the [ C]-methyl isocyanate, in its break-seal con-
tainer as received from the supplier, is reacted with the appropriate
oxime or phenolic intermediate without exposing the isocyanate to
atmospheric moisture. Results are presented for the insecticides
carbaryl, carbofuran, aldicarb and methomyl demonstrating consistent
yields of 85 to 95% using a procedure of uncommon simplicity.
Carbon-14 labeling of the carbonyl group in carbamate anticholinester-
ase toxicants provides an excellent tool for studying the disposition
of these chemicals in various biological systems. In particular,
knowledge of the metabolism of methylcarbamate insecticides has greatly
increased because of the use of this and other radiolabeling techniques
(Kuhr and Dorough, 1976). In addition to the accountability and ease
of quantitation afforded by radiotracer techniques, 1'tC-carbonyl label-
ing of the carbamates gives rapid data relative to an animal's ability
to directly detoxify methylcarbamates through hydrolysis of the ester
linkage, as this detoxication results in expiration of [14C]-carbon
dioxide (Krishna and Casida, 1966).
One widely used method of producing ll+C-carbonyl methylcarbamates
involves esterification of the appropriate phenolic or oxime starting
material with [ll*C-carbonyl]-methyl isocyanate. However, unless
extreme caution is used in handling the volatile, moisture-labile
methyl isocyanate, the simple one-step reaction can be frustrating as
well as expensive due to loss of the desired radiocarbon. The
purpose here is to report a facile, efficient means of synthesis of
aromatic and aliphatic methylcarbamates which circumvents many of the
problems encountered in handling small quantities of radioactive methyl
123
-------
1 4
isocyanate. By this method C-carbonyl labeling was accomplished
with the insecticides carbaryl (1-naphthyl methylcarbamate), carbo-
furan (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate), aldi-
carb (2-methyl-2-(methylthio)propionaldehyde 0-(methylcarbamoyl)oxime,
and methomyl (S-methyl-N-[(methylcarbamoyl)-oxy] thioacetimidate.
Materials and Methods
Phenolic and oxime starting materials for syntheses were obtained by
alkaline hydrolysis of analytical grade methylcarbamates used in
residue analysis. Subsequently, thin-layer chromatography (TLC) on
silica gel plates (Silgel F-254, Brinkman Instruments, Des Plains,
111. 60016) as detailed in Table 35 afforded purification. In this
manner, 1-naphthol, carbofuran phenol (2,3-dihydro-2,2-dimethyl-7-
hydroxybenzofuran), aldicarb oxime (2-methyl-2-(methylthio)propional-
dehyde oxime) and methomyl oxime (S-methyl-N-hydroxy thioacetimidate)
were isolated using systems A, B, C and D, respectively. [11+C-
Carbonyl]-methyl isocyanate, 5.38 mCi/mM, was obtained from New
England Nuclear, Boston, Mass. 02118. Reagent grade triethylamine
was used as a catalyst. Benzene, dried over sodium, served as the
solvent for the reaction.
Reactions of the various phenols or oximes with the radiolabeled methyl
isocyanate were carried out in the glass break-seal vial (similar to
K-892750, Kontes Glass Co., Vineland, N.J. 08360), as supplied by New
England Nuclear. This vial consisted of two compartments. Compartment
A contained 1.06 mg of the radioactive methyl isocyanate. Compartment
B was dried with dessicated air and a twofold molar equivalent of the
proper phenolic or oxime reactant was added along with 0.01 ml of
triethylamine and 0.5 ml of dry benzene. In addition, a 1 cm section
of 0.5 cm diameter glass rod was carefully inserted and compartment B
was tightly sealed with a teflon-covered cork. Compartment A was
immersed in a dry ice-acetone bath which served to condense the methyl
isocyanate and create a reduced pressure within compartment A. Holding
124
-------
Table 35. R VALUES OF STARTING MATERIALS AND PRODUCTS ISOLATED
BY SILICA GEL TLC SYSTEMS.
Solvent system
Compound
Carbaryl
Naphthol
Carbofuran
Carbofuran phenol
Aldicarb
Aldicarb oxime
Me thorny 1
Methomyl oxime
A
.58
.84
.57
.88
.36
.90
.11
.57
B
.46
.69
.41
.65
.24
.65
.13
.34
C
.58
.81
.58
.84
.38
.84
.14
.55
D
.74
.86
.71
.83
.61
.89
.36
.72
A - 4:1 diethyl ether - hexane
B - 7:3 benzene - diethyl ether
C - 9:1 diethyl ether - hexane
D - 9:1 diethyl ether - acetone
125
-------
the tube horizontally so that the tip of the break-seal was immersed
in the benzene solution, the seal was easily broken by the glass rod
upon a sudden movement of the vial. Due to the negative pressure,
the benzene solution containing the nonradioactive reactant and the
catalyst was rapidly withdrawn into compartment A. When this transfer
was essentially complete, the reaction vial was placed in a water bath
at 45°C for about 18 hours. The tube was then opened and the 14C-
carbonyl methylcarbamate isolated by silica gel TLC using the solvent
systems previously described. Radioactive products were located on
the plates by radioautogrpahy. These were quantitated by direct
counting of the silica gel in 3a70B scintillation cocktail (Research
Products International Corp., Elk Grove Village, IL. 60007) by means
of a Packard Model 3380 liquid scintillation spectrometer.
Mass spectra of the purified synthetic products were obtained using a
Finnigan Model 1015-C quadrupole instrument. Samples were introduced
via the solid probe inlet and were ionized with a 70 eV electron beam.
Results and Discussion
The radiopurity of the crude reaction mixtures of synthesized methyl-
carbamates were 94 and 92% for carbaryl and carbofuran, and 88 and 86%
for aldicarb and methomyl, respectively. The reproducibility of this
synthetic method was demonstrated by yields of carbaryl ranging from
93.9 to 94.6% radiopurity in 4 separate attempts. Radioactive im-
purities occurred as 3 distinct products with only slight TLC molibity
(R <.10) in the indicated solvent systems.
In addition to thin layer chromatography with authentic standards, mass
spectra of the synthetic products confirmed their authenticity. Major
fragment ions in these spectra are indicated in Table 36. The spectra
of all compounds indicated ions of m/e 58, corresponding to Ct^NHCO
which originated from the methylcarbamyl moiety. With the exception of
aldicarb, the spectrum of each compound exhibited molecular and phenol-
126
-------
Table 36. MAJOR IONS IN THE MASS SPECTRA OF THE SYNTHESIZED
METHYLCARBAMATES. PERCENT ABUNDANCES ARE LISTED IN PARENTHESES.
Compound Mass spectra
Carbaryl *201(3), 144(100), 127(3), 116(33), 115(43), 89(6),
63(6), 58(5) and 57(5).
Carbofuran *221(6), 164(100), 149(74), 131(18), 123(19), 122(21),
121(13), 91(12), 77(12), 58(11) and 57(10).
Aldicarb 144(47), 100(30), 89(21), 87(50), 86(100), 85(63),
76(37), 58(62), 55(32) and 41(90).
Methomyl *162(1), 105(76), 88(29), 58(100), 47(30), 44(29),
42(37), and 41(33).
* Molecular ions.
127
-------
ic or oxime ions. In contrast, the thiomethylene group of aldicarb
is labile apparently in preference to molecular ionization or decarba-
mylation.
All spectra were identical, except for the minor [ C] contribution,
to reference spectra of authentic compounds taken in this laboratory.
In addition, the spectrum of carbaryl coincided with that previously
published (Damico, 1965). The mass spectrum of aldicarb differed in
base peak (and consequently in abundance of other ions) from spectra
reported elsewhere (Damico et al., 1969). Such variance was judged
to be due to differences in ionization or mass separation techniques.
In conclusion, the above method offers an uncommonly simple means of
obtaining radiolabeled methylcarbamates. This method should be of
wide utility due to the extensive use of methylcarbamate compounds as
drugs and pesticides. Moreover, the potential for synthesis of small
quantities of these materials at low cost should extend the availabil-
ity of this radiolabel to any interested investigator.
128
-------
THE INFLUENCE OF DOSE ON THE DETOXICATION AND ELIMINATION OF CARBAMATES
IN RATS
Studies of methylcarbamate insecticide metabolism typically deal with 2
general types of chemical reactions in vivo, oxidation and hydrolysis.
The latter process deserved more attention than it has received to date
since it provides a single-step direct detoxication mechanism, is ob-
served extensively in both animals and plants, occurs rapidly, and be-
gins almost immediately within an organism after exposure. This report
represents an attempt to focus on hydrolysis of the anticholinesterase
insecticide Croneton (2-ethylthiomethylphenyl methylcarbamate) in the
rat in order to examine parameters governing the efficacy of this form
of detoxication under differing conditions of animal exposure.
One such parameter to be considered in any situation of animal-toxicant
interaction is the level of exposure. Since hydrolysis of 11+C-carbonyl
labeled methylcarbamate insecticides is known to yield 11+CO2 (Ruhr and
Dorough 1976), it was felt that changes in hydrolysis could be examined
by monitoring the expired air of rats treated with [11+C-carbonyl]-Crone-
ton. In addition, excretion from rats of such radiocarbon by urinary
and respiratory routes is essentially quantitative within 3 days (Nye
et al. 1976). This knowledge, combined with the observation that nearly
90% of radioactivity from 1£tC-labeled sodium carbonate given i.p. ap-
peared in exhaled air within one hour of administration (Krishna and
Casida 1966), suggested that the kinetics of hydrolysis could be ade-
quately inferred from kinetics of 1'4C02 expiration following [^C-carbon-
yl]-Croneton dosing. This is further suggested by the finding that less
than 1% of urinary radioactivity excreted from rats treated with [14C-
carbonyl]-Croneton sulfoxide was released upon acidification of urine
with 1 N HC1 (Hurst and Dorough 1976), as urinary [14C]-bicarbonate
would be. With this implicit assumption, an examination has been under-
taken of the elimination and detoxication of various doses of Croneton
ranging from small quantities causing no overt toxicity to those re-
sulting in severe poisoning.
129
-------
Materials and Methods
Chemicals - Stock [14C-carbonyl_]-Croneton (5.88 mCi/mM) , synthesized by
esterif ication of 2-ethylthionvethylphenol with [ll4C-carbonyl]-methyl
isocyanate, was used in preparation of animal doses. This material was
diluted with nonradioactive Croneton (Chemagro Division of Mobay Chemical
Corporation, Kansas City, MO.) to specific activities of 2.2, 0.44, 0.044,
and 0.0044 mCi/mM for doses of 100, 20, 2.0, and 0.2 mgAg animal weight,
respectively. These doses were prepared on the day of treatment by dis-
solving appropriate quantities in Tween-80 (Fisher Scientific Company,
Fair Lawn, N.J.) such that each dose would contain about 106 dpm in a
preset volume of vehicle (0.5 ml/250 g rat).
Animals and Treatment - All rats used were female Cox-S. D. albinos
(Laboratory Supply Company, Indianapolis, IN.) weighing about 250 g each.
All animals were starved overnight prior to weighing and dosing with a
feeding needle. Atropinized rats were injected i.p. with 50 mg/kg atro-
pine sulfate in 0.2 ml distilled water immediately before oral treatment.
Immediately following gavage, animals were placed in glass metabolism
cages (Delmar Scientific Glass Products of Coleman Instruments, Maywood,
IL.) designed for separate collection of urine, feces, and respiratory
gases.
Analysis - Following treatment, all animals were observed periodically
and evident symptoms of poisoning were noted.
Air from each metabolism cage was withdrawn by vacuum pump through 2
CC>2 traps connected in series containing 1 N KOH. Airflow was maintained
at 400 ml by periodic adjustment of a needle-valve manifold. Pairs of
20 ml traps for each cage were changed at 2 hr intervals during the ini-
tial 24 hr post-treatment, after which 200 ml traps were substituted for
the 2 subsequent 24 hr intervals. Replicate 2 ml aliquots were taken
from each trap, diluted with 2 ml of distilled water, and counted using
15 ml of 3a70B liquid scintillation cocktail (Research Products Inter-
130
-------
national Corporation, Elk Grove Village, IL.) in a Packard Model 3380/
544 counter.
Urine was collected at intervals of 12, 18, 24, 48, and 72 hr after
treatment. Each sample was assayed by direct counting and was partitioned
using a slight modification of the procedure of Dorough (1971). In this,
urine aliquots were diluted with saturated sodium chloride solution and
then extracted with acetonitrile and chloroform. Organic and aqueous
phases were then radioassayed by liquid scintillation for phase distribu-
tion of radiocarbon.
Feces were allowed to accumulate throughout the 72 hr experimental period.
These were weighed and representative aliquots were air-dried overnight
prior to combustion in a Packard Model 306 sample oxidizer. Combusted
samples were then radioassayed as above.
Transformation of ^CO? Data - All data from radioassays were calculated
and tabulated in terms of percent of the dose administered to each ani-
mal. In addition, the kinetics of Croneton hydrolysis as determined by
1I+CO2 evolution were examined by conventional methods involving trans-
formation of exponential funcitons into their linear equivalents (Riggs
1963). For this, the total lt+CC>2 expiration occurring in the 3-day
period was obtained. Then the percent of the 3-day total trapped in
each successive interval throughout the first 24 hr was calculated.
These data, as cumulative total excretion with time, were subtracted
from 100% at each interval yielding a decreasing exponential function
representing the percentage of 3-day hydrolyzed material remaining in
the animal body with time. Plots of the natural logarithm of this de-
creasing percentage were biphasically linear with time. Regression
lines were fitted to each phase by the method of least squares. Excel-
lent line fits (typically r2=.98 or better) were obtained in this manner.
Half-times (ti/2) for the initial (a) phase, which typically lasted for
about 12 hr after dosing, and the later (g) phase were calculated from
the line slopes. Calculations were performed for individual rats and
are expressed as mean ± standard error values at each dosage level for
131
-------
comparison of variation due to dose effects with that due to individual
differences.
Results and Discussion
Symptomatology evident after the experimental poisonings with doses
ranging from 0.2 to 100 mg/kg is listed in Table 37. The visible con-
sequences of administration of 0.2 and 2.0 mg of Croneton per kg rat
weight can be seen to be slight or unapparent. With increasing dose,
toxicity was moderate at 20 mg/kg ranging to severe at 100 mg/kg. The
time of functional recovery, according to the subjective interpretation
of this observer, was also considerably lengthened at the high dose. In
each case symptoms were typical of those seen in situations of animal
poisoning with anticholinesterase agents.
With increased dose, the relative rate of hydrolysis in terms of the
quantity administered (relative hydrolysis) was observed to decrease dur-
ing the first 12 hr. (It should be made clear at this point that the
absolute or molar rates were obviously increased with increased dosage.
However this discussion centers on rates relative to the quantity to be
detoxified in order to examine the animals effective ability to maintain
or return to homeostasis.) This decrease in relative hydrolysis may be
examined in its different forms according to the method utilized in pre-
sentation of the data. Table 38, in which is tabulated the mean propor-
tion hydrolyzed per time interval at the various doses, indicates a de-
crease in the percentage of dose undergoing hydrolysis in a given time
with increased quantities. In addition, relative peak rates occur later
with increased administration. These effects combine to produce a
flattened ^CC^ cumulative elimination curve at higher doses when the
data tabulated in Table 39 are plotted with time. This flattenting of
elimination in terms of dose occurs to the extent that hydrolysis pro-
ceeds almost linearly (about 1.5 yM/m) between 4 and 16 hr after treat-
ment in animals receiving 100 mg/kg. Thus, peak quantities of approxi-
mately 3.1 x 10~6 moles at hour 10, 9.6 x 10~7 moles at hour 8, 2.1 x
132
-------
Table 37. RELATIVE SYMPTOMS APPARENT IN RATS GIVEN VARIOUS
DOSES OF [1^C-CARBONYL]-CRONETON
Dose (mg/kg) Symptoms
100 Extensive tremors, ataxia, and prostration for several
hours; excessive salivation to the extent that the
entire underside of the animal is wet; eyes wide and
bulging, exuding reddish tears several hours after dos-
ing; death in 1 of 4 animals dosed, those surviving
recovered relatively normal feeding and grooming habits
by 12 hours after dosing.
20 Moderate tremors and ataxia; little or slight saliva-
tion to the extent that the chin was wet, ceasing with-
in 3 hours; recovery of normal mannerisms 3-6 hours
after dosing.
2.0 Very slight tremors; no overt excessive salivation;
otherwise asymptomatic; minimal disturbance in behavior
patterns.
0.2 No apparent symptoms; animals maintain normal explora-
tory, feeding, and grooming habits throughout interval.
133
-------
Table 38. MEAN ELIMINATION FROM RATS OF 14C02 PER TIME INTERVAL
AS A FUNCTION OF DOSE OF [14C-CARBONYL]-CRONETON
Percent of dose eliminated per
b
Hour
2
4
6
8
10
12
14
16
18
20
22
24
48
72
0.
7.
13.
10.
6.
3.
2.
1.
0.
0.
0.
0.
0.
2.
1.
2 mg/kg
5 ± 1.
7 + 1.
8 + 1.
2 ± 0.
9 ± 0.
0 + 0.
2 ± 0.
8 ± 0.
6 ± 0.
5 ± 0.
4 ± 0.
4 ± 0.
9 ± 0.
2 ± 0.
0
4
6
4
08
06
09
03
06
09
0
0
3
3
2
5.
9.
7.
5.
2.
2.
1.
0.
0.
0.
0.
0.
2.
0.
mg/kg
9 + 0.
3 + 0.
2 ± 0.
1 ± 0.
6 ± 0.
1 ± 0.
1 ± 0.
7 ± 0.
6 ± 0.
4 + 0.
4-0.
3 + 0.
1 ± 1.
9 ± 0.
7
5
3
7
4
3
2
1
2
2
2
1
1
4
interval as 14CO2a
20 mg/kg
1.
3.
3.
4.
3.
2.
1.
0.
0.
0.
0.
0.
3.
1.
4 ± 0.
3 ± 0.
9 ± 0.
3 ± 0.
4 ± 0.
2 ± 0.
4 ± 0.
9 ± 0.
7 ± 0.
5 ± 0.
5 ± 0.
5 ± 0.
2 ± 1.
5 + 0.
3
7
8
5
4
1
3
2
2
2
2
2
0
4
100 mg/kg
0.6
1.8
2.3
2.3
2.8
2.6
2.6
2.3
2.0
1.6
1.2
1.0
4.4
1.9
± 0.07
± 0.1
± 0.2
± 0.3
± 0.7
± 0.5
± 0.5
± 0.6
± 0.4
± 0.2
+ 0.03
± 0.03
± 0.6
± 0.3
Mean values ± standard error for 3 rats per dose.
Time between dosing and end of 2 or 24 hour interval.
134
-------
Table 39. MEAN CUMULATIVE ELIMINATION OF 1UCO2 FROM RATS
ADMINISTERED VARYING DOSES OF [1^C-CARBONYL]-CRONETON
Cumulative percent of
Hour
2
4
6
8
10
12
14
16
18
20
22
24
48
72
0.
7.
21.
32.
38.
42.
44.
45.
46.
46.
47.
47.
47.
50.
52.
2 mg/kg
5 ±
2 ±
0 ±
2 ±
0 +
1 ±
3 ±
1 ±
7 ±
2 ±
5 ±
9 ±
8 ±
0 ±
1.0
2.4
4.0
4.4
4.4
4.4
4.3
4.3
4.2
4.2
4.2
4.1
3.9
3.8
2
5.
15.
22.
28.
30.
32.
34.
34.
35.
35.
36.
36.
38.
39.
mg/kg
9 ±
2 ±
4 ±
2 ±
8 +
9 ±
1 ±
8 ±
4 ±
8 ±
2 ±
5 ±
6 ±
7 ±
0.7
1.1
1.3
1.0
1.1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
2.7
3.2
dose as 1LfCO? per dose
20
1.
4.
8.
12.
16.
18.
20.
21.
21.
22.
22.
23.
26.
27.
mg/kg
4 ± 0
7 ± 1
6 ± 1
9 ± 2
3 ± 2
5 ± 2
0 ± 2
0 ± 2
6 ± 2
1 ± 2
7 ± 2
2 ± 2
3 ± 2
8 ± 2
.3
.0
.8
.3
.6
.5
.4
.2
.2
.1
.2
.3
.6
.8
100
0.
2.
4.
7.
9.
12.
14.
16.
18.
20.
21.
22,
27.
29.
mg/kg
6 ±
4 ±
7 ±
0 ±
5 ±
0 ±
5 ±
9 +
8 ±
5 ±
9 ±
8 ±
1 ±
0 ±
0.05
0.2
0.3
0.4
0.6
0.9
1.4
1.9
2.3
2.5
2.5
2.5
2.3
2.4
Mean values ± standard error for n=3 rats per dosage level.
Time after single oral dose.
135
-------
10"7 moles at hour 4, and 3.0 x 10~8 moles at hour 4 were hydrolyzed
per 2 hr interval by animals receiving 100, 20, 2 or 0.2 mg/kg- These
data suggest some degree of saturation at high doses of the mechanisms
responsible for hydrolysis and respiratory elimination.
Further evidence of the effect of increased dose on relative hydrolysis
can be observed with the increase in the mean a phase half-time
of hydrolysis as shown in Table 40. These half-times of occurring
hydrolysis range from about 4 hours with doses of 0.2 and 2 mg/kg to an
extreme of 14.5 hr with doses of 100 mg/kg. An analysis of variance
performed with data from the 12 nonatropinized rats indicated a highly
significant effect (p<.001) due to dose. Further comparisons using the
least significant difference (L.S.D.) test among the dose means (Sokal
and Rohlf 1969) indicated no statistical difference at p=.05 level for
doses of 0.2, 2, and 20 mg/kg. In contrast, the mean half-time of oc-
curring hydrolysis in animals dosed with 100 mg/kg differed to a highly
significant degree (p<.001) when compared with mean half-times occurring
after lower doses. No influence on these ti/2 values could be observed
following pretreatment of rats with atropine.
The increase in half-time of the a-phase of occurring hydrolysis cor-
relates well with the appearance and time course of overt symptoms of
poisoning. At doses of 0.2 and 2 mg/kg, a-phase half-times are very
similar (about 4 hr) and symptoms are minor or unapparent. With an in-
crease of the dose to 20 mg/kg, this mean half time exhibits an apparent
(though not statistically significant) increase and cholinergic symptoms
become more prominent. The symptoms and half-times of the individual
rats within the 20 mg/kg dosage and provides further correlation. Two
of these rats, from which individual a-phase half-times of 4.3 and 6.0
hr were calculated, exhibited tremors and ataxis, but did not salivate
excessively. The third animal dosed at 20 mg/kg was ataxic, exhibited
tremors, and had salivated excessively to the point that the fur around
the face was wet. These symptoms continued for about 3 hr after dosing.
The a-phase of hydrolysis half-time was calculated to be 9.3 hr. At the
136
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Table 40. HALF TIMES OF HYDROLYSIS (lkC02 EXCRETION) FROM RATS
ADMINISTERED VARYING ORAL DOSES OF [1V-CARBONYL]-CRONETONa
Dose (mg/kg)
0.2
0.2 Ab
2.0
20
100
100 A
Values expressed are
per group.
b
a
3.
4.
4.
6.
14.
14.
the mean ±
ti/2 ±
phase
7 ± 0.3
0
0 ± 0.5
5 ± 1.5
5 ± 0.7
2
standard
S.E. (hr)
3 phase
12.8 ± 1.0
9.5
10.9 ± 2.2
14.0 ± 0.6
8.5 ± 1.4
11.0
error calculated from 3 rats
Doses labeled A represent single animals receiving in addition to the
dose 50 mg/kg atropine sulfate i.p.
Approximate duration of 12 hr following dose.
137
-------
100 mg/kg level, the animals exhibited much more pronounced symptoms
(Table 37) and the calculated mean half-time was 14.5 hr. Symptoms per-
sisted considerably longer, also. Thus, the a-phase of hydrolysis tj/2
may be a measurable correlate of the intensity and duration of poisoning
by methylcarbamate anticholinesterases. Certainly, knowing the time
course of such poisoning in which recovery or death occurs within the
first few hours, the a-phase duration (0-12 hr post treatment) coin-
cides temporally while the g-phase (14-24 hr post treatment) occurs
later. Too, little or no dose correlation can be seen with the latter
phase t1//2 (Table 40) which fluctuates.
Recovery of the dose during the 3-day study was excellent at levels of
0.2, 2, and 20 mg/kg as indicated in Table 41. Urinary and respiratory
elimination of the radiocarbon accounted for greater than 90% of the
administered doses. At the 100 mg/kg level, however, recovery was con-
sistently poorer and averaged about 62% of the dose. Examination of the
percentage of dose collected during the period 48-72 hr after dosing re-
vealed that substantial elimination was still occurring.
Significantly less respiratory elimination (and thus hydrolysis) occurs
as the dose is increased from 0.2 to 20 mg/kg (L.S.D. tests between
means, p<.05). Increasing the dose to 100 mg/kg did not significantly
alter the proportion of the dose hydrolyzed. This trend of decreasing
proportion hydrolyzed with increasing dose is manifest during the
first 24 hr of the experiment. During this time, differences in
the percent of dose excreted in urine are apparent also. Urinary
excretion increases with the dose from 0.2 to 2 mg/kg. This might
be interpreted as urinary compensation for overload of the respiratory
route. With further increases in the dose to 20 mg/kg, urinary
radiocarbon is maintained at about 70% of the dose while respiratory
elimination declines. At the high dose, however, the percentage of uri-
nary excretion has been reduced drastically while the respiratory pro-
portion is maintained at about 25%, and substantially reduced total
138
-------
Table 41. ELIMINATION OF RADIOCARBON FROM RATS ADMINISTERED
VARYING DOSES OF *^C-CARBONYL-CRONETON
Excretory route and
hr after treatment
C-Carbon dioxide
6
12
18
24
48
72
Urine
12
18
24
48
72
Feces
72
Total
72
Cumulative
0.
32.
43.
46.
47.
49.
52.
36.
41.
44.
47.
48.
4.
104.
2
0
9
6
9
9
0
9
8
8
3
0
9
9
2.
22.
32.
35.
36.
38.
39.
50.
62.
65.
66.
68.
1.
109.
% of dose /mg/kg dose
0
4
9
4
5
6
5
8
5
2
9
1
8
4
20
8.
18.
21.
23.
26.
27.
56.
63.
60.
63.
67.
6.
101.
6
5
6
2
3
8
9
3
7
3
1
9
8
100
4.
12.
18.
22.
27.
29.
6.
14.
17.
23.
26.
6.
61.
7
0
9
8
1
0
1
3
4
2
0
5
5
Values represent mean of results obtained from 3 animals per dose.
139
-------
recovery occurs.
Comparison of the total 1I+C excretion of atropinized rats (Table 42) in-
cludes a slightly slowed urinary elimination in the rat given 0.2 rag/kg.
This was apparently due to decreased urine flow from the animal. In
contrast, the atropinized rat given 100 mg/kg exhibited substantially
increased urine flow (>20 ml/24 hr compared to 3-15 ml/24 hr in rats
treated with 100 mg/kg Croneton only). This apparently enabled greater
urinary excretion of radiocarbon (44% vs a mean of 26% in 72 hr) and a
substantially more complete recovery of the radioactivity. These ob-
servations raise questions concerning the interaction in vivo of varying
quantities of anticholinesterase and anticholinergic compounds on urine
formation, and the possibility of using diuretic agents to promote ex-
cretion of a methylcarbamate such as Croneton which is known to form a
relatively water soluble toxic metabolite (Croneton sulfoxide).
From the partitioning characteristics of the urinary radiocarbon (Table
43), information is available regarding the time course of the metabolic
processes responsible for production of water soluble metabolites. In
the case of Croneton, the formation of hydrophilic metabolites is appar-
ently slow relative to hydrolysis as indicated by the small proportion
of the early 14C excretion which is water soluble. This water soluble
fraction of excretion per interval appears to increase with time. How-
ever if the decreased percentage of the dose excreted in each succeeding
interval is considered, it can be seen that organosoluble materials de-
cline dramatically while excretion of hydrophilic material is more near-
ly constant.
As a brief recall of the salient trends discussed, hydrolysis expressed
as a function of the dose of Croneton is slowed and diminished with in-
creasing dosage. The degree of slowing is quite pronounced at high
doses and is reflected in the increased half-time of * CC>2 elimination.
Such increases in half-time correlate well with observed toxicity as
manifest by excessive cholinergic stimulation. (Salivation serves as a
140
-------
Table 42. CUMULATIVE ELIMINATION OF RADIOCARBON FROM RATS
ADMINISTERED [*4C-CARBONYL]-CRONETON AFTER RECEIVING
50 MG/KG ATROPINE SULFATE I.P.
Dose (mg/kg) Hour
0.2 6
12
18
24
48
72
100 6
12
18
24
48
72
Cumulative
CO2
28.6
42.9
47.2
48.7
51.7
42.9
5.5
12.5
18.5
21.6
27.3
29.6
percent
Urine
-
1.2
35.4
36.5
39.1
40.0
-
23.2
31.7
34.7
42.2
44.0
of dose excreted per animal
Feces Total
28.6
44.1
82.6
85.2
90.8
7.3 100.2
5.5
35.7
50.2
56.3
69.5
9.9 83.5
141
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Table 43. PARTITIONING CHARACTERISTICS OF URINARY RADIOCARBON
FROM RATS AS A FUNCTION OF TIME AND DOSE OF [14C-CARBONYL]-CRONETON
Percent distribution between phases
Hour Phase0 0.2 mg/kg 2.0 mg/kg 20 mg/kg 100 ing/kg
12 0 87.4 ± 2.2 88.0 ± 3.0 79.2 ± 8.6 67.7 + 12.7
w 12.6 12.0 20.8 32.3
(%)d 36.9 ± 1.1 38.2 ± 13.2 47.0 ± 9.7 6.1 ± 3.1
18 0 68.0 ± 4.5 62.4 ± 14.3 61.5 ± 5.0 32.2 ± 4.3
w 32.0 37.6 38.5 67.8
(%) 4.9 ± 0.4 11.7 ± 3.5 3.4 ± 0.3 8.2 ± 2.0
24 0 49.4 ± 11.6 37.9 ± 9.6 45.4 ± 11.5 14.6 ± 1.0
w 50.6 62.1 54.6 85.4
(%) 2.9 ± 1.3 2.7 ± 1.4 4.1 ± 2.6 3.1 ± 0.8
48 0 22.6 ± 6.9 13.7 ± 2.1 20.8 ± 3.0 9.4 ± 2.9
w 77.4 86.3 79.2 90.6
(%) 2.6 ± 0.4 1.7 ± 0.3 2.9 ± 1.4 5.9 ± 1.2
72 0 10.3 ± 1.4 11.1 ± 3.5 12.2 ± 4.8 8.8 ± 3.8
w 89.7 88.9 87.8 91.2
(%) 0.7 ± 0.06 1.2 ± 0.5 0.9 ± 0.2 2.8 ± 0.7
Mean ± standard error for 3 rats per dosage level.
Time of urine collection after single oral dose.
£
W - saline, 0 - acetonitrile-chloroform phase.
Mean ± standard error (n=3) percent of dose represented in total sample.
142
-------
good indicator.) Diminished relative hydrolysis of Croneton with in-
creased doses below levels producing apparent poisoning symptoms (0.2-
2 rag/kg) appears to result in increased (compensatory) urinary elimina-
tion. With further increases in the dose to levels producing overt
toxicity (20-100 mg/kg), relative hydrolysis diminishes to an apparent
asymptote of about 25% of the dose and urinary compensation becomes in-
creasingly less effective. Consequently at high doses, 3-day recovery
of the administered radioactivity ceases to be quantitative. Adminis-
tration of moderate doses of atropine sulfate does not alter hydrolysis,
but may affect urinary excretion by possible influences on urine flow.
143
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FATE OF 1-NAPHTHYL N-HYDROXYMETHYLCARBAMATE IN RATS
A major mode of metabolism of foreign substances in mammals is oxida-
tion. In the case of aryl methylcarbamates, both N-methyl and nuclear
hydroxylation are known to occur. Many studies have indicated that
N-hydroxymethyl carbaryl is an important metabolite in the metabolism
of carbaryl in both plants and animals (Kuhr and Dorough, 1976). For
this reason, the metabolism of N-hydroxymethylcarbaryl in rats was
examined in order to estimate the fate of this material when ingested
as residues in the diet of man.
Materials and Methods
Chemicals - l-Naphthyl-l-^C carbaryl (15.2 mCi/mMol) , carbonyl-ll*C
carbaryl (5.38 mCi/mMol), and metabolites, 5,6-dihydro-5,6-dihydroxy
carbaryl, N-hydroxymethylcarbaryl, desmethyl carbaryl, and 1-naphthol
were obtained from stocks maintained in our laboratory.
Chromatography - The purity of the llfC-labeled and authentic compounds
was determined by thin-layer chromatography. The ether extractable
metabolites were separated on tic (Brinkman precoated silica gel 60-F-
0254, 250 x 250 mm2) using the following solvent systems: A - Ether-
hexane, 4:1 (v/v); B - Ether-hexane, 9:1 (v/v); C - Chloroform-
acetonitrile, 2:1 (v/v).
Biosynthesis of l-naphthyl-l-llfC N-hydroxymethylcarbaryl and carbonyl-
If*C N-hydroxymethylcarbaryl were obtained from in^ vitro metabolism of
1 Coring and carbonyl-ll*C labeled carbaryl by rat liver microsomes. &
ten gram sample of fresh rat liver was obtained from mature male albino
rats (Cox-SD, Laboratory Supply Company) weighing approximately 200 g
each. The liver was homogenized with a Teflon pestle in a glass homo-
genizer containing 40 ml of 0.1 M phosphate buffer solution (pH 7.4).
The homogenate was centrifuged at 15,000g for 15 min and the 15,000g
supernatant fraction centrifuged at 105,000g for 1 hr to obtain the
144
-------
microsomal fraction.
In in vitro experiments l-naphthyl-^C carbaryl or carbonyl-1 "*C
carbaryl was deposited on the surface of the reaction vessel by
evaporation of a benzene solution. Then, 3.0 ml of the microsomal
fraction were added and mixture shaken for 30 min at 37°C with NADPH
(1.115 mg), magnesium chloride (6.41 rag), methyl nicotinamide (6.34
mg), glucose-6-phosphate (3.92 mg), glucose-6-phosphate dehydrogenase
(2 units), and glutathione (1 mg) in a total volume of 4.0 ml. Control
incubations were identical except that the liver enzymes were initially
heated for 30 min at 90°C.
Analysis of incubation mixture - Sodium chloride (^2 g) was added to
each incubation mixture and extracted with ether to remove carbaryl
and the remaining unconjugated materials. The ether layer was con-
centrated to a volume which allowed direct application to silica gel
thin-layer plates. Solvent systems A and B were used to resolve the
radioactive components of the ether extract.
Detection of the radioactive zones on tic plates was accomplished by
means of autoradiography. Extraction of each component from the plates
was by removal of the gel, sonication in acetone and then concentrating
the acetone solution. Identification of each component was based on
its tic behavior and confirmed by mass spectral analysis.
In some of the experiments, rat liver homogenates were obtained from
rats which had been given intraperitoneal injections of sodium pento-
barbital (40 mg/kg) daily for 4 days in order to induce high levels of
drug-metabolizing enzymes.
Stability of N-hydroxymethylcarbaryl - The stability of N-hydroxy-
methylcarbaryl during various experimental procedures used for
evaluating the metabolism of the carbaryl in animals was determined.
Because many of the procedures described herein could also cause
145
-------
1 4
degradation, this aspect was considered using ring C-labeled N-
hydroxymethylcarbaryl obtained from the in vitro study. The compound
was subjected to 1 N HC1, pH 5.8 buffer and neutral conditions for
24 hr. The degree of stability was determined by the amount of
degradation products revealed utilizing tic and autoradiography.
Treatment - Albino rats (Cox-SD, Laboratory Supply Co.) weighing
approximately 200 g each were used in all metabolism experiments. In
1 4
the single oral dose studies, female animals were treated with C-
i "*
ring and C-carbonyl-labeled carbaryl and N-hydroxymethylcarbaryl at
a dosage level of 0.5 mg/kg. The specific activity of the compounds
was adjusted to 6 mCi/mMol and delivered to the animals in 0.5 ml of
30% Tween 80 via a feeding needle. Two rats were treated so that all
data collected represented the results obtained from these two rats.
Sample collection and radioassay - Following treatment, the animals
were placed in metabolism cages allowing for separation of urine and
14
feces. Those treated with the C-carbonyl-labeled materials were
placed in cages modified for collection of exhaled gases. Urine
samples were radioassayed by direct scintillation counting. Feces
samples were collected at 48 hr after end of treatment and radioassay-
ed by combustion using a Packard Model 306 Sample oxidizer. The
14
COa in the exhaled gases of rats were collected by drawing air from
the cages through 100 ml of 1.0 N potassium hydroxide. This system
was tested before the experiment using C-sodium bicarbonate and
found to collect 95+% of the carbon dioxide. Samples were taken for
radioassay at hourly intervals for 4 hr post treatment, then at 2 hr
intervals for the next 10 hr, 3-hr intervals for the next 6 hr and at
different intervals for the remainder of the 48 hr experiment. Radio-
assay was accomplished by direct scintillation counting of 1.0 ml of
the trap solution.
Analysis of urinary radiocarbon - Urine was extracted with ether and
then lyophilized to dryness and the solid residue washed with methanol
146
-------
repeatedly until no radioactivity was detected in the wash. This
process was quantitative in the recovery of the urinary radiocarbon.
The methanol wash was evaporated to dryness, the solid residue disolv-
ed in 6 ml of distilled water and then extracted thoroughly with ether.
Portions of each of the water phases were incubated with 1 N HC1 at
40°C for 24 hr. The incubations were conducted in a sealed tube con-
taining 4 ml of ether. Periodic shaking of the tubes extracted the
hydrolyzed exocons (or non-polar products) into the ether layer to
prevent further decomposition. The ether extracts were combined and
analyzed by tic.
Enzyme preparations also were utilized to cleave possible conjugated
water-soluble metabolites. The enzymes 3-glucuronidase (bacterial,
powder) and arysulfatase (powder) were purchased from Sigma Ltd.
(St. Louis, Mo.). 3-Glucuronidase hydrolysis of the conjugates (1.0
ml) was conducted in 0.1 M phosphate buffer pH 6.2 (2 ml) containing
10 units of the enzyme. The reaction mixture was incubated for 24 hr
at 37°C. Arylsulfatase hydrolysis was effected by incubation of
arylsulfatase (10 units) with 1 ml of the conjugates and 0.1 M sodium
acetate-acetic buffer, pH 5.2 (2 ml), for 24 hr at 37°C.
Results and Discussion
In vivo metabolism of carbaryl - Degradation studies of carbaryl using
subcellular liver preparation have played a major role in determining
the metabolic pathway of this carbamate in animals. The results from
this investigation showed that N-hydroxymethylcarbaryl and 5,6-dihydro-
5,6-dihydroxycarbaryl were the major metabolites, 27 and 14% of total,
found in the incubation mixture (Table 44). Almost identical amounts
of these two metabolites were found using carbonyl-^C carbaryl as the
substrate. The microsomal fraction from rats which received sodium
pentobarbital metabolized about 15% more of the carbaryl and this was
reflected primarily as increased quantities of N-hydroxymethylcarbaryl
147
-------
Table 44. RADIOACTIVE COMPONENTS RESULTING FROM THE METABOLISM OF
l-NAPHTHYL-l-^C CARBARYL BY RAT LIVER MICROSOMES.
Metabolite
Unknown origin material
5 , 6-Dihydro-5 , 6-dihydroxy 1-naphthyl
N-methylcarbamate
1-Naphthyl N-hydroxymethylcarbamate
Carbaryl
Water-soluble metabolites
Total 1£|C recovery
% lkC in
a
Untreated
4.7
14.4
27.3
35.7
10.1
92.2
sample
b
Treated
5.1
20.1
37.4
20.1
11.4
94.1
Microsomal fraction obtained from rats which did not receive sodium
pentobarbital treatments.
Microsomal fraction obtained from rat treated with sodium pento-
barbital.
148
-------
and 5,6-dihydro-5,6-dihydroxy carbaryl.
Stability of N-hydroxymethylcarbaryl - When ring-1'*C-labeled N-hy-
droxymethylcarbaryl was incubated with 1 N HC1 for 24 hr at 37°C, 3
radioactive materials were produced (Table 45) Two radioactive
materials were identified as desmethyl carbaryl (19%) and 1-naphthol
(3.7%). The other material remained at the tic origin and accounted
for only 2% of the total radiocarbon. In acetate buffer solution
(pH 5.2), N-hydroxymethylcarbaryl was more stable than in 1 N HC1
when incubated under identical conditions. However, 6% desmethyl
carbaryl was produced under these conditions. In water, only a small
amount (1.2%) of desmethyl carbaryl was formed. In all of the stabili-
ty studies, desmethyl carbaryl was the major decomposition product.
This information makes it clear that desmethyl carbaryl would be
produced if the hydroxymethyl metabolite was subjected to acid
conditions normally used to cleave conjugated materials.
Excretion - The recovery of radioactivity from rats treated with
C-ring-labeled carbaryl and N-hydroxymethylcarbaryl (orally, 20
mg/kg) is presented in Table 46. Total lV-recovery after 48 hr from
rats dosed with 14C-ring-labeled carbaryl was 88%, with 82% in the
urine and 6% in the feces. For * Boring-labeled N-hydroxymethylcarba-
ryl, total 1J|C recovery after 48 hr was 86%; 72% was in the urine and
13% in the feces (Table 46). The amount of elimination of urinary
metabolites in the carbaryl- and N-hydroxymethylcarbaryl-treated rats
was distinctly different during the first 10 hr. Approximately 38%
as compared to 64% was excreted in the urine of rats dosed with carba-
ryl and N-hydroxymethylcarbaryl, respectively. The difference in
C excretion in the urine during the first 10 hr after treatment
probably resulted from the hydroxy group on the methyl group of the
metabolite. This hydroxy group would likely form conjugates very
rapidly and, thereby, facilitate the excretion of N-hydroxymethyl
carbaryl.
149
-------
Table 45 . THE CHEMICAL STABILITY OF RING-l "*C-N-HYDROXYMETHYLCARBARYL
STORED UNDER DIFFERENT CONDITIONS FOR 24 HR AT 37°C.
% of radiocarbon as indicated product
pH 5.2 pH 7.0
Products 1 N HC1 acetate buffer water
Unknown origin product 2.1 1.0 0.0
N-Hydroxymethyl carbaryl 70.6 91.3 97.7
Desmethyl carbaryl 19.4 6.0 1.2
1-Naphthol 3.7 0.0 0.0
Total 95.8 98.3 98.9
150
-------
Table 46. URINE AND FECAL ELIMINATION OF RADIOCARBON BY RATS
TREATED WITH l^C-RING-LABELED CARBARYL OR N-HYDROXYMETHYLCARBARYL.
Cumulative % of
Substrate
and
hours
Urine
5
10
20
30
48
Feces
48
Total
48
Carbaryl
A
18.
35.
75.
81.
83.
6.
90.
B
8
3
3
1
9
4
3
20
39
72
82
82
5
87
.0
.4
.1
.0
.1
.7
.8
Avg
19.
37.
78.
81.
83.
6.
89.
4
4
7
6
0
0
0
dose
N-Hydroxymethylcarbaryl
C
47.
65.
70.
71.
74.
14.
88.
7
7
6
4
2
2
4
D
50.
63.
69.
70.
72.
11.
83.
8
5
9
7
1
2
3
Avg
49.
64.
70.
71.
73.
12.
85.
2
6
2
0
2
7
9
Animals A and B treated orally with 20 mg/kg carbaryl (1.1 x 106 dpm)
while C and D were treated with N-hydroxymethylcarbaryl at the same
dose (1.5 x 106 dpm).
151
-------
Administration of l^C-carbonyl-labeled carbaryl and N-hydroxymethyl-
carbaryl to rats as a single oral dose resulted in rapid elimination
of the radiocarbon as carbon dioxide (Table 47). An average of 26%
of the dose was exhaled as COz in rats treated with 1^C-carbonyl-
labeled carbaryl, as compared to 53% of the dose in rats treated with
ll*C-carbonyl-labeled N-hydroxymethylcarbaryl. This difference
occurred predominantly within the first 20 hr after treatment, and
demonstrated that the hydroxyl group on the N-methyl portion of
carbaryl increased its susceptibility to hydrolytic attack.
Elimination of urinary radiocarbon in rats dosed with carbonyl-ai*C-
labeled carbaryl and N-hydroxymethylcarbaryl was 60% and 30% of the
doses, respectively (Table 48). The difference occurred mainly within
the first 20 hr after treatment. This difference resulted from the
comparatively more hydrolytically stable carbaryl which reduced the
loss of the carbonyl carbon as carbon dioxide. When all routes of
elimination were considered (Table 49), there was no difference
between carbaryl and hydroxymethylcarbaryl. In each case, 90% of the
orally administered doses was eliminated from the body after 48 hr.
Analysis of urine - Ether extraction of the urine removed less than
1% of the 1!*C materials. Therefore, no further analyses were attempted
on this phase. Because most of the 1'*C-residues remained in the urine
after extraction with ether, emphasis was placed on the identification
of the water-soluble metabolites. Table 50 shows the results of the
conversion of water-soluble materials in the urine of rats dosed with
1'*C-ring- and 14C-carbonyl-labeled N-hydroxymethylcarbaryl to ether-
soluble materials by enzyme and acid hydrolysis.
3-Glucuronidase and sulfatase treatments of the polar metabolites re-
leased 25% and 4% ether-soluble materials, respectively, from the urine
of rats dosed with ring- and 14C=O N-hydroxymethylcarbaryl. The
exocon released from the urine of rats treated with carbonyl-1 C
N-hydroxymethylcarbaryl by glucuronidase was all N-hydroxymethyl-
152
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lit
Table 47. RECOVERY OF 14CO2 FROM RATS TREATED ORALLY (20 mg/kg) WITH
CARBONYL-lV-CARBARYL OR CARBONYL-l^C-N-HYDROXYMETHYLCARBARYL.
Cumulative % of orally administered
Carbaryl
Hours
1
2
3
4
6
8
10
12
14
17
19
22
26
32
48
A
0.5
1.5
1.6
2.9
7.0
11.1
13.8
17.2
19.4
21.8
22.5
23.9
25.0
25.6
26.4
B
1.1
2.1
3.5
5.0
8.2
12.1
14,6
17;8
19.6
21.7
22.6
23.7
24.5
25.0
25.6
Average
0.8
1.8
2.5
3.9
7.6
11.6
14.2
17.5
19.5
21.8
22.5
23.8
24.7
25.3
26.0
dose
Hydroxymethylcarbaryl
C
4.3
10.8
14.6
17.5
21.3
25.8
29.5
36.5
40.0
47.1
49.0
51.0
51.1
51.7
51.9
D
4.4
8.6
11.3
14.1
17.8
22.5
27.6
37.0
41.7
45.5
50.0
53.5
53.6
53.9
54.1
Average
4.4
9.7
12.9
15.8
19.6
24.1
28.6
36.7
40.8
46.3
49.0
52.2
52.3
52.8
53.0
A and B rats were dosed with carbonyl-1'*C carbaryl (2,375,250 dpm
each), while C and D rats received carbonyl-l **C N-hydroxymethyl-
carbaryl (1,253,040 dpm each).
153
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Table 48. EXCRETION OF 1^C IN THE URINE OF RATS TREATED ORALLY
1 *,
(20 MG/KG) WITH CARBONYL- 4C CARBARYL OR
CARBONYL- l "* C N-HYDROXYMETHYLCARBARYL.
Cumulative '
% of orally administered dose
of rat
Carbaryl Hydroxymethylcarbaryl
Hours
5
10
14
19
30
48
Table
A B
15.0 15.2
27.3 24.5
38.2 36.2
47.9 45.8
60.1 56.3
61.2 58.4
49. ELIMINATION OF
Average C D
15.1 6.8 5.8
25.9 13.2 10.9
37.2 16.5 18.5
46.8 20.2 26.2
58.2 26.4 29.6
59.8 28.8 31.2
Average
6.3
12.1
17.5
23.2
28.0
30.0
RADIOACTIVITY FROM RATS TREATED ORALLY
(20 MG/KG) WITH CARBONYL- 1 4C CARBARYL OR
CARBONYL- l k
C N-HYDROXYMETHYLCARBARYL.
a
Animal
A
B
C
D
Cumulative
"coz
26.4
25.6
51.9
54.1
% of orally administered dose
Urine Feces
59.9 4.3
59.7 3.9
28.8 7.6
31.2 6.5
after 48 hr
Total
90.6
89.2
88.3
91.6
A and B rats dosed with carbonyl-1 "*C carbaryl (2,375,250 dpm each),
while C and D rats received carbonyl-14C N-hydroxymethylcarbaryl
(1,253,040 dpm each).
154
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Table 50 . ATTEMPTED CLEAVAGE OF WATER SOLUBLE METABOLITES IN URINE OF
RATS TREATED ORALLY WITH l CORING- OR l ^C-CARBONYL-LABELED
N-HYDROXYMETHYLCARBARYL
% distribution after treatment/label
* "c-Carbonyl
Treatment
Glucuronidase
Sulfatase
0.5 N HC1
1.0 N HC1
Ether
25.1
4.1
63.5
74.2
Water
73.2
94.5
31.8
19.1
Total
98.3
98.6
95.3
93.3
Ether
8.6
1.1
20.0
30.4
Water
90.2
97.0
61.9
51.9
Total
98.8
98.1
81.9
82.3
155
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carbaryl.
Acid hydrolysis of the urinary metabolites from rats dosed with ring-
1"*C hydroxymethylcarbaryl yielded exocons which were recognized as
1-naphthol (97%), and a small amount of N-hydroxymethylcarbaryl (2%)
and desmethyl carbaryl (1%). Very small quantities, about 5%, of
N-hydroxymethyl and desmethylcarbaryl were detected in the ether frac-
tion after acid hydrolysis of urine from rats treated with carbonyl-
14C N-hydroxymethylcarbaryl. The remainder of the radioactivity in
the ether, up to 25% of the total 1I+C in the sample, was lost during
evaporation and/or tic analysis. Since this loss was not apparent
with the ring-labeled ether solubles, it is evident that the volatile
and/or labile carbonyl-labeled product did not contain the ring moiety.
These data suggest that the N-hydroxymethylcarbamate fragment of the
molecule formed upon hydrolysis does not degrade entirely to carbon
dioxide as does the carbamate group from carbaryl, but: to a compound
having sufficient stability and polarity for urinary elimination.
156
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BIOAVAILABILITY OF BOUND AND WATER-SOLUBLE CARBAMATE INSECTICIDE
RESIDUES
The presence of chemical residue in food and nonfood crops resulting
from the commercial use of pesticides is well documented (Pimentel,
1971). Most residue analysis methods utilize apolar solvents for ex-
traction and are designed to detect the parent compound and perhaps
one or two metabolites. The polar and bound products are largely over-
looked. However, countless plant metabolism studies with radiolabeled
compounds have revealed many examples where a considerable portion of
the plant pesticide residue burden is of a polar and bound nature
(Casida and Lykken 1969, Shumabukuro et al. 1971, Kuhr and Dorough 1976).
This is particularly applicable to such widely used chemical classes of
pesticides as the carbamates, thiocarbamates, and s-triazines. Recent-
ly, more interest has been directed toward evaluating the toxicological
significance of the bound and water-soluble residues, and the possibil-
ity that they might possess harmful biological activity has been recently
pointed out (Dorough 1976).
In order for the bound and polar metabolites to express any toxic action,
though, they must first be available for absorption by biological sys-
tems. Only a few studies exist in the literature which have examined
this parameter in mammals. The water-soluble metabolites of carbaryl
naphthyl-[ll*C]-treated bean plants were shown to be bioavailable after
oral administration to rats (Dorough and Wiggins, 1969). No investiga-
tion was carried out on the unextractable metabolites of carbaryl.
Paulson et al. (1975) concluded that the water-soluble products of pro-
fam phenyl-[11+C] from alfalfa plants were bioavailable when fed to rats,
and that the bound residue was not. The unavailability of the bound
metabolites was based on 86% of the dose being eliminated via the feces
in four days, however, the role of biliary excretion of metabolites in-
to the feces was not determined. Eighty-nine percent of a dose of
propanil phenyl-[ lt+C] unextractable metabolites from rice plants to
rats was eliminated via the feces and 11.5% was found in the urine
157
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(Sutherland 1976). Only .05% of a similar lkC dose to biliary fistu-
lated mice and dogs appeared in the bile, thus the bound residue of
propanil was deemed to be of ]ow toxicological concern.
A need exists for complete studies which include the role of biliary
excretion in determining the bioavailability of bound and polar resi-
dues of several chemical classes of pesticides. This paper reports the
results of such investigations in rats on the residues from bean plants
treated with 11+C-labeled preparations of a series of structurally di-
verse carbamate insecticides.
Throughout this text the terminology defined by Dorough (1976) at the
ACS sponsored symposium on bound and conjugated pesticide residues is
followed. "Water soluble metabolites" (WSM) and "conjugates" are used
interchangeably, as are "unextractable metabolites" (UM) and "bound
residue". "Endocon" refers to that portion of a conjugate derived from
an endogenous compound, and "exocon" to that portion derived from an
exogenous or foreign compound. "Bioavailable" means capable of being
absorbed from the gastrointestinal tract following oral administration
to animals.
Materials and Methods
Chemicals - The radiochemicals used in this study (Table 51) were car-
baryl ring-[14C] (19.7 mCi/mM), carbofuran ring-[14C] (2.85 mCi/mM),
Croneton ring-[14C] (7.05 mCi/mM), Croneton carbonyl-[14C] (5.88 mCi/
mM) , and aldicarb thiomethyl- [11+C] (2.03 mCi/mM) . Ring-[luC] carbo-
furan was purified by streaking a Brinkman precoated thin layer chro-
matography (tic) plate (silica gel 60-G-254) with the radioactive prep-
aration and developing it in 7:3 benzene:ether with a carbofuran stand-
ard. The parent band was scraped off and extracted from the silica gel
with acetone, utilizing an Artek sonic dismembrator. Aldicarb thio-
methyl- [lt+C] was purified on a microcolumn packed with Mallinckrodt
SilicAR cc-7 silica gel and eluted with 3:1 ether:acetone. Radiopu-
158
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Table 51. RADIOACTIVE INSECTICIDES USED IN THE TREATMENT OF
BEAN PLANTS
Chemical identity and
position of carbon-14
Designation
Sp. act.
(mCi/mMol)
1-Naphthyl- [
carbamate
methyl-
2,3-Dihydro-2,2-dimethyl-7-
benzo- [1^C]-furanyl N-meth-
ylcarbamate
2-Ethylthiomethylphenyl-
[11+C] N-methylcarbamate
2-Ethylthiomethylphenyl
N-methylcarbamate-carbonyl-
2-Methyl-2-(methyl-[ll+C]-
thio) propionaldehyde 0-
(methylcarbamoyl) oxime
carbary1 ring-[1^C]
carbo furan r ing-[1^C]
Croneton ring-[1^C]
Croneton carbonyl-[1LfC]
aldicarb thiomethyl- [ll+C]
19.70
2.85
7.05
5.88
2.03
159
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rity of aldicarb was then confirmed by tic with a standard using 6:3:2
ether:hexane:acetone as the solvent system. Radiopurity of carbaryl
ring [ll+C], Croneton ring and Croneton carbonyl [1LfC] was determined
by tic using the following solvent systems: carbaryl, 4:1 ether:
hexane and Croneton, 6:5 ace tone-.hexane.
The insecticide metabolite standards used for tic were obtained from
existing stocks in our laboratory. These standards were either syn-
thesized previously in our lab or obtained from the developer. Their
purity was checked utilizing the appropriate solvent systems outlined
above. 3-Glucosidase, B-glucuronidase Type V-A, and sulfatase Type III
were acquired from Sigma Chemical Company, and glusulase was purchased
from Endo Laboratories, Inc.
Animals - Albino rats (Cox-SD variety) weighing about 200 to 400 g
were obtained from the Laboratory Supply Co., Indianapolis, Indiana.
They were housed in Hoeltage suspended wire cages and given Purina Lab-
oratory Rat Chow and water ad libitum until used in the experimental
investigations.
Radioassay - A Packard scintillation spectrometer, model 3380 equipped
with a Packard 544 absolute activity analyzer, was used in quantifying
radioactivity. Direct scintillation counting was accomplished using
scintillation cocktail type 3a70B (Research Products International).
Other samples were combusted in a Packard Model 306 sample oxidizer;
the evolved lt4CO2 trapped with Carbosorb (Packard) , and counted after
dispensing in Permafluor 25X liquid scintillator.
Chromatography - Thin layer chromatography (tic) was carried out using
Brinkman precoated tic plates with a 250 micron thickness of silica
gel 60-F-254. All tic was performed using the appropriate standards
described in the chemicals section. Identity of carbofuran-[ C] me-
tabolites was made by developing the tic plates twodimensionally,
first in 7:3 benzene:ether, and second in 4:1 dichloromethane:acetoni-
160
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trile. Similarly, carbaryl-[14C] metabolites were analyzed by tic two
dimensionally in 4:1 ether:hexane and 2:1 chloroform:acetonitrile, or
single dimensionally in 4:1 etherrhexane. Aldicarb- and Croneton-
[14C] metabolites were analyzed single dimensionally by tic in 3:1
ether:acetone and 6:5 acetone:hexane, respectively. Radioactive spots
were located by radiography on Kodak X-ray film. Carbofuran, carba-
ryl, and Croneton standards were located by spraying the developed tic
plates with a 1% methanolic solution of p-nitrobenzenediazonium fluobo-
rate to detect phenols as yellow spots. The plates were then sprayed
with a 5% methanolic solution of KOH to hydrolyze the carbamate-type
standards, then sprayed again with the p-nitrobenzenediazonium fluobo-
rate to detect the resulting phenols as yellow spots. Aldicarb stand-
ards were visualized by spraying the tic plates with a 1% aqueous so-
lution of potassium permanganate.
Quantitation of the radioactive spots was carried out by scraping the
radioactive area of tic plates into a scintillation vial containing 2
ml of methanol, pulverizing the silica gel on a sonic dismembrator,
and analyzing directly by liquid scintillation counting.
Treatment of bean plants and collection of metabolites - Bean plants,
ten days old, were stem injected by puncturing a small hole with a nee-
dle at the base of the plant, then administering the 35-yl dose in a
second hole approximately 8 cm above the soil level with a Hamilton 50
yl syringe. Ten plants were treated with carbaryl ring [14C] (5.0 x
106 dpm per plant, 23 yg) and 10 with carbofuran ring [14C] (5.0 x 106
dpm per plant, 176 yg). Twenty plants were treated with Croneton car-
bonyl [11+C] (2.5 x 106 dpm, 43 yg) , 20 with Croneton ring [14C] (2.5 x
106 dpm, 36 yg) , and 20 with aldicarb thiomethyl [^C] (2.5 x 106 dpm,
106 yg). The plants treated with any one of the insecticides are re-
ferred to as a group. Some phytotoxiciy was noted in the carbofuran
group due to the acetone vehicle. All plants were maintained in a
greenhouse for 20 days after treatment in a peat moss:soil mixture con-
taining plant nutrients.
161
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Fifteen 10-day-old bean plants were stem injected with 500 yg of car-
baryl (100 yl of a 5 mg carbaryl/ml of 5:1 water-.acetone) which con-
tained a small amount of carbaryl ring [ll+C] as a tracer. The speci-
fic activity of the preparation was .00850 mCi/mM and each plant re-
ceived 46,500 dpm. Eight control plants were included in this study.
All plants were maintained in an environment chamber (10 hour light
cycle at a temperature of 27°C, and a 14 hour dark cycle at 18°C) for
20 days thereafter in a peat moss:soil mixture containing plant nutri-
ents.
At 20 days post-treatment, each plant was cut at the soil level, sealed
in a plastic bag, and stored in a freezer until extracted. For ex-
traction, the bean plants treated with each insecticide were divided
into three subgroups and extracted separately. This was done to de-
termine the reproducibility of the extraction procedure. One of the
subgroups from each treated group of plants was further divided into
epicotyl leaves, the remaining leaves, and stems and each of these
plant parts were extracted separately. This was done to determine the
distribution of radioactivity within the plants.
The plant material of each subgroup or plant part was cut into small
pieces and homogenized thoroughly in about 100 ml of acetone. This
was then filtered, re-extracted once with acetone, filtered, and re-
extracted with chloroform. The filtrates were combined in a separa-
tory funnel with 25 ml of water, and shaken thoroughly with 300 ml of
chloroform. After separation of the phases, the organic solvent layer
was drained into a clean separatory funnel and washed twice with 15 ml
of water. Samples of the organoextractable (containing free metabo-
lites) and water soluble fractions (containing WSM) were removed for
radioassay. The plant solids were then subjected to a Soxhlet extrac-
tion overnight (about 16 hours) with 150 ml of methanol. Prior to
radioassay of the Soxhlet extract, the solvent was centrifuged for 5
minutes to remove the particulate material. The amount of unextract-
able [1£tC] (UM) in the plant solids was determined by oxidizing about
162
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100 mg of the solids in a Packard Tri-Carb 306 combuster and assaying
the collected 14C02.
Sorghum plants were treated in the field by Chemagro Chemical Company
five times at 10-day intervals. Sampling occurred 14 days after the
last treatment. Ring-labeled C-Croneton was applied with an aerosol
spray apparatus as an emulsifiable liquid formulation in water with a
specific activity of 3.63 mCi/mmol. The application rate was eight
ounces Al/acre.
The sorghum leaves were each coarsely chopped in a Waring blender.
They were then weighed and homogenized at high speed for 5 min in a
Virtis 45 homogenizer containing a 100 ml mixture of 8:2 acetonitrile
and water. The homogenate was filtered under vacuum using Whatman
No. 2 filter paper. The solid residue was re-homogenized in the same
solvent mixture using a polytron homogenizer (Brinkman) and filtered.
Aliquots of both the organic and water layers were radioassayed by
liquid scintillation counting. The solids were dried, combusted in a
Packard Model 306 Oxidizer and radioassayed.
Treatment of rats and collection of radiocarbon - Female rats were
housed one animal per cage in Delmar metabolism cages which separate
urine and feces and allow for the collection of expired gases. Before
administration of WSM to rats, one animal was treated with the concen-
trated water solubles of five untreated 20-day-old bean plants, and
observed for 24 hours to determine if any lethal or sublethal toxicity
from this fraction existed. The rats were acclimated to the metabolism
cages and deprived of feed overnight, but given water ad libitum. The
WSM of carbaryl ring-[ C] (1.0 x 10° dpm, 4.6 yg) , carbofuran ring-
[14C] (1.0 x 106 dpm, 35.2 yg), Croneton ring-[14C] (1.0 x 106 dpm,
14.4 yg), Croneton carbonyl-[lkC] (1.1 x 105 dpm, 1.9 yg), and aldi-
carb thiomethyl-[lkC] (1-0 x 106 dpm, 42.4 yg) were concentrated by
lyophilizing to dryness and administered orally via a stomach tube in
an aqueous solution that did not exceed 2 ml.
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The UM of carbaryl ring-[ll|C] (1.53 x 106 dpm, 7.0 pg) and carbofuran
ring-[1I*C] (6.5 x 106 dpm, 22.9 yg) from bean plants were fed to rats
and the fate of the radiocarbon examined. Four rats were deprived of
feed for 18 hours. They were then acclimated to a feeding schedule of
2 grams of laboratory chow mixed with 0.5 grams of dried, non-treated
bean plant solids per day. This was done for 2 days. On the third
day, the respective UM were mixed into the 2 grams of feed for each
rat.
lt+CO2 was trapped in a 1.0 N KOH solution which was changed every 12
hours to prevent saturation. Samples of the KOH were taken every 6
hours and assayed for radiocarbon content by direct scintillation
counting. Urine was collected every 6 hours and 0.5 ml samples as-
sayed by direct counting. Feces were collected at each 6 hour inter-
val after treatment if they were available. Each fecal sample was
weighed, emaciated, and a 0.5 gram portion combusted for radioassay.
Cannulation of bile ducts was carried out by anesthetizing the rats
with ether, shaving the ventral mid-body, and making a midline abdom-
inal incision. The common bile duct was located, and after removing
the adhering tissue, a small incision was made in the duodenal portion
of the duct with iris scissors. A 12 in length of sterile polyethyl-
ene tubing (.038 O.D.) fit with a 25 G 1/2 in blunt needle was intro-
duced approximately 3 mm into the bile duct and secured by ligature.
A stab incision was made in the tail approximately 1 in from the base
with a 6 in 12 G stainless steel luer-lok needle fit with a stylus,
which was then thrust forward subcutaneously to the midline incision.
The bile duct cannula was then fed through the needle, and hence
emerging from the stab incision in the tail after removal of the needle.
The abdominal incision was then sutured, and the skin closed with Clay
Adams 9 mm stainless steel staples. These rats were then placed in
modified Hoeltoge plastic metabolism cages, which allowed for restrain-
ing of the animal by leading the tail through an enlarged hole in the
feeder. The tail was then secured in this position by affixing a No.
164
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7 stopper to the tail with Eastman 910 Adhesive and strapping adhesive
tape aroung the tail just caudally to the stopper. Bile was collected
on an hourly basis using an Isco model 1200-Pup fraction collector.
Shanvoperated animals were carried through all the surgical manipula-
tions with the exception of cutting and ligating the bile duct. All
experimental animals were restrained and housed in the same manner as
the bile duct cannulated rats.
Each biliary fistulated or sham-operated rat was treated with one of the
following: carbaryl ring-[ll+C] WSM (3.2 x 105 dpm, 1.5 yg) , carbofuran
ring-[ll4C] WSM (3.0 x 105 dpm, 10.6 yg) , aldicarb thiomethyl- [ lkC] WSM
(5.8 x 105 dpm, 24.7 yg) , Croneton ring-f^C] UM from sorghum (3.0 x
105 dpm, 8.5 yg) , carbaryl ring-[1'*C] UM from bean plants (5.4 x 10s
dpm, 2.6 yg) , or carbofuran ring-[11+C] UM from bean plants (3.6 x 105
dpm, 12.7 yg).
Metabolite Analysis - The water soluble fractions from the treated bean
plants were lyophilized to dryness and the solid residue washed repeat-
edly with methanol until no radioactivity was detected in the wash.
This process was quantitative in the radiocarbon recovery of the WSM,
while leaving much of the interfering plant material behind. The meth-
anol wash was then concentrated to about 10 ml and stored in a freezer.
For enzyme treatment of the WSM, a portion of the concentrated methanol
wash, amounting to 200,000 dpm, was removed and evaporated to dryness
under a gentle stream of nitrogen in 15 ml centrifuge tubes. Five ml
of acetate buffer (pH=5.6) was added to each tube,, as was 20 units of
glucosidase or 20 units of sulfatase. The tubes were then incubated
for 24 hours at 37°C in a shaking water bath. The released exocons
were extracted from the buffer with three equal volumes of ether (5 ml)
and samples removed for radioassay. The ether extracts were then con-
centrated under a gentle stream of nitrogen and applied to silica gel
plates for tic analysis.
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Portions of the plant solids containing the UM of the insecticides and
amounting to 500,000 dpm each were removed for treatment with acid.
The solids were finely pulverized with a mortar and pestle, and mixed
into about 30 ml of 1.0 N HC1 in 50 ml test tubes. The test tubes
were then placed into a boiling water bath and the contents mixed pe-
riodically for 2 hours. The released exocons were extracted with three
equal volumes of ether (30 ml) and samples taken for radioassay. The
ether extracts were then concentrated under a gentle stream of nitrogen
and applied to silica gel plates for tic analysis. The solids were
removed from the acid mixture by centrifugation and the aqueous portion
radioassayed.
The samples of urine from each rat were pooled, extracted with 20 ml
of chloroform, and lyophilized to dryness in 250 ml round bottom flasks.
The solid residue was washed repeatedly with methanol until no radioac-
tivity was detected in the wash. This process left many of the inter-
fering urinary components behind, and where it proved quantitative in
the recovery of urinary radiocarbon, the methanol was concentrated and
samples removed that contained 200,000 dpm each for enzyme and acid
treatments. The urine from rats treated with Croneton carbonyl [14C]
WSM contained only 80,000 dpm, therefore the samples for enzyme and acid
treatments contained only 20,000 dpm. The methanol was evaporated un-
der a gentle stream of nitrogen. The methanol extraction of lyophi-
lized urine from rats treated with WSM of carbaryl ring [1<+C] was not
efficient at removing all the radiocarbon, thus the residue from this
urine was washed twice with 5 ml of distilled water. The methanol
wash of the carbaryl WSM urine was evaporated to dryness and the water
wash was combined with this radiocarbon. Four samples were removed
that contained 200,000 dpm each and added to 15 ml centrifuge tubes.
To the urinary radiocarbon samples of Croneton ring [ll+C] WSM, Croneton
carbonyl-[11+C] WSM, carbaryl ring-[11+C] WSM, carbofuran ring-[14C] WSM,
and aldicarb thiomethyl-[14C] WSM treated rats were added 20 units of
glucosidase in 5 ml acetate buffer (pH=5.6), 20 units of glucuronidase
166
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in 5 ml acetate buffer, 20 units of sulfatase in 5 ml of acetate buf-
fer, or 10 ml of 1.0 N HC1. The samples of urinary radiocarbon from
rats administered carbofuran ring-[ll+C] UM were treated with 20 units
of glucuronidase, or 20 units of sulfatase. The sample of urinary ra-
diocarbon from rats administered carbaryl ring-[11+C] UM was treated
with 0.5 ml of glusulase. The enzyme treated samples were incubated
in 15 ml centrifuge tubes in a shaking water bath at 37°C for 24 hours.
The acid treated samples were placed in a boiling water bath in 25 ml
test tubes for 1 hour. The released exocons were extracted with three
equal volumes of ether (5 ml) and radioassayed. The ether extracts
were then concentrated under a gentle stream of nitrogen and applied
to silica gel plates for tic analysis.
The feces from rats treated with carbaryl and carbofuran UM were ex-
tracted twice with 100 ml of acetonitrile and filtered. The filtrate
was partitioned between 50 ml of water and 50 ml of chloroform, then
the chloroform-acetonitrile fraction was washed twice with 15 ml of
water. The aqueous portions were combined and all fractions were ra-
dioassayed.
Results and Discussion
Fate of insecticides in bean plants - An investigation of the radiocar-
bon distribution in treated bean plants revealed that the epicotyl
leaves of plants treated with carbofuran ring- [llfC] , Croneton ring-
[14C], and aldicarb thiomethyl-[1LfC] contained 70, 90, and 90% of the
dose, respectively. Since the bound and water-soluble residue from
these insecticides were to be administered to rats, it was desirous to
obtain preparations of these metabolites that were high in specific ac-
tivity with a minimum of extraneous plant material. Therefore, only
the epicotyl leaves of plants treated with the three above mentioned
preparations were extracted and used in this study. Likewise, 78% of
the radiocarbon recovered from five plants treated with Croneton car-
bonyl-[11+C] was found in the epicotyl leaves, thus all subsequent ex-
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tractions and tests involving bean plants containing equivalents of
this preparation were carried out with the epicotyl leaves only. In
contrast to the other insecticides studied, a sample of five carbaryl
ring-[14C] treated plants had a radiocarbon distribution of 21.2% of
the dose in the epicotyl leaves, 17.8% in the remaining leaves, and
60.9% in the stems. Thus, the entire bean plant of all those treated
with carbaryl ring-[ C] was extracted and used in this study.
Table 52 illustrates the nature of the radiocarbon extracted from bean
plants. The tendency for bound residue to accumulate varied markedly
with a startling 50.6% of the dose for the carbaryl ring-[11+C] treated
plants, while the amount found with the remaining insecticide applica-
tions were well below this figure at 7.0, 3.3, 1.3, and 2.2% for carbo-
furan ring [^C], Croneton ring-[lt+C], Croneton carbonyl [1J*C], and
aldicarb thiomethyl-[1LfC] , respectively. WSM accounted for 30.5, 56.6,
40.6, 2.6, and 29.3% of the dose for carbaryl, carbofuran, Croneton
ring-[lltC], Croneton carbonyl-[ll|C] , and aldicarb, respectively. It
is evident that the combined UM and WSM from the ring-labeled aryl
carbamates was about equal to or greater than the organosoluble or
free metabolites. However, the aldicarb data presented a different
picture with the organosoluble metabolites amounting to almost twice
the UM and WSM. These results are not unlike those seen in other
studies conducted on the plant metabolism of carbamate insecticides
(Dorough 1968, Dorough and Wiggins 1969, Coppedge et al. 1967, Hartley
et al. 1970). The low radiocarbon recovery of carbonyl-[14C] Croneton
(17.8% of the dose) as compared with that of ring-[14C] Croneton (89.9%)
indicated hydrolysis of the carbamic acid ester in the plants and loss
of the ltfC label as 1<+C02.
Fate of bean plant residues in rats - When the UM of carbaryl and car-
bofuran were administered to rats as a dietary supplement, 99.5 and
78.8% of the dose was eliminated via the feces in 48 hours, while the
urinary excretion amounted to 1.3 and 8%, respectively (Table 53).
In the same time span, only 0.3 and 1.4% of an identical UM dose was
168
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Table 52. NATURE OF RADIOCARBON IN BEAN PLANTS TREATED
WITH [14C]-LABELED CARBAMATE INSECTICIDES3
Mean percent of total injected and
standard error
water-
organo-
Insecticide Treatment
Carbaryl ring- [ l ^C]
Carbofuran ring-[ll*C]
Croneton ring- [11*C]
Croneton carbony 1- [ 1 ^C]
Aldicarb thiomethyl- [14C]d
soluble
19.2
14.5
45.9
13.9
57.9
± 1.9
+ 0.6
± 1.3
± 2.0
± 3.2
soluble
(WSM)
30.5
56.6
40.7
2.6
29.3
± 1.2
± 3.4
+ 0.2
± 0.2
± 1.1
bound
50.6 ±
7.0 ±
3.3 ±
1.3 ±
2.2 ±
(UM)
7.3
0.3
1.0
0.4
0.4
Data compiled from bean plants extracted 20 days after stem injec-
tion of the respective insecticide preparation.
Combined results on three groups of plants with each insecticide.
Whole plant extracted due to radiocarbon distribution.
Epicotyl leaves only extracted due to radiocarbon distribution.
169
-------
Table 53. ELIMINATION OF RADIOCARBON FROM RATS TREATED WITH
CARBARYL, CARBOFURAN, AND CRONETON UNEXTRACTABLE
METABOLITES FROM PLANTS
Cumulative % of dose
metabolites
Carbaryl ring- [14C]a
Carbofuran ring-[ll*C]
Croneton ring- [ * ^C]
Hours
-> fi-czV
treatment
12
24
48
12
24
48
12
24
48
r1
Intact rat"
Urine
0.9
1.2
1.3
4.5
6.5
8.0
8.2
14.8
16.4
Feces
57.1
82.7
99.5
36.5
67.6
78.8
27.2
51.2
90.8
Biliary fistulated rat
Bile
0.1
0.2
0.3
0.4
0.7
1.4
0.5
1.7
2.5
Urine
0.4
0.8
1.3
0.0
1.6
4.4
3.4
9.2
15.2
Feces
0.3
32.2
69.0
0.0
10.8
44.2
0.0
12.6
35.0
Unextractable metabolites from bean plants administered as a dietary
supplement. Values for intact rats are the mean of four animals.
Unextractable metabolites from sorghum administered as a dietary
supplement.
Respiratory gases were monitored for ll|C02, but none was detected.
170
-------
excreted in the bile of biliary fistulated rats. A similar pattern of
elimination was seen when the UM of Croneton from sorghum was fed to
rats. After 2 days 90.8% of the dose was found in the feces and 16.4%
in the urine, while the bile of a bile duct-cannulated rat contained
only 2.5% of the dose. Thus, the UM were bioavailable only to a slight
extent.
Monitoring biliary radiocarbon was a vital aspect of these studies,
since the relative quantities of urinary and fecal elimination of for-
eign compounds is not a good indication of what is or is not absorbed
from the gut (Kleassen, 1975). For example, Houston et al. (1975)
demonstrated that 6 hours after a single I.V. dose of carbaryl car-
bonyl-f^C] to rats, up to 40% of the administered radiocarbon was ex-
creted into the bile when the common bile duct was cannulated. As
shown with intact rats, though, only 3% of an I.V. dose is eventaully
eliminated in the feces. On the other hand, from an unpublished study
on a endosulfan, we found that only 14% of an oral dose was eliminated
in the urine in 48 hours, while 35% was voided via the feces. These
data would lead one to conclude that a endosulfan is slowly absorbed
from the gastrointestinal tract. However, up to 58% of an oral dose
was found in the bile of bile duct cannulated rats in the same time
period. Other examples of this same phenomena are the polycyclic hy-
drocarbons 1:2-benzanthracene and 3,4-benzopyrene, which undergo ex-
tensive biliary excretion and are eventually eliminated in the feces
(Falk 1963, Boyland and Sims 1964, Levine 1970).
It should be noted in Table 53 relative to the elimination of UM that
the total excretion of Croneton UM from intact rats in 48 hours was
107% of the dose, for carbaryl UM, 100%, and for carbofuran UM, 86%.
In contrast, only 50%, 71%, and 50% of the respective UM dose was eli-
minated by biliary fistulated rats in the same time period. When the
study on a bile duct-cannulated rat treated with carbaryl UM was ex-
tended to 3 days, the total elimination of radiocarbon increased to
99.3% of the dose, or an amount equivalent to that observed in intact
171
-------
rats in 2 days. This slower elimination seems to be a characteristic
attributable to the surgical manipulations of the animal. Such a con-
clusion is supported below by a study on the fate of carbaryl WSM in
rats.
Six rats were treated with carbaryl WSM; one with the bile duct cannu-
lated, one sham-operated, and four intact. The results are shown in
Table 54. The four intact rats gave very similar results as well as
good radiocarbon accountability. After 24 hours, about 85% of the dose
was found in the urine and 7% in the feces. However, the sham-operated
and cannulated rats demonstrated a much slower elimination capacity in
the first 24 hours with total excretion amounting to 51 and 35% of the
dose, respectively. This investigation was carried out to 66 hours
post-treatment, and by the end of this period the total elimination of
carbaryl WSM for the three types of rats had drawn quite close togeth-
er. For the bile duct cannulated, sham, and intact rats the recover-
ies were 89.2, 94.6, and 99.6% of the dose. Thus, surgical manipula-
tion of rats obviously decreased the rate of elimination of carbaryl
WSM, although the vast majority of radiocarbon is eventually excreted
within a 3 day period as is normally seen with intact rats.
To further investigate these effects on excretion kinetics due to sur-
gery, a slightly different approach was taken when six rats were treat-
ed with aldicarb WSM. As with the data discussed above, there was one
biliary fistulated rat, one sham-operated and four intact, but dosing
of the "operated" rats was delayed until 4 hours post-surgery. As can
be seen in Table 54, only in the first 12 hours post-treatment is there
any noticeable differences in the total amount of radiocarbon excreted.
At this time the intact rats had eliminated 78.8% of the dose, while
that from the sham-operated amounted to 23.6%, and for the bile duct
cannulated 64.1%. By 24 hours the respective quantities were 86.8,
69.0, and 79.0%. These data on the fate of aldicarb WSM in rats and
that on carbaryl WSM tend to support the conclusion that the slower
elimination observed in surgical animals is caused by a transient re-
172
-------
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covery period immediately following the operation, and not factors at
play throughout the entire test period. The effect is possibly due to
decreased absorption caused b^ a temporary loss of gastrointestinal
motility following surgery. Gastrointestinal motility markedly affects
absorption of substances from the gut (Parsons 1975) .
It is evident from the rat excretion data in Table 54 and 55 that WSM
of the four carbamate insecticides were readily available for absorp-
tion, since the bulk of oral doses was recovered in the urine. In 36
hours the urinary radiocarbon of intact rats accounted for 87.7, 87.5,
81.2, and 64.8% of the WSM dose for carbaryl ring-[ll+C], aldicarb thio-
methyl-[ll+C] , Croneton ring-[14C], and carbofuran ring [lkC] . Feces
contained 9.7, 1.0,3.7, and 8.3% of the dose, respectively. In the
same time period, bile of biliary fistulated rats treated with WSM of
aldicarb, carbofuran, and carbaryl comprised of 20.7, 17.2, and 12.9%
of the dose. The quantity of aldicarb WSM radiocarbon found in the
bile after 36 hours is about ten times greater than the amount elimi-
nated in the feces of an intact rat in 72 hours (2.2% of the dose). This
suggests an enterohepatic circulation of these metabolites. Even at
that, however, aldicarb WSM as well as those of the other insecticides
studied are rapidly eliminated from rats.
Since the WSM are bioavailable, the chemical nature of these metabo-
lites and their fate in rats prior to elimination becomes an important
toxicological question. Moreover, when WSM of Croneton carbonyl- [ C]
were administered orally to rats (Table55) 30.0% of the dose was found
14
as COz, 39.2% in the urine and 6.1% in the feces. These data from a
carbonyl label demonstrated that WSM with an intact carbamate moiety
were readily capable of being absorbed. Such an observation lends even
more weight to the need for defining sequential biochemical pathways of
these insecticides from plants through animals. Thus, an analysis of
the WSM and UM from treated bean plants and the urinary radiocarbon from
rats administered these metabolites was undertaken.
174
-------
Table 55. ELIMINATION OF RADIOCARBON FROM RATS TREATED WITH
CRONETON WATER-SOLUBLE METABOLITES FROM BEAN PLANTS
Water-soluble
a
metabolites
Croneton ring-[11+C]
Croneton carbonyl- [ **C]
Hours after
treatment
6
12
20
26
30
36
6
12
18
24
30
36
Cumulative % of
^C02
0.0
0.0
0.0
0.0
0.0
0.0
23.1
30.0
30.0
30.0
30.0
30.0
Urine
66.6
76.6
78.8
79.8
80.7
81.2
31.2
34.5
37.5
38.4
39.0
39.2
dose
Feces
0.0
0.2
2.8
3.4
3.6
3.7
0.1
0.2
3.8
5.6
5.7
6.1
Administered via oral intubation.
Mean data from three rats.
175
-------
The WSM of all four insecticides were treated with glucosidase, since
the major conjugating mechanism in plants is glucoside conjugation.
Based on the enzyme treatment, the glucosides of carbaryl, carbofuran,
aldicarb, Croneton ring-[11+C], and Croneton carbonyl-[11+C] amounted
to 29, 39, 14, 51, and 15% of the WSM, respectively.
Attempts to release the UM from the bean plant solids using acid hydro-
lysis was, for the most part, unsuccessful. The radiocarbon released
as chloroform soluble amounted to only 1.2, 0.7, 0.4, 0.3, and 0.2% of
the dose for carbaryl, carbofuran, aldicarb, Croneton ring-[11+C], and
Croneton carbonyl-[14C], respectively.
The exocons tentatively identified by tic analysis from glucosidase
treatment of the WSM and acid hydrolysis of the plant solids are pre-
sented in Table 56. The majority of the Croneton ring-[11+C] equiva-
lents released by glucosidase were hydrolytic products, primarily
Croneton phenol sulfoxide (52.4% of the exocons) and phenol sulfone
(39.8%). The glucosidase released exocons shown with Croneton carbon-
al-[ C] in Table 56 are, of course, only products with the carbamate
intact. Hydrolysis of the carbamic acid ester with carbonyl [ C]
carbamates results in loss of the radioisotope as CO2- The gluco-
sides of Croneton carbonyl-[C] amounted to only 0.2% of the dose,
while the glucosides of Croneton ring-[ C] were 24.6%. Overall, as
evidenced by these results on the two [ C]-labels, glucosidation of
Croneton was predominantly with the phenolic metabolites.
With aldicarb 80% of the glucosidase released radiocarbon remained at
the origin of tic plates (Table 56). The two identified metabolites
were aldicarb sulfoxide (7.5% of the radioactivity present), and ox-
ime sulfoxide (5%). When the carbaryl glucosidase released exocons
were analyzed by tic, 5.8, 7.0, 12.8, 11.6, and 3.5% of the radiocar-
bon present was identified as N-hydroxymethyl carbaryl, 4-hydroxy
carbaryl, 5-hydroxy carbaryl, carbaryl, and 1-naphthol. When carbo-
furan WSM were treated with glucosidase, the exocons released and
176
-------
Table 56. THIN-LAYER CHROMATOGRAPHY OF RADIOCARBON RELEASED BY
ACID AND ENZYME HYDROLYSIS OF THE BOUND AND WATER-SOLUBLE RESIDUE
FROM BEAN PLANTS TREATED WITH CARBAMATE INSECTICIDES
% of radiocarbon
released
Water solubles Unextractables
Insecticide/Metabolite
Carbaryl ring-^C]
Origin
3 , 4-dihydroxy
5 , 6-dihydro-dihydroxynaphthol
N-hydroxymethyl
4-hydroxy
5-hydroxy
parent
1-naphthol
unknown
Carbofuran ring-[1LfC]
Origin
3-hydroxy
3 -hydr oxypheno 1
3-keto
3 -ketophenol
phenol
unknown
Croneton ring-[ltfC]
Origin
sulf oxide
phenol sulf oxide
sulfone
phenol sulfone
parent
phenol
unknown
Croneton carbonyl- [1LfC]
Origin
sulf oxide
sulfone
parent
unknown
Aldicarb thiomethyl- [lkC]
Origin
sulf oxide
oxime sulf oxide
nitrile sulfoxide
unknown
Glucosidase
released
36.0
0.0
0.0
5.8
7.0
12.8
11.6
3.5
23.3
1.0
83.0
6.8
0.0
3.9
1.9
3.4
0.5
0.8
52.4
0.8
39.8
1.2
0.0
4.5
2.4
17.1
4.9
0.0
75.6
80.0
7.5
5.0
0.0
7.5
Acid
hydro ly zed
54.2
0.0
13.3
0.0
0.0
0.0
0.0
12.5
20.0
7.2
47.9
30.4
0.0
11.6
2.9
0.0
3.3
20.0
46.7
6.7
20.0
3.3
0.0
0.0
5.0
75.0
15.0
0.0
5.0
11.6
0.0
34.9
39.5
14.0
177
-------
identified as hydrolytic products were minor in amount as compared to
those exocons identified as oxidation products of the insecticide.
Eighty-three percent of the radiocarbon cleaved was 3-hydroxy carbo-
furan. Other exocons found were 3-hydroxy carbofuran phenol (6.8%),
carbofuran phenol (1.9%), and 3-keto carbofuran phenol (3.9%).
It is difficult to draw many inferences from the results on tic of
the radiocarbon released by acid hydrolysis of the plant solids
(Table 56). Very little of the radioactivity released was organo-
soluble and, thus, conducive to chromatographic analysis. In gener-
al, the principal exocons that were identified from the plant solids
were the same as those found with the glucosidase released exocons of
the WSM. The major exceptions to this generalization were the aldi-
carb nitrile sulfoxide amounting to 39.5% of the exocons released
from the solids, and the 5,6-dihydro-dihydroxynaphthol, a metabolite
from the solids containing carbaryl UM, amounting to 13.3%. Neither
of these exocons were identified from among the aglycones of the res-
pective WSM.
Very little of the radiocarbon recovered in the urine of WSM treated
rats was organosoluble. Aldicarb was the only insecticide that re-
sulted in any significant partitioning of urinary radiocarbon into
chloroform, and this accounted for only 12.7% of the administered WSM.
The other four groups of WSM treated animals had considerably less
organosoluble urinary radiocarbon. The values were 2.3, 2.5, 1.0, and
0.4% of the dose for carbaryl, carbofuran, Croneton ring-[ C] , and
Croneton carbonyl-[ C], respectively. Thus, there were very few free
metabolites in the urine from any of the animals administered WSM, but
such results are not surprising when one considers the water soluble
nature of the inoculum.
Enzyme hydrolysis (glucosidase, glucuronidase, and sulfatase) of this
urine released radiocarbon that was organoextractable in amounts e-
qual to 45.1, 23.3, 11.7, 46.8, and 11.7% of the dose for carbaryl,
178
-------
carbofuran, aldicarb, Croneton ring-[ll+C], and Croneton carbonyl-
[11+C] , respectively. When urine from WSM treated rats was subjected
to an acid hydrolysis, more exocons were released than that resulting
from enzyme cleavage, except for urine containing Croneton equiva-
lents. Specifically, 47.8, 42.0, 19.9, 31.8, and 2.0% of the WSM
dose was released by acid hydrolysis of urine containing equivalents
of carbaryl, carbofuran, aldicarb, Croneton ring-[11+C] , and Croneton
carbonyl-[11+C] , respectively. Acid hydrolysis of the Croneton carbon-
yl-fl^C] urine resulted in a loss of radiocarbon equivalent to 23.2%
of the dose. Presumably, the loss of radioactivity from this urine
is the result of released volatile products which escaped into the
atmosphere.
Table 57 shows the results of a tic analysis undertaken on exocons
released in the acid and enzyme hydrolysis of rat urine discussed
above. No data on Croneton carbonyl-[11+C] is presented in this table,
because attempts to identify the low levels of radiocarbon released
by acid and enzyme hydrolysis of rats treated with the WSM were un-
successful. The entire dose to rats amounted to only 110,000 dpm.
From the urine containing carbofuran equivalents, three major exocons
were found that accounted for 90% or more of the radiocarbon released
by the three enzyme treatments and acid hydrolysis. These were 3-
hydroxy carbofuran, 3-hydroxy carbofuran phenol, and 3-keto carbofu-
ran phenol.
1-Naphthol was the major exocon identified from the urine of rats
treated with carbaryl WSM, amounting to over 50% of the combined ra-
diocarbon present as glucosides, glucuronides, sulfates, and acid
hydrolyzed metabolites. From the glucosidase released carbaryl exo-
cons out of rat urine, 25% was identified as 5,6-dihydro-5,6-dihy-
droxy carbaryl, and 29, 17, and 13% of the sulfates were identified
as 5-hydroxy carbaryl, 3,4-dihydroxy carbaryl, and 5,6-dihydro-5,6-di-
hydroxy carbaryl, respectively. These were minor metabolites, though,
179
-------
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because all the glucosides and sulfates combined from the carbaryl
WSM treated rats amounted to only 9% of the dose.
Aldicarb sulfoxide and oxime sulfoxide accounted for 60% of the radio-
carbon released by the three enzyme treatments and acid hydrolysis of
urine from the aldicarb WSM treated rats. About 37% of the acid hy-
drolyzed exocons was the aldicarb nitrile sulfoxide, whereas only a
small quantity of this metabolite, amounting to less than 1% of the
dose, was isolated following enzyme treatments of the urine. It is
possible that the oxime sulfoxide was converted to the nitrile sulf-
oxide under acid conditions.
Croneton sulfoxide, phenol sulfoxide, and phenol sulfone were the
major exocons identified from the urine of Croneton ring-[1LfC] WSM
treated rats. Although 46% of the sulfates was the Croneton phenol,
this was equivalent to only 1.3% of the dose, and could not be consid-
ered a major identified product. The combined glucosides and glucuro-
nites accounted for 44.2% of the dose, thus the identified products
resulting from these two enzyme treatments were a better indication
of the major exocons present in rat urine. No Croneton sulfoxide was
found following any of the enzyme treatments of urine, but 24.5% of
the acid released exocons were so identified, amounting to 7.8% of
the dose.
The tentatively identified exocons from rat urine with the carbamic
acid ester intact were 3-hydroxy carbofuran (9.2% of the enzyme and
acid released radiocarbon combined) 3,4-dihydroxy carbaryl (1.3%),
5,6-dihydro-5,6-dihydroxy carbaryl (1%), 4-hydroxy carbaryl (2.9%),
5-hydroxy carbaryl (3.8%), aldicarb sulfoxide (10.1%), aldicarb sul-
fone (0.6%), and Croneton sulfoxide (9.9%). With the exception of 3,
4-dihydroxy carbaryl, 5,6-dihycro-5,6-dihydroxy carbaryl, and aldi-
carb sulfone, all of these carbamate exocons were found in the WSM
from bean plants (Table 57) in equal or greater amounts than in the
urine of rats treated with the WSM. In only three instances were the
181
-------
intact carbamate exocons a relatively significant proportion of the
enzyme and acid released urinary radiocarbon. These were 3-hydroxy
carbofuran, aldicarb sulfoxide, and Croneton sulfoxide. For the most
part, then, rats degraded the potentially toxic plant conjugates.
The majority of radiocarbon released by enzyme and acid hydrolysis of
urine was phenolic derivatives, unknowns, and polar origin material
with all four insecticides studies.
From the data discussed above it is apparent that upon absorption of
WSM by rats, the metabolic degradation of the insecticides begun in
bean plants was continued. This was most clarly demonstrated in the
carbofuran study. In plants treated with that insecticide, a pre-
dominance of oxidation was seen at the three position of the benzo-
furanyl ring to form 3-hydroxy carbofuran (Table 56). This metabolite
accounted for 83% of the aglycones, while phenolic products of carbo-
furan amounted to only about 13%. After administration of the carbo-
furan WSM to rats, only 9.2% of the urinary exocons was 3-hydroxy
carbofuran, whereas 14% was the 3-hydroxy carbofuran phenol and 63%
the 3-keto carbofuran phenol. Furthermore, almost 21% of the carbo-
furan WSM were glucosides, but when the WSM were administered to rats
only 6.8% of the dose was eliminated in the urine as glucosides.
Glucuronides and sulfates amounted to 16.5% of the dose. Presumably
from this data, there was cleavage of 3-hydroxy carbofuran glucoside
within the animal, followed in some order by oxidation of the 3-hydroxy
to a keto, hydrolysis of the carbamate, and conjugation with either
glucuronic or sulfuric acid prior to elimination.
It is difficult to make assessments, such as the one above, based
solely on enzyme analyses since enzyme preparations are not altogeth-
er specific. However, a study utilizing a naphthyl-[^C] glucoside
preparation and a naphthyl glucoside-[14C] label showed that only 20%
of the conjugate was excreted intact as administered, while 24% was
eliminated as a glucuronide (Dorough et al. 1974). The use of two
labels enabled these workers to ascertain that the glucuronide
182
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was not formed by oxidation of 1-naphthyl-glucoside, but resulted
from the cleavage of the glucoside linkage to form 1-naphthol, which
was then conjugated with endogenous glucuronic acid. It was demon-
strated that the administration of free 1-naphthol, a metabolite of
carbaryl, resulted in a more rapid excretion than its glucoside.
This suggests that the cleavage occurs in the gut since the water sol-
uble nature of a glucoside would be a deterrent to absorption.
When the carbaryl WSM were administered to rats only 3.5% of the agly-
cones in the dose was 1-naphthol (Table 57). However, based on en-
zyme and acid hydrolysis of urinary radiocarbon, almost 60% of the
dose was eliminated as conjugates of 1-naphthol, and less than 2% of
these were glucosides. A continuing degradative process of WSM in the
rat is readily apparent. In contrast, little difference was seen be-
tween the aglycones of Croneton ring-[^C] WSM and the exocons of
urine from rats treated with these WSM. In both cases, the Croneton
phenol sulfoxide and phenol sulfone were the major identified metabo-
lites. Unlike the other carbamates, Croneton was quickly hydrolyzed
by bean plants, and this would account for the similarity of products
found in plants and rats.
Not many inferences can be drawn on data generated with aldicarb, be-
cause so little of the WSM dose (4%) in plants was cleaved by gluco-
sidase, and 80% of this remained on the origin of tic plates. The
major identified aglycones of the WSM were aldicarb sulfoxide and ox-
ime sulfoxide. Similarly, these were found in high quantities among
the exocons of urine from rats administered aldicarb WSM at 10.1 and
37.6% of the radiocarbon present, respectively.
An analysis of the urine from rats administered carbaryl and carbo-
furan UM resulted in just 0.7 and 1.1% of the dose partitioning into
chloroform, respectively. Enzyme treatments freed 19% of the radio-
carbon in the urine of the rat fed carbofuran UM, and 17% of that in
the urine of carbaryl UM treated rats. Acid hydrolysis was more ef-
183
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fective in releasing exocons from urine, but this still amounted to
only 4.9% of the carbofuran UM dose, and of 0.5% of the carbaryl UM
dose. Thin layer chromatography on exocons released from the urine
of carbaryl and carbofuran UM treated rats was inconclusive, due to
the low radioactivity and large amount of interfering material ex-
tracted with the exocons. When carbofuran and carbaryl plant solids
were treated with acid, 10% and 2% of the radiocarbon present was re-
leased into chloroform, respectively. These are roughly the same
percentages of bound residues administered to rats that were recovered
in the urine, and might be the same metabolites available for absorp-
tion. Thin layer chromatography of the acid released exocons from
the plant solids (Table 56) showed that 3-hydroxy carbofuran was the
only liberated product with intact carbamate. The remaining were
phenolic derivatives and unidentified materials with both the insecti-
cides. However, it has been reported that six days after a stem in-
jection to bean plants of carbaryl carbonyl-[^C] , 35% of the radio-
carbon was bound to plant solids (Abdel-Wahab et al. 1966). The data
indicated that the majority of the carbaryl unextractables were in-
tact carbamate. Aside from the tic mentioned above, support of this
conclusion is evident from the results of the present study using the
ring label, where a similar quantity of UM were detected. Likewise,
Kuhr and Casida (1967) obtained evidence that the UM were carbamate-
type metabolites. No sound conclusions can be drawn on the nature of
the small quantities of carbaryl and carbofuran UM that were available
for absorption.
Two main processes govern the absorption of drugs and foreign com-
pounds from the gastrointestinal tract (Aguiar 1975). One is the
build-up of an effective concentration of the chemical in solution at
the absorption site, and the other is the rate of permeation through
the gastrointestinal barrier. When a compound is of an insoluble na-
ture, such as the UM investigated, the accumulation of an effective
concentration at the site governs the rate of absorption. It was evi-
dent from a close examination of the feces from rats treated with UM
184
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that little physical change had taken place in the plant solids con-
taining the UM during their course through the alimentary tract. This
lack of dissolution of the administered material helps explain the un-
availability of the UM studied.
On the other hand, when drugs or foreign compounds are administered in
solution, as was the case with the WSM, the permeation rate is assumed
to be the controlling step in the absorption sequence (Aguiar 1975).
Permeation of the WSM was rapid and extensive as demonstrated by the
urinary excretion data on rats treated with these metabolites. Since
rapid absorption of a chemical is generally associated with a high
lipid solubility (Parke 1968), the bioavailability of the very polar
carbamate insecticide WSM appears to be a complex phenomena. Gastro-
intestinal motility, physiological and metabolic properties of the
intestinal epithelia, and presence of gastrointestinal microflora cer-
tainly could all play a significant role (Kurz 1975). An investigation
of these parameters and their contribution to the bioavailability of
carbamate insecticide WSM would be of toxicological interest.
185
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FURTHER STUDIES ON THE CHEMICAL NATURE OF RADIOACTIVE WATER SOLUBLE
AND BOUND RESIDUES IN PLANTS TREATED WITH l4C-CARBARYL
Materials and Methods
Chemicals - 1-Naphthol-l- C (15.2 mCi/mM) was obtained from Amersham
Searle Company, Des Plaines, IL. , while naphthalene-14C (5.1 mCi/mM)
was purchased from California Bionuclear Corporation, Sun Valley, CA.
Nicotamide adenine dinucleotide phosphate (NADPH2), uridine-5-diphos-
phoglucose (UDPG), tris(hydroxymethyl)aminomethane (Tris), and 3-D-
glucoside glucohydrolase (EC No. 3.2.1.21) were supplied by Sigma Chem-
ical Company, St. Louis, MO. Triethyl amine was obtained from Matheson,
Coleman and Bell, Chicago, IL.
Synthesis - Radiolabeled carbaryl (naphthyl-l-11+C-carbaryl) 15.2 mCi/
mM) was synthesized essentially by the procedure of Dorough and Casida
(1964) except that triethylamine was added as a catalyst to the reac-
tants l-naphthol-l-lt+C and methyl isocyanate, to increase the yield.
1-Naphthyl-l- C-glucoside was prepared with crude fly homogenate us-
ing the procedure of Kumar and Dorough (1974).
l-Methoxynaphthalene-l-11+C was prepared from l-naphthol-l-11+C by the
diazomethane procedure of Pyrek and Achmetowicz (1970). All of the
above radiolabeled compounds were purified by thin-layer chromatography
on silica gel plates (Brinkman 254, 0.24 mm). The preparation of 1-
naphthyl glucoside has previously been described (Dorough et al. 1974).
The intermediate 1-naphthyl tetra-0-acetyl-B-D-glucopyranoside was
characterized by its nmr (acetone d6), 62.00 (S, 12H, acetyl CH), 62.78
(d, 1H aliphatic CH), 64.19 (m, 2H, CH ), 65.20-5.58 (m, 4H aliphatic-
CH) and 67.0-8.0 (m, 7H aromatic CH). The melting point of the naph-
thyl glucoside was 171-175°C.
Generation of Carbaryl Metabolites by Plants - Dwarf Horticulture (Tay-
lor) bush beans were grown in the greenhouse in sterilized soil to re-
186
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duce microbial growth during the summer months in the greenhouse, and
transferred to environmental growth chambers 9 days after germination.
The plants were watered daily with Hoaglands nutrient solution and kept
at 27°C during the day and 23°C at night with a 12-hr photo period.
Treatment occurred when the plants were 12 days old. Twenty-two yg of
naphthyl-l-^C-carbaryl were injected in 75 yl as a 3:1 water-acetone
solution into the stem above the soil level. The stem was punctured 2
cm above the point of injection in order to prevent back pressure dur-
ing injection.
To extract radioactive compounds individual plants were homogenized in
a Virtis 45 homogenizer in 75 ml:25 ml acetone-water at high speed for
five min. The homogenate was filtered under vacuum using a Buchner
funnel and Whatman No. 4 filter paper. The solids were rehomogenized
twice more for 5 min in 40 ml:40 ml:40 ml acetone-methanol-ethyl acetate
and filtered. This solvent mixture eliminated soxhlet extraction of the
solids, which extracted an additional 5% radioactivity as tried in ear-
lier experiments. The combined filtrates were evaporated until only the
water remained, transferred to separatory funnels and washed 3 times
with hexane to remove lipids and chlorophyll. The hexane wash contained
no radioactivity. The water layer was then extracted three times with
100 ml chloroform to separate the water-soluble metabolites from the
parent compound. Both water- and organo-fractions were radioassayed.
The total solids containing the "unextractable" metabolites of each
plant were combusted for radioassay in 2-3 portions at 95% efficiency
on a Packard 306 Tricarb Oxidizer.
Aglycones from plant-generated ^C-carbaryl water soluble metabolites
were prepared by adjusting the water soluble carbaryl metabolites to
1.0 N HC1 and heating for 1 hr in a boiling water bath. After cooling,
2 ml of saturated NaCl in water were added to the 4 ml reaction and
extracted 4 times with 10 ml of chloroform. The chloroform was evap-
orated and the aglycones dissolved in 3:1 water:acetone prior to treat-
ment of bean plants or bean plant homogenates.
187
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In order to generate aglycones by enzyme hydrolysis, carbaryl water
solubles were freeze-dried and resuspended in 10 ml of 1 M acetate
buffer, pH 5. This solution, containing approximately 6,200,000 dpm
of 1^C-carbaryl water-solubles was transferred to a 25 ml Erlenmeyer
flask and placed into a water bath shaker at 37°C. Two mg of 3-D-
glucoside glucohydrolase in 2 ml of buffer were added and incubated for
24 hr. A control containing about 200,000 dpm of water solubles was
set up in the same manner without the enzyme. After terminating the
reaction with chloroform, the aqueous phase was extracted 4 times with
50 ml chloroform. Thirty ml of saturated sodium chloride solution were
added prior to extraction.
In vitro Metabolism - Ten grams of 12-day-old bean plants were cut into
small pieces, sealed into a plastic bag and quick frozen in dry ice-
acetone. The plant material was then ground with a pre-frozen mortar
and pestle to a very fine powder and suspended up to 100 ml with 0.5 M
phosphate buffer, pH 7.2, containing 0.15 M KC1. In one experiment with
14C-carbaryl NADPH (1 mg/ml reaction mixture) and NaCN (25 yg/ml) were
added in an attempt to increase the metabolism of plant enzymes and to
inhibit phenol oxidases, respectively.
The suspended plant homogenate was stirred on a magnetic stirrer until
equilibrated to 25°C. The radiolabeled compound was then added im-
mediately while stirring continuously. Ten ml aliquots were taken af-
ter the following time intervals: 1 min, 2.5 min, 15 min, 30 min, 1 hr,
and 2 hr. The aliquots were pipetted into 500 ml separatory funnels
containing 30 ml of distilled water and 100 ml of chloroform. After
shaking and separating the two phases, the water layer was extracted
twice more with 100 ml of chloroform. The combined organo extracts
were evaporated to a low volume, transferred to graduated centrifuge
tubes and radioassayed by liquid scintillation. The plant emulsion
which occurred during extraction was separated by centrifugation. The
plant solids were added back to the water layer remaining in the sepa-
ratory funnel and 100 ml of acetone were added, shaken, and allowed to
188
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precipitate for 15 min. The solutions were then filtered under vacuum,
the solids washed with 1:1:1 f^O-acetone-ethyl acetate. After evapora-
tion of the solvents, the water layer was radioassayed and the solids
combusted. The following radiolabeled compounds were subjected to this
in vitro system: ll4C-carbaryl (15.2 mCi/mM) ; l-naphthol-l-lt+C (15.2
mCi/mM) , l-naphthol-l-ll+C-glucoside (15.2 mCi/mM) ; naphthalene-lkC
(5.1 mCi/mM) and l-methoxynaphthalene-^C (15.2 mCi/mM) .
Results and Discussion
The distribution of carbaryl equivalents in 12-day-old bean plants 0 to
25 days after stem injection with 1 ug of naphthyl-l-ll4C-carbaryl is
shown in Table 58. 14C-Carbaryl equivalents were rapidly distributed
between the water soluble and solid phases one day after treatment with
a greater proportion of the dose being incorporated into the water sol-
uble phase. The distribution of radiolabel in the two phases remained
essentially the same until day 15 post-treatment at which time there was
a noticeable decline in water soluble metabolites at the expense of an
increase in "bound" metabolites. This trend continued for the remainder
of the experiment. Earlier studies by Dorough and Wiggins (1968) had
shown a similar redistribution of C-carbaryl metabolites in bean
plants 10-20 days after treatment with 25 yg lltC-carbaryl. This sug-
gested that water soluble metabolites, possibly the glycoside conjugates
of phase I carbaryl metabolism, were the immediate precursors to unex-
tractable metabolites (Kuhr and Dorough, 1976). However, the distribu-
tion data from plants treated with 1 yg of carbaryl indicate that
another mechanism for formation of unextractable metabolites may exist.
This was indicated by the rapid incorporation of radiolabel into the
water-soluble and "unextractable" plant fractions one to two days after
treatment followed by a period of 10-12 days with essentially no change
in residue levels in the two fractions. As indicated in Table 58, in-
jecting plants with 25 pg 14C-carbaryl effectively masks the distribu-
tion pattern seen at the lower dose.
189
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Table 58. EXTRACTION AND PARTITIONING CHARACTERISTICS OF
CARBARYL AND METABOLITES AFTER INJECTION INTO BEAN PLANTS
Treatment
and fraction
ll+C-Ring carbaryl
1 yg/plants
Organosolubles
Water-solubles
Bound
25 ug/plant
Organosolubles
Water-solubles
Bound
% Distribution/days
0
98
0
2
95
1
4
1
8
62
30
56
13
21
3
7
57
36
40
33
27
5
7
56
37
21
45
34
7
12
58
30
10
49
41
after treatment
9 11 16 20
0000
61 62 50 30
39 38 50 70
2 - - -
52
46
25
0
25
75
-
-
-
^C-Naphthol
25 yg/plant
Organosolubles
Water-solubles
Bound
1 4C-Naphthylglucoside
1 pg/plant
Organosolubles
Water-solubles
Bound
84
10
6
0
95
5
8
13
79
10
48
42
9
14
77
15
54
31
7
11
82
14
60
26
0 - -
Q _
92
14 18 25
61 60 55
25 22 20
35
50
15
190
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The rapid incorporation of ^C-carbaryl equivalents into the unextract-
able fraction the first one to two days after treatment suggested to us
that the phase I, hydroxylated carbaryl derivatives were the proximate
intermediates in the formation of "bound" residues and not the conjugat-
ed derivatives of phase I products. More specifically, the formation
of conjugated and "bound" metabolites proceeds by two different pathways
each competing for a common substrate, e.g., hydroxylated carbaryl
derivatives.
If hydroxylated derivatives are proximate intermediates in the formation
of "bound" residues, then administering these radiolabeled materials to
plants should lead to a more rapid and extensive incorporation of radio-
activity into the "bound" residues than would be the case if water-sol-
uble metabolites were administered to the plants. Indeed, when 12-day-
old bean plants excised at ground level were allowed to take up carbaryl
aglycones (acid released from lltC-ring-carbaryl water soluble metabolites
obtained from carbaryl treated bean plants), there was rapid and extensive
incorporation of radiolabel into the "bound" fraction and water soluble
fraction as indicated by the data in Table 59. It was also observed
that l-naphthol-l-ll4C, a phase I metabolic product of ^C-carbaryl, was
rapidly and extensively incorporated into unextractable fraction after
stem injection with only about 10% of the applied dose being distributed
to the water soluble phase (Table 58). The results with naphthol are
consistant with those obtained by Kuhr and Casida (1967).
When excised bean plants were allowed to take up water soluble metabo-
lites derived from carbaryl treated plants, only a small fraction of the
applied radioactivity was incorporated into the unextractable fraction
even after 25 days post-treatment as shown by the data in Table 59.
Stem injecting bean plants with 1 yg of l-naphthol-l-11+C-glucoside, a
water-soluble carbaryl metabolite, lead to a distribution pattern dif-
ferent from that obtained with the total crude water soluble metabolites.
As indicated in Table 58, there was a rapid decrease in radioactivity
associated with the water-soluble fraction and a corresponding increase
191
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Table 59. EXTRACTION CHARACTERISTICS OF RADIOCARBON IN EXCISED
BEAN PLANTS FOLLOWING UPTAKE VIA THE STEM OF CARBARYL
WATER-SOLUBLE METABOLITES (BEAN PLANT-GENERATED) AND
AGLYCONES RESULTING FROM ACID HYDROLYSIS
and fraction
1LfC-Ring-Carbaryl water
Organosolubles
Water-solubles
Bound
11+C-Aglyconesa
Organosolubles
Water-solubles
Bound
% Distribution/days after treatment
4
soluble
15
82
3
17
55
28
5
metabolites
14
74
12
12
40
48
8
13
71
16
5
48
47
10 20
10 10
82 78
8 12
6
61
33
25
11
76
13
-
-
-
ll+C-Metabolites cleaved from carbaryl watersolubles from bean plants
by acid treatment.
192
-------
in the radiolabel associated with the unextractable phase. Also, there
was a gradual increase in radioactivity associated with the organo-sol-
uble fraction. The nature of the organo-soluble fraction is unclear.
The data would suggest something other than 1-naphthol, since, as we
have seen previously (Table 58), injecting 1-naphthol-l- C into bean
plants leads to almost complete incorporation of the radiolabel into
the unextractable phase and to a much lesser extent into the water-
soluble phase. The preceeding results obtained with carbaryl water-
soluble metabolites, their acid-released aglycones, and 1-naphthol
indicate that phase I or hydroxylated carbaryl metabolites serve as
proximate intermediates to unextractable metabolites. However, it
could be argued that the lack of incorporation of water soluble-metabo-
lites into bound residues, the strongest evidence for their not being
considered as proximate intermediates to this fraction, may be due to
the inaccessibility of these applied metabolites to the cellular sap.
That is, because of their polarity, water soluble metabolites may be un-
able to penetrate the lipoidal cellular membrane and are thus unavail-
able for enzymatic or nonenzymatic reactions within the cell. There-
fore, an in vitro system, using a bean plant homogenate suspended in
phosphate buffer (pH 7.2) and 0.15 M KC1, was used in order to eliminate
cellular barriers. Table 60 presents the results obtained in such a
system using naphthol-1-1^C and l4C-carbaryl aglycones (acid released)
as the substrate. A time dependent incorporation of label into the
polar and unextractable fraction is evident for both substrates. Heat-
ing of the plant homogenate for 30 min at 100°C and cooling to room
temperature prior to addition of radiolabeled naphthol effectively
blocked its incorporation into the polar and unextractable fractions.
The heating process presumably inactivated enzymes involved in the re-
action of 1-naphthol- C, although it is possible that reactive sites
not necessarily involving enzymes may have been changed due to this
drastic treatment. THe rather large percentage (34%) of the l^C-
carbaryl aglycone equivalents associated with the polar phase 1.0 min
after addition of the radiolabel suggests that some reaction other than
enzymatic is involved with this substrate. It is also interesting to
193
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Table 60. IN VITRO METABOLISM OF CARBARYL AND RELATED COMPOUNDS
BY BEAN PLANT HOMOGENATES
% Distribution/min of incubation
Substrate and fraction
14C-Ring-carbaryl
Organosolubles
Water-solubles
Bound
14C-Naphthol
Organosolubles
Water- solubles
Bound
ll+C-Naphthalene
Organosolubles
Water-solubles
Bound
1 ^C-Methoxynaphthalene
Organosolubles
Water-solubles
Bound
1 ^C-Naphthy Iglucoside
Organosolubles
Water-solubles
Bound
11+C-Carbaryl water-solubles
Organosolubles
Water-solubles
Bound
m b
C-Aglycones
Organosolubles
Water-solubles
Bound
1
99
1
0
94
2
4
96
4
0
90
10
0
11
89
0
5
95
0
62
34
4
2.5
99
1
0
_
-
-
97
3
0
-
-
-
13
87
0
4
95
1
_
-
15
99
1
0
93
9
8
98
2
0
79
21
0
13
87
0
5
94
1
64
30
6
30
99
1
0
14
54
32
97
3
0
73
36
1
10
90
0
6
94
0
59
34
7
60
98
2
0
8
62
30
95
5
0
82
17
1
14
86
0
5
94
1
50
39
11
120
99
1
0
_
-
-
-
-
-
90
10
0
9
91
0
5
94
1
-
-
a ^C-Ring-carbaryl water-solubles from bean plants injected with
carbaryl.
b a
Aglycones from released by acid treatment.
194
-------
note that the unextractable metabolites did not appear to be formed at
the expense of the apolar fraction (14C-carbaryl aglycones). Based on
work with the herbicide cisanilide (cis-2,5-dimethyl-l-pyrrolidinecarbo-
anilide), Frear (1967) suggested that the phenolic intermediates in
cisanilide metabolism were further oxidized and served as precursors of
an insoluble residue fraction in plants. He also suggested that the
phenol oxidase system in plants could mediate the formation of these
terminal pesticide residues.
The behavior of a number of substrates in the in vitro system are sum-
marized in Table 60. The fact that the radiocarbon from 1-naphthyl-
C-glucoside and C-carbaryl water soluble metabolites was not incor-
porated into the unextractable fraction suggests that these materials
are not precursors to the bound residues in the intact plant (Table 58).
It appears that compounds which do not have a free hydroxyl group (see
also naphthalene and methoxynaphthalene, Table 60) do not form signifi-
cant amounts of bound residues.
That carbaryl metabolism was not occurring in the bean plant homogenate
even in the presence of NADPH (1 mg/ml reaction mixture) and NaCN (25
yg/ml) was indicated by the lack of incorporation of radioactivity into
the water and solid phases. Sodium cyanide was included in the reaction
medium to reduce phenol oxidase activity which may inactivate oxidative
enzymes via phenol oxidation products (Frear et al. 1969). However,
when the chloroform fractions were subjected to thin-layer chromato-
graphy, only carbaryl was present. Without initial hydroxylation, the
production of conjugates and bound residues obviously does not proceed.
The data obtained thus far indicate that the hydroxylated carbaryl me-
tabolites may be the proximate intermediates in the formation of bound
residues initially formed in carbaryl treated bean plants. That such
hydroxylated derivatives of pesticides may be the proximate intermedi-
ates has been suggested by the work of others. For example, Briggs et
al. (1974) reported that the p-hydroxyphenyl derivative of carboxin
195
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(5,6-dihydro-2-methyl-l,4-oxathiin-3-carboxanilide), the major metabo-
lite of this fungicide in plants, was bound to the lignin fraction of
growing barley plants. Frear and Swanson (1975) reported quantitative
differences in the metabolism of the experimental herbicide cisanilide
in cell cultures and excised leaves. Cell cultures when compared to
excised leaves showed a reduced capacity to form secondary glycoside
conjugates and an increased ability to form methanol-insoluble residues.
The major metabolic pathway for this herbicide in carrots and cotton was
via hydroxylation at the 4-position of the phenyl ring and the 3-posi-
tion of the pyrrolidine ring. Furthermore, these authors found that
carrot cell suspension cultures treated with a radiolabeled aglycone of
cisanilide plant metabolism, 2,5-dimethyl-l-pyrrolidine-4-hydroxy-car-
boxanilide, rapidly incorporated the radiolabel into an extracellular
methanol insoluble residue. Preliminary studies indicated that the ra-
dioactivity was bound to a carbohydrate protein thought to be associated
with the cell wall surface.
196
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RULE OF GLUTATHIONE CONJUGATION IN THE METABOLISM OF CARBARYL IN RATS
While the importance of glutathione conjugation of pesticides has not
been fully elucidated, there is evidence that such conjugation may con-
stitute major routes of detoxification. It is known that glutathione
is involved in the transfer of alkyl groups during the metabolism of
organophosphorus insecticides in animals and insects (Hutson 1972).
Moreover, glutathione conjugation of carbaryl is a proposed metabolic
route for in vitro studies using rat liver homogenate, although the
glutathione conjugate was not isolated (Bend et al., 1971). It was
proposed that conjugation began by oxidation of carbaryl across the
5,6-positions of the naphthalene ring to form the epoxide which reacts
with glutathione. Then, the glutathoine conjugate was excreted in the
bile or subsequently converted to the mercapturic acid. However, the
proposed structures of the glutathione and mercapturic conjugates of
carbaryl were based solely on indirect evidence.
The purpose of this investigation was to determine the significance of
glutathione and mercapturic acid conjugate formation in the detoxifica-
tion of certain aromatic chemicals in rats. Naphthalene was selected as
a model compound and the insecticide carbaryl was studied to see if
these very similar compounds undergo the same metabolic conversion to
glutathione conjugates.
Materials and Methods
Chemicals - Naphthalene-1-1 4C, 1-naphthol-l-1 "*C, and glutathione-3 5S
were purchased from Amersham Searle Company (Chicago, IL) while
bromobenzene-ring-UL-l4C was purchased from California Bionuclear Corp.
(Sun Valley, CA). These chemicals had specific activities of 5.1
mCi/mmol, 15.2 mCi/mmol, 25.7 mCi/mmol and 8.75 mCi/mmol, respectively.
1-Naphthyl-l-14C Npmethylcarbamate (1-naphthyl-l-14C carbaryl) was
prepared from methylisocyanate and 1-napthol- C. Deacetoxydibutyltin
was used as a catalyst (Nye et al., 1976). The 1-naphthyl-l-14C
197
-------
carbaryl, purified by tic, had a specific radioactivity of 15.2 mCi/iranol.
Cochromatography in two tic solvent systems showed that the radioactive
chemical was pure. The carbonyl- C label was synthesized by the
reaction of 1.5 to 1 ratio of 1-naphthol and methyl isocyanate-CO-1'*C
in dry benzene. Two drops of diacetoxydibutyltine was also used as a
catalyst.
For use as metabolite standard for conjugates, S-(1-naphthyl)-glutathione,
S-(1-naphthyl)cysteine, and N-acetyl-S-(1-naphthyl)-cysteine (also called
1-naphthyl mercapturic acid) were synthesized by a modification of the
methods of Park and Williams (1951) and Booth et al. (1960) in which
diazotied 1-naphthylamine and cuprous mercaptide were utilized. The
mercapturic acid was obtained by acetylation of the S-(1-naphthyl)-
cysteine. S-(1-naphthyl)glutathione was also confirmed by heating to
110°C in 6 N HC1 for 24 hrs. The products were examined on tic with
solvent system butanol:acetic acid and water (11:4:5 v/v). Glycine,
glutamic acid, and S-(1-naphthyl)cysteine were detected as ninhydrin-
positive spots having R values of 0.23, 0.28, and 0.74, respectively.
S-(2,4-Dinitrophenyl)glutathione, S-(2,4-dinitrophenyl)cysteine and N-
acetyl-S-(2,4-dinitrophenyl)cysteine were prepared from 2,4-dinitro-
fluorobenzene and their relative S-amino acid derivatives (Sokoloovsky
et al., 1964 and Lamourex, 1975). The reaction mixtures were run in
saturated sodium bicarbonate and 2 ml of ethanol solution. The crude
products were obtained by adjusting reaction mixture to pH 2. The
desired products were recrystallized several times from the crude
products. Structural confirmation for the above compounds was performed
by nuclear magnetic resonance and mass spectrometry. For the NMR spec-
tra, samples were dissolved in DaO containing 1% tetramethylsilane and
measured with a Model-T-60 spectrometer (Varian Associates). Mass
spectra were taken at 70 ev using a Finnegan Model 1015 C quadrupole
mass spectrometer.
198
-------
Radioassay and Chromatography - Radioactive samples were assayed for
14C and 35S by liquid scintillation counting using a Packard Tri-Carb
scintillation spectrometer, Model 3314, equipped with automatic external
standardization. The ether extractable and water soluble C-metabo-
lites were separated on tic (Brinkman precoated silica gel 60-F-254,
250 mm) using the following solvent systems: (A) Ether:hexane 9:1;
(B) Ether:hexane 3:7; (C) Chloroform:acetonitrile 2:1; (D) Hexane:
benzene 1:1; (E) Benzene:acetic acid 95:5; (F) Butanol:acetic acid:
water 11:4:5.
In Vitro Metabolism - Ten samples of fresh rat liver were homogenized
with a Teflon pestle in a glass homogenizer containing 40 ml of 0.1 M
adequate buffer pH. The homogenate was centrifuged at 15,000 g for
10 minutes. One-half of 15,000 g supernatant fraction was centrifuged
at 105,000 for 1 hour to obtain the microsomal fraction and soluble
supernatant. Three 4-ml samples of each fraction were used as the enzyme
source. Control incubations were identical except that the homogenate
was initially heated 30 minutes at 90°C. The incubation mixtures were
then extracted with ether to remove unchanged substrates and unconjugated
metabolites.
Animal Treatment - Six female Sprague-Dawley albino rats (average wt.
200 g) were used for each compound tested. The rats were given intra-
peritoneally a single dose of naphthalene-l-llfC or 1-naphthyl-l-1 "*C
carbaryl at a concentration of 100 mg/kg and 25 mg/kg, respectively.
The animals were housed in glass metabolism chambers where urine and
feces were collected daily.
Analysis of Incubation Mixture and Urinary Radiocarbon - The incubation
mixture was extracted with ether 3 times and the extracts concentrated
to a volume which allowed direct application to silica gel thin-layer
plates. Two-dimension solvent systems were used to resolve the radio-
active components of the urine. Cochromatography with synthetic com-
199
-------
pounds and mass spectroroetry were used to identify the metabolites.
The aqueous layer was lyophilized to dryness and the solid residue
washed with methanol solution (contained 20% of water) repeatedly until
no radioactivity was detected in the wash (10 ml x 3). The methanol
wash was then concentrated for tic analysis. Solvent system F was
used to resolve the radioactive components of the aqueous layer.
Extraction of each component detected by autoradiography from the plates
was by scraping off the radioactive zone, sonicating with methanol
solution, and then centrifuging to remove the gel.
Urine samples were extracted with ether and the aqueous layer divided
into two fractions. The first fraction was handled as above while the
second fraction was cooled to 10°C, adjusted to pH 2 with cold 2 N HC1
and extracted with five equal volumes of ethyl acetate. The ethyl
acetate was concentrated and chromatographed on tic plates with solvent
systems E and F.
Acid and Basic Hydrolysis of Water-soluble Metabolites - Portions of
each water-soluble metabolite were incubated in 1 N HC1 at 40°C for
24 hrs. The incubations were conducted in a sealed tube containing 4 ml
of ether. Periodic shaking of the tubes extracted the hydrolyzed exocons
(or non-polar products) into the ether layer to prevent further decom-
position. The ether extracts were combined and analyzed by tic. The
aqueous solution was lyophilized to dryness and the solid residue washed
with methanol solution. The methanol solution was then concentrated,
and applied to tic. Solvent system F was used to resolve the decomposi-
tion products.
Acid hydrolysis of glutathione conjugates was conducted in 50 yl of
6 N HC1, containing 20 yg of sample (glutathione conjugate or peptide
amino acid). The mixture was contained in a 10-ml sealed tube and
heated to 110°C for 24 hrs (Frear et al. , 1973). Following hydrolysis,
the reaction mixture was taken to dryness under reduced pressure. Basic
200
-------
hydrolysis was effected by heating the glutathione conjugates in 1 N
NaOH solution (2 ml) at 40°C for 2 hr. In all hydrolysis, the exocons
(or non-polar products) were isolated by ether extraction and subjected
to tic analysis.
Enzymatic Hydrolysis - Enzyme preparations were utilized to cleave
possible conjugated water-soluble metabolites. The enzymes (?~glucuron-
idase bacterial (powder), arylsulfatase (powder) and acetylase (from
hog kidney) were purchased from Sigma Ltd. (St. Louis, Mo.). (3-Gluc-
uronidase hydrolysis of the conjugates (1.0 ml) was conducted in 0.1
M phosphate buffer pH 6.2 (2 ml) containing $-glucuronidase (10 units).
The reaction mixture was incubated for 24 hr at 37°C.
Arylsulfatase hydrolysis was effected by incubation of 10 units for 24
hr at 37°C with the conjugates (1.0 ml in water) and 0.1 M sodium
acetate-acetic buffer, pH 5.2 (2 ml). Acylase was handled in 0.1 M
phosphate buffer pH 7.4 (2 ml) containing 5 units of acylase and
conjugates. The reaction mixture was incubated for 16 hr at 37°C.
Amino Acid Determination - Ninhydrin reagent and the amino acid analyzer
were used to detect amino acid or peptide conjugates. The ninhydrin
reagent contained 300 mg of ninhydrin in 100 ml of 1-butanol. After
the ninhydrin had dissolved, 3 ml of glacial acetic acid were added to
the 1-butanol solution. After spraying the plates, color was developed
by heating the plates for 10 to 20 min at 100°C. The TechnicorT*
Sequential Multi-Sample Amino Acid Analyzer (TSM) was used to quantitate
the amino acids, glutamic acid and glycine. The column operating
conditions were following: flow rate 0.5 ml/min, temperature 60°C,
resin bed 23.0-23.5 cm, resin type C-3.
Determination of Sulfur - Sulfur-containing compounds were located on
tic by spraying 0.1 M potassium dicromate and acetic acid (1:1 v/v),
followed by 0.1 M silver nitrate (Knight and Young, 1958). The sulfur-
containing compounds appeared as yellow spots on the plates.
201
-------
Results and Discussion
Glutathione and Mercapturic Asid Conjugation of Naphthalene and Carbaryl
in the Rat - Optimum substrate concentration and cofactors used in the
metabolism of naphthalene-l-llfC and l-naphthyl-l-lifC carbaryl in vitro
were obtained from previous studies (Jerina et al. , 1970; Bend et al.,
1971) . The amount of substrates used was 512 ug and 40 Ug for naphtha-
lene-1- "*C and 1-naphthyl-l- C carbaryl, respectively. Cofactors were
the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH,
0.115 mg), magnesium chloride (MgCla, 6.41 mg), methylnicotinamide
(6.34 mg), glucose-6-phosphate (G-6-P, 3.92 mg), glucose-6-phosphate
dehydrogenase (G-6-PDH, 1 unit) and glutathione (1.0 mg).
The pH effect on conjugation was determined by the total amount of
radioactivity transferred to the water layer after 30 min of incubation.
Therefore, this assay included all types of conjugation and not just
that attributed to glutathione. Different regions of optimum pH were
observed for naphthalene-l-^C (pH 8.0-8.5 for tris HC1 buffer and
pH 7.6-8.0 for phosphate buffer) and for 1-naphthyl-l-lkC carbaryl
(pH 6.9-7,7 for phosphate buffer). This indicated that two different
kinds of enzymes were involved in the conjugation of naphthalene and
carbaryl.
The subcellular distribution of enzyme effective in the metabolism of
naphthalene and carbaryl was investigated in an attempt to locate the
fraction which processed the maximum enzymatic conjugative activity
(Table 61 ). Two centrifugal fractions, one containing the 15,000 g
supernatant and the other the 105,000 g precipitate plus 105,000 g
supernatant, formed the highest amount of conjugation (above 60%) as
compared to crude homogenate, microsomal and soluble fractions. About
15% conjugation was obtained in the incubation of naphthalene with
microsomes as compared to 3.5% in the control incubation mixture; only
6.2% conjugation occurred with carbaryl as compared to 4.2% of the
control incubation mixture. The 15% conjugation of naphthalene by
202
-------
Table 61. CONJUGATION OF NAPHTHALENE-1-l^C AND CARBARYL BY
DIFFERENT SUBCELLULAR FRACTIONS OF RAT LIVER HOMOGENATE3.
b
% Conjugation
Centrifugal fraction Naphthalene Carbaryl
Control 3.5 4.1
Crude homogenate 47.6 38.2
15,000 g Supernatant 72.2 64.7
105,000 g Precipitate
(microsomal) 15.5 6.2
105,000 g Supernatant
(soluble) 3.6 4.5
105,000 g Precipitate
plus 105,000 g
c
supernatant 68.4 62.1
a
Substrates: naphthalene 512 Ug, carbaryl 40 Ug, cofactors: NADPH
0.115 mg, magnesium chloride 6.41 mg, methylnicotinamide 6.34 mg,
glucose-6-phosphate 3.92 mg. Glucose-6-phosphate dehydrogenase
1 unit, glutathione 1000 yg.
Conjugation was taken as that quantity of radiocarbon converted to
a water soluble form during incubation.
2 ml of 105,000 g precipitate and 2 ml of 105,000 g supernatant
were used in the incubation mixture.
203
-------
microsomes probably was catalyzed by GSH-S-transferase which was not
completely removed from pellet.
The 15,000 g supernatant converted naphthalene and carbaryl to
conjugation products to a greater extent than did the other fractions.
This system was routinely used as a source for conjugation in addition-
al studies. Attempts to obtain even greater conjugation by increasing
the incubation time beyond 30 min were unsuccessful.
Formation of naphthalene conjugates by the 15,000 g supernatant increas-
ed from 34% to 74% upon addition of from 0-600 yg of glutathione, where-
as formation of carbaryl and 1-naphthol conjugates was independent of
additional glutathione. This indicated that glutathione conjugation
occurred with naphthalene but not with carbaryl. It was also clear
that glutathione reacted with a metabolite of naphthalene produced by
the microsomes and not with the parent compound per se.
Metabolites Formed by Rat-Liver Enzymes - In order to determine the
nature of metabolites from glutathione conjugation, the cross-labeled
preparations of substrates were incubated separately with the 15,000 g
supernatant for 10 minutes. The cross-labeled preparations of substrates
contained five different sets of substrates in the experiment (Table
62 ). By using these combinations, it was possible to determine the
presence or absence of the glutathione and naphthol moieties in the
metabolites. The radiocarbon in each preparation was separated into
three fractions and the water-soluble radiocarbon further separated by
tic. Eight different radioactive bands were observed. Incubation with
naphthalene-l-^C plus nonradioactive GSH and naphthalene plus GSH-35S
showed one metabolite (III) with the same R value and was, therefore,
considered as a GSH conjugate. No metabolite with the same Rf value
in the cross-labeled preparations of carbaryl and glutathione was
observed; thus, no GSH metabolite of the carbamate was evidenced.
204
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Identification of the ether-soluble metabolites was attempted first,
since information gained might help in the identification of conjugates.
As shown in Table 62, 32% of the radiocarbon was extracted into ether
from the incubation mixture using naphthalene-1- **C and GSH. Four
metabolites (two-dimensional solvent systems A and C) were detected in
this extract (Table 63). The l,2-dihydro-l,2-dihydroxy naphthalene
accounted for 40% of the ether-soluble metabolites. The mass spectrum
of this compound was complex but similar to a spectrum of 1-naphthol.
The major parent ion, m/e 162, suggested incorporation of 2 oxygens and
2 hydrogens into the naphthalene ring. An ion of m/e 144, the molecular
ion for 1-naphthol, and another ion at m/3 129 which showed the same
abundance as m/e 128 suggested one more hydrogen relative to naphthal-
ene. This indicated that the ion at m/e 129 was a hydro form of the
naphthalene ring. 1-Naphthol (85%) and 2-naphthol (10%) were obtained
after acid hydrolysis of l,2-dihydro-l,2-dihydroxy naphthalene, indi-
cating that a water molecule was removed from the original compound.
The existence of l,2-dihydro-l,2-dihydroxy naphthalene as a metabolite
was consistent with previous findings (Jerina et al., 1970).
Ether-soluble metabolites obtained from incubation mixtures of 1-
naphthyl 1-1'*C carbaryl plus nonradioactive GSH were primarily 5,6-
dihydro-5,6-dihydroxy 1-naphthyl N-methylcarbamate (28%) and 1-naphthyl
N-hydroxymethylcarbamate (40%). Equal amounts of these two metabolites
were also found by using carbonyl-1'*C carbaryl plus nonradioactive GSH.
Water-soluble Metabolites - The identification of water-soluble meta-
bolites I to VIII (Table 62) were attempted by enzymatic and acid
hydrolysis (Table 64). Exocons released from the enzymatic and acid
hydrolysis were identified by comparison of their tic behavior and mass
spectra with those of authentic standards. The characteristics of
each water-soluble metabolite I to VIII are described individually as
follows.
206
-------
Table 63. RADIOACTIVE COMPONENTS OF THE ETHER FRACTION FROM
IN VITRO METABOLISM OF NAPHTHALENE-1-J "*C AND 1-NAPHTHYL-l- 1 4C CARBARYL
% of C in sample
Metabolite Naphthalene Naphthyl-1- Carbonyl-
1- C C carbaryl 1 C carbaryl
Unknown origin material 3.1 2.0 1.8
5,6-Dihydro-5,6-
dihydroxy 1-naphthyl
N-methylcarbamate - 28.3 30.1
l,2-Dihydro-l,2-
dihydroxy naphthalene 40.6
1-Naphthyl N-hydroxy-
methylcarbamate - 40.5 41.4
Carbaryl
1-Naphthol
Naphthalene
Total recovery
6.2
43.7
93.6
20.6 26.3
-
-
91.4 99.6
Ether fraction constituted 32.0%, 24.7%, 26.5% of the radiocarbon
added for naphthalene-l-llfC, naphthyl-l-^C carbaryl, carbonyl-14C
carbaryl, respectively.
- Indicates that no radiocarbon was detected.
207
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I: This was tic origin material. Obtained only from S-labeled GSH
plus naphthalene and carbaryl, and did not release ether extractable
radiocarbon when subjected to enzymatic and acid hydrolysis.
II: Obtained only from 35S-labeled, and not converted to ether-soluble
materials by enzymatic and acid hydrolysis. It gave a positive response
to ninhydrin reagent, and had a R value of 0.23 in solvent system P,
which corresponds to that reported for oxidized glutathione (GSSG) by
Shishido et al., 1972. Therefore, metabolite II was considered to be
an oxidized form of glutathione.
i ^
III: Obtained from naphthalene-1- C plus nonradioactive GSH and could
not be separated from the metabolite III (by tic in solvent system F)
obtained from naphthalene plus GSH- S when equal amounts of radioacti-
vity from both sets of substrates were mixed together. This indicated
that both metabolites obtained from different labels ( C and S) were
identical.
The C-metabolite III treated with glucuronidase and sulfatase convert-
ed no radioactivity to ether-soluble materials, and, thus, was not a
glucuronic acid or a sulfate conjugate. A positive response to ninhyd-
rin reagent was obtained as was a positive test to the KzC^Ov-AgNOa
reagent, indicating that a free a-amino and sulfur were contained in the
molecule. The metabolite chromatographed identically with synthetic
S-(1-naphthyl)glutathione when treated with mild acid. This indicated
that metabolite III was an acid labile compound, probably S-(2-hydroxy-
1,2-dihydro 1-naphthyl)glutathione which was proposed by Jerina et al.,
1970.
Tic analysis of the 6N HC1 hydrolysis mixture of metabolite III showed
5 radioactive compounds, plus 2 nonradioactive products, glutamic acid
and glycine (Table 65). The major hydrolytic product, S-(1-naphthyl)-
cysteine, was identified by comparison of its tic behavior and mass
spectra with that of authentic standards. S-(1-Naphthyl)cysteinyl
209
-------
Table 65. PRODUCTS FORMED UPON TREATMENT OF 2-HYDROXY
I^-DIHYDRO-S-CNAPHTHYL-I-^OGLUTATHIONE (iu)a WITH
6 N HCL AT 110°C FOR 24 HR.
Rf of product
1.00
0.74
0.60
0.51
0.46
0.25°
0.16C
Total 1>*C
% of 14C
applied to
tic
4.4
52.2
7.4
13.1
7.3
-
-
90.4
Tentative identification
1-Naphthol
S- (1-Naphthyl) cysteine
S- (1-Naphthyl) cysteinyl
glutamic acid
S- (1-Naphthyl) cysteinyl
glycine
S- ( 1-Naphthyl) glutathione
Glutamic acid
Glycine
See Table 62 for position on tic and source of metabolite.
b
Reaction mixture was applied to 250 mm silica gel chromatoplates
and developed in solvent system F.
£j
Products were located by ninhydrin spray.
210
-------
glutamic acid. These two resultant products showed the incomplete
cleavage of the peptide bond in the glutathione moiety. These charac-
teristics were also found in the hydrolysis of the synthetic compound
S-(1-naphthyl)glutathione. From the combined information, the chemical
structure of metabolite III was considered to be S-(2-hydroxy-l,2-di-
hydro 1-naphthyl)glutathione.
14 14
IV: Obtained from 1-naphthyl-l- C carbaryl and carbonyl- C carbaryl
and showed no separation of radioactivity by tic in solvent system F
when equal amounts of radioactivity from ring and carbonyl labeled
were mixed together. Acid and enzyme hydrolysis gave 1-naphthyl N-
hydroxymethylcarbamate. The radiocarbon which could not be hydrolyzed
by acid was concentrated and reapplied to tic. Solvent system F was
used and unreacted original metabolite IV was obtained. The amount of
hydrolysis by $-glucuronidase and sulfatase was similar (21% with B-
glucuronidase and 15% with sulfatase). Therefore, it was concluded
that metabolite IV consisted of glucuronide and sulfate esters of
1-naphthyl N-hydroxymethylcarbamate.
V: The metabolite was also obtained with carbonyl-1 C carbaryl (Table
62 ). This indicated that the carbamate ester was still intact as
with metabolite IV. Acid and enzyme hydrolysis released the same
exocon as did metabolite IV, i.e., 1-naphthyl N-hydroxymethylcarbamate.
The radiocarbon which could not be hydrolyzed by acid was reapplied on
tic. The results showed that metabolite IV was observed in addition to
unreacted original metabolite V. This evidence indicated that metabo-
lite IV was derived from V by partial cleavage of the conjugate. While
not confirmed experimentally, metabolite V may have been the N-hydroxy-
methyl analog conjugated both on the nitrogen and on the hydroxymethyl
moiety. Thus, complete cleavage of the conjugate would give the same
exocon as obtained with metabolite IV. But, cleavage of just the N-
conjugate would yield metabolite IV directly.
211
-------
VI: Metabolite VI derived from naphthalene was hydrolyzed by &-gluc-
uronidase (79%) and sulfatase (18%) (Table 64) - The exocon released
from enzymatic hydrolysis was identified as l,2-dihydro-l,2-dihydroxy
naphthalene which was chromatographically and mass spectrally identical
to l,2-dihydro-l,2-dihydroxy naphthalene obtained as an ether-soluble
metabolite. Acid hydrolysis gave two products, 1,2-dihydro-l,2-di-
hydroxy naphthalene and 1-naphthol. Their identities were confirmed by
mass spectral analysis. Radiocarbon left in the aqueous layer after
acid hydrolysis of metabolite VI showed two radioactive metabolites.
They cochromatographe'd with the original metabolite VI and 1-naphthyl
glucuronide. From the above evidence, metabolite VI was identified as
l,2-dihydro-2-hydroxy 1-naphthyl glucuronide.
VII: Metabolite VII was obtained from l-naphthyl-l-1'*C carbaryl plus
1 U-
nonradioactive GSH but was not obtained from carbonyl- C carbaryl.
This indicated that the structure of metabolite VII did not contain
the carbamate ester. Enzyme and acid treatment of metabolite VII
yielded an exocon which chromatographed with 5,6-dihydro-5,6-dihydroxy
naphthol. Its identity was confirmed by mass spectral analysis. The
amounts of exocon released by treatment with glucuronidase and sulfa-
tase were not significantly different (68% and 61% respectively).
Therefore, the metabolite VII was considered to be 5,6-dihydro-5,6-
dihydroxy 1-naphthyl glucuronide or sulfate.
VIII: Incubation of metabolite VIII with 3-glucuronidase and sulfatase
separately for 24 hr at 37°C cleaved 60% and 57% of the conjugate,
respectively. Acid treatment yielded 96% ether-soluble materials which
was in contrast to a control incubation containing all constituents
except the acid where only 3% ether-soluble materials were found. The
exocon released from the above hydrolysis was identified as 5,6-dihydro-
5,6-dihydroxy 1-naphthyl N-methylcarbamate.
In summary, analysis of the water-soluble metabolites III and VI
6btained from the metabolism of naphthalene-l-^C in vitro showed that
212
-------
glutathione conjugation was a major type of conjugation in the metabo-
lism of naphthalene. On the contrary, glucuronic acid and sulfate
conjugation were the major types of conjugation in the metabolism of
carbaryl. Even though naphthalene and carbaryl have a similar phase I
or primary metabolic pathway, formation of an epoxides and hydroxyla-
tions, their conjugation processes are quite different.
In Vivo Studies: Excretion - In the first 24 hr, the ^C found in the
urine was 62% of the administered carbaryl dose, while it was 24% for
naphthalene. Total ll*C recovery from the rats dosed with naphthalene-
l-^C was 74% after 72 hr, with 60% in the urine and 14% in the feces.
For l-naphthyl-l-ll*C carbaryl total 14C recovery after 72 hr was 84%;
80% was in the urine and 4% in the feces.
Urinary llfC Metabolites - Approximately 6% and 16% of the radiocarbon
could be extracted by ether from the urine of rats dosed intraperiton-
eally with naphthalene-l-1'*C and l-naphthyl-l-14C carbaryl respective-
ly. In two dimension solvent system A and C, 3 metabolites from
naphthalene-1-14C and 8 metabolites from l-naphthyl-l-^C carbaryl were
detected in ether extract (Table 66). 1,2-Dihydro-1,2-dihydroxy
naphthelene (28%) and 1-naphthol (60%) were major ether-soluble meta-
bolites in urine of rats dosed with naphthalene-l-llfC. 5,6-Dihydro-
5,6-dihydroxy naphthol (27%), 4-hydroxy 1-naphthyl N-methylcarbamate
(12%) and 5,6-dihydro-5,6-dihydroxy 1-naphthyl N-methylcarbamate (12%)
were highest in the ether extract of urine of rats dosed with 1-naph-
i k
thyl-1- C carbaryl. The occurrences of these ether-extractable
metabolites in the urine of rats dosed with naphthalene-1-14C and
1-naphthyl-l- C carbaryl were consistent with the previous studies
(Boyland et al., 1958 and Dorough, 1970).
Four radioactive water-soluble metabolites (IX-XII) were isolated from
the urine of rats given naphthalene-1-14C intraperitoneally (Table 67 )
Metabolite XI was the major metabolite, accounting for 65% of total
radiocarbon in the water-soluble fraction. Seven radioactive water-
213
-------
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214
-------
Table 67. WATER-SOLUBLE RADIOACTIVE METABOLITES IN THE URINE
EXCRETED DURING A 72-HR PERIOD AFTER RATS WERE TREATED
WITH NAPHTHALENE- l-^C.
"/
1.00
0.87
0.81
0.72
Total Recovery
Designation
IX
X
XI
XII
% of total radiocarbon
in the water soluble
fraction
5.2
8.0
65.0
16.8
95.0
Solvent system F was used
Water soluble fraction contained 94.0% of radiocarbon in the total
urine.
215
-------
soluble metabolites were obtained from the urine or rat dosed with 1-
naphthyl-l-li+C carbaryl (Table 68). The amount of each metabolite (XIII-
XIX) ranged from 7% to 17%, but metabolite XVII was the major one. The
identification of water-soluble metabolites IX to XIX was by comparison
of the tic behavior and mass spectra of exocons released by enzyme or
acid hydrolysis (Table 69 and 70) with those of authentic standards.
IX: This metabolite(s) remained at the tic origin and accounted for
only 6% of the water-soluble fraction. No attempt was made to identify
the product.
X: This metabolite was hydrolyzed by arylsulphatase; 77% of the
conjugate was an exocon chromatographically and mass spectrally iden-
tical to l,2-dihydro-l,2-dihydroxy naphthalene. Acidic hydrolysis of
the metabolite X gave two exocons which cochromatographed with 1,2-
dihydro-l,2-dihydroxy naphthalene and 1-naphthol in solvent system A.
The radiocarbon which could not be hydrolyzed by acid was unreacted
metabolite X. Based on the above evidences, metabolite X was apparently
a sulfate ester of 1,2-dihydro-l,2-dihydroxy naphthalene.
XI: No ether-soluble exocon was released from this metabolite by
3-glucuronidase and arylsulphatase, but ether-soluble radiocarbon (87%)
was released by 3 N HC1 (Table 69). Three compounds were obtained by
using solvent system F and chromatography and mass spectroscopy showed
that these three compounds corresponded to 1-naphthol, N-acetyl-S-(1-
naphthyDcysteine (R 0.90), and S-(1-naphthyl) cysteine (R 0.74) with
percentages of 2%, 84% and 10%, respectively. The same compounds were
obtained by basic hydrolysis, but with different percentages of each
compound, 25%, 37% and 34%, respectively. The endocons which stayed in
the water-soluble fraction after acid and base hydrolysis were located
by spray agent. N-Acetylcysteine and cysteine were recognized as two
endocons in the reaction mixture after metabolite XI was treated with
acid and base. Acylase treatment of metabolite XI (ninhydrin negative)
released an unknown compound with R o.72 which was ninhydrin positive.
216
-------
Table 68. WATER-SOLUBLE RADIOACTIVE METABOLITES IN URINE EXCRETED
DURING A 72-HR PERIOD AFTER RATS WERE TREATED WITH
1-NAPHTHYL-1-*"c CARBARYL.
*fa
0.98
0.90
0.88
0.84
0.81
0.75
0.60
Total
Designation
XIII
XIV
XV
XVI
XVII
XVIII
XIX
% of the radiocarbon in
water-soluble fraction*3
12.0
8.0
14.0
7.0
17.4
16.3
15.7
90.4
a
Solvent system F was used in this experiment.
Water-soluble fraction contained 84% of radiocarbon in the total
urine.
217
-------
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This unknown compound (R 0.72) was converted into S-(1-naphthyl)cyste-
ine by acid hydrolysis. From the above evidence, this unknown (R 0.72)
compound probably was S-(l,2~dihydro-2-hydroxy-l-naphthyl)cysteine which
was proposed by previous study (Boyland et al., 1958).
Further evidence for the identification of metabolite XI was obtained
from the mass snectral data (Table 71). The mass spectra of meta-
bolite XI analogs, such as N-acetyl-cysteine, cysteine, N-acetyl-S-
(1-naphthyl)cysteine, and S-(1-naphthyl)cysteine were also obtained
iii order to assist in assigning the spectrum of metabolite XI. The
relative abundance of M and the prominent fragments, over m/e 40, of
those analogs are listed in Table 71. The molecular ion of metabolite
XI (m/e 264) was not seen. The major breakdown under electron impact
was either loss of water to yield N-acetyl-S-(1-naphthyl)cysteine or
loss of the N-acetyl-cysteine to yield naphthol (m/e 144, 12%). The
resultant product, N-acetyl-S-(1-naphthyl)cysteine, was very unstable
under electron impact. Two principle cleavages of N-acetyl-S-(1-naph-
thyl) cysteine under electron impact was proposed from the mass spec-
trum of N-acetyl-S-(1-naphthyl)cysteine and its analogs, N-acetyl-
cysteine and S-(1-naphthyl)cysteine. The first cleavage was between
the naphthalene and sulfur bond, to yield ions N-acetylcysteine m/e
163 (2%) and naphthyl ring m/e 128 (40%). The second cleavage was
alpha to the naphthalene ring, to yield the 1-naphthalenethiol m/e 160
(30%) arising via a hydrogen transfer process. The prominent ion, at
m/e 60, in the mass spectrum of metabolite XI was associated with
fragmentation of N-acetylcysteine molecule.
From a previous study (Boyland et al., 1958), the structure of meta-
bolite XI was not fully elucidated. It was generally identified by
comparing the tic behavior of its acid hydrolytic product with synthe-
tic compound N-acetyl-S-(1-naphthyl)cysteine. Based on the above in-
formation, metabolite XI was identified as S-(l,2-dihydro-2-hydroxy
1-naphthyl)cysteine.
220
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XII: Metabolite XII was identical to metabolite VI (l,2-dihydro-2-
hydroxy 1-naphthyl glucuronide), obtained from metabolism of naphthal-
ene in vitro.
The water-soluble metabolites (XIII to XIX) obtained from the urine
of rats dosed with 1-naphthyl carbaryl were analyzed (Table 70) using
the same techniques as those from the urine of rats dosed with naph-
thalene-l-^C. The data obtained relative to metabolite XIII to XIX
are described as follows:
XIII: 5,6-Dihydroxy-5,6-dihydroxy naphthol accounted for 50% of the
radiocarbon in the ether fraction and 3,4-dihydro-3,4-dihydroxy
naphthol accounted for 50%. The amounts of exocons released by $-
glucuronidase and sulfatase (26% and 32% respectively) were similar.
Therefore, metabolite XIII was tentatively identified as mixtures of
glucuronide and sulfate conjugated forms of 5,6-dihydro-5,6-dihydroxy
naphthol and 3,4-dihydro-3,4-dihydroxy naphthol.
XIV: Enzymatic and acid hydrolysis of metabolite XIV resulted in
23% ether-soluble materials with 3-glucuronidase, 69% with sulfatase
and 50% with acid. Two exocons in equal amounts were released from
the hydrolyses and were identified as 4- and 5-hydroxy 1-naphthol N-
methylcarbamate. Therefore, metabolite XIV was identified as glucuro-
nide and sulfate conjugates of 4- and 5-hydroxy 1-naphthyl N-methyl-
carbamate.
XV: Metabolite XV was identical to metabolite VIII (5,6-dihydro-5,6-
dihydroxy 1-naphthyl N-methylcarbamate glucuronide) obtained from the
metabolism of 1-naphthyl-l-llfC carbaryl in_ vitro.
XVI: 1-Naphthyl sulfate was determined as the identity of metabolite
XVI by identical tic mobility of metabolite XVI and 1-naphthyl sulfate.
Sulfatase hydrolysis (50% of conjugate) of standard 1-naphthyl sulfate
and metabolite XVI substantiated that metabolite XVI was 1-naphthyl
222
-------
sulfate.
XVII: This metabolite was identified as the glucuronide of 1-naphthol
by cochromatography with 1-naphthyl glucuronide. The exocon released
from enzymatic and acid hydrolysis was 1-naphthol.
XVIII: Metabolite XVIII was identical to metabolite VII (5,6-dihydro-
5,6-dihydroxy 1-naphthyl glucuronide and/or sulfate) obtained from the
metabolism of carbaryl in vitro.
XIX: Metabolite XIX was identical to metabolite IV (1-naphthyl N-hy-
droxymethylcarbamate glucuronide/sulfate, obtained from the metabolism
of carbaryl in vitro.
Results of acid hydrolysis of water-soluble metabolites XIII through
XIX obtained from the urine of rats dosed with l-naphthyl-l-1'*C car-
baryl substantiated those obtained by enzyme hydrolysis. Each water-
soluble metabolite which remained in the aqueous layer after treatment
with acid and enzyme was identified as the original water soluble
metabolite. This indicated that mercapturic acid was not formed in
the urine of rats dosed with carbaryl.
Previously, the mercapturic acid conjugate of bromobenzene was
successfully isolated from rat urine after being adjusted to pH 2
(Gillham, 1968). The same procedure was used for detecting the mer-
capturic acid in the urine of rats treated with naphthalene-l-ll+C and
l-naphthyl-l-^C carbaryl. The results showed that N-acetyl-S-(1-naph-
thyl) cysteine, called 1-naphthyl mercapturic acid, was successfully
isolated from the urine of rats treated with naphthalene-I-1V (Table
72 ). The results also showed that the free dihyrodiol compounds, plus
1-naphthyl glucuronide and 1-naphthyl sulfate were extractable from the
pH 2 urine of rats treated with l-naphthyl-l-ll*C carbaryl. This
evidence indicated that no mercapturic acid derivatives of carbaryl
were present in the urine of rats treated with the carbamate.
223
-------
Table 72. RADIOACTIVE COMPONENTS IN THE ETHYL ACETATE OF URINE
(ADJUSTED TO PH 2) EXCRETED DURING A 72-HR PERIOD AFTER RATS TREATED
WITH NAPHTHALENE- l-^C AND 1-NAPHTHYL-l-l "*C CARBARYL
Naphthalene
Carbaryl
Metabolite
% of total
radioactivity
in the urine
Metabolite
% of total
radioactivity
in the urine
N-Acetyl-S-
(1-naphthyl)-
cysteine
1-Naphthyl
glucuronide
Sub Total
EtOAC Extract
before adjust
to pH 2
Remaining
as water
soluble
Total
56.0
12.0
68.0
6.0
18.2
92.2
5,6-Dihydro-5,6-dihy-
droxy 1-naphthyl
N-methylcarbamate
3,4-Dihydro-3,4-dihy-
droxy naphthol
5,6-Dihydro-5,6-dihy-
droxy naphthol
1-naphthyl sulfate
1-naphthyl glucuronide
Sub Total
2.4
6.5
8.7
3.8
6.7
26.1
16.7
47.0
89.8
224
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In summary, the mercapturic acid conjugate was not found in the meta-
bolism of carbaryl. Bend et al. (1971) claimed that when the water-
soluble metabolites in the urine of rats dosed with carbaryl were
treated with acid, the water-soluble metabolite left in the aqueous
layer after acid hydrolysis was a thioether amino acid conjugate. The
detailed procedure of Bend's was followed to generate the compound
reported to be a thio amino acid conjugate. The compound gave an
identical R value on tic in solvent system F with metabolite XVIII.
Hydrolysis of this "thioamino acid conjugate" to the extent of about
19% occurred with 3-glucuronidase, about 17% occurred with sulfatase,
and about 24% occurred with acid. 5,6-Dihydro-5,6-dihydroxy 1-naphthyl
N-methylcarbamate (76%) and its hydrolysis product, 5,6,-dihydro-5,6,-
dihydroxy naphthol (22%) were detected by chromatographic analysis of
the extracts from enzyme and acid hydrolysis. These data proved that
Bend's metabolite was not a thioether amino acid conjugate.
225
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V PUBLICATIONS
1. Dorough, H. W., J. P. McManus, S. S. Kumar and R. A. Cardona.
1974. Chemical and metabolic characteristics of 1-naphthyl-B-D-
glucoside. J. Agr. Food Chem. 22(4): 642-45.
2. El-Shourbagy, N. A., and H. W. Dorough. 1974. Glycoside conjuga-
tive activity in different insect and vertebrate species. J. Econ.
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3. Lin, T., and H. W. Dorough. 1974. Influence of insecticide expo-
sure to rats on in vivo and in vitro metabolic activity. Arch.
Environ. Contam. and Toxicol. 2(4): 365-77.
4. Kumar, S. S. , and H. W. Dorough. 1974. Glucosylation by housefly
microsomes and effect of monoamine oxidase inhibitors. J. Insect
Biochem. 5: 1-9.
5. Dorough, H. W., and Y. H. Atallah. 1975. Cigarette smoke as a
source of pesticide exposure. Bull. Environ. Contam. and Toxicol.
13(1) : 101-107.
6. Atallah, Y. H., and H. W. Dorough. 1975. Insecticide residues in
cigarette smoke: transfer and fate in rats. J. Agr. Food Chem.
23(1): 64-71.
7. Dorough, H. W., and J. H. Thorstenson. 1975. Analysis for carba-
mate insecticides and metabolites. J. Chromat. Sci. 13(5): 212-
224.
8. Atallah, Y. H., H. W. Dorough and J. H. Thorstenson. 1975. Nature
and fate of insecticide residues inhaled by rats in cigarette smoke.
Drug Metabolism and Disposition 3(6): 513-519.
9. Nye, D. E., and H. W. Dorough. 1976. Fate of insecticides admin-
istered endotracheally to rats. Bull. Environ. Contam. Toxicol.
15(3): 291-96.
10. Nye, D. E., H. E. Hurst, and H. W. Dorough. 1976. Fate of Crone-
ton (2-ethylthiomethylphenyl N-methylcarbamate) in rats. J. Agr.
Food Chem. 24(2): 371-77.
11. Marshall, T. C., H. W. Dorough and H. E. Swim. 1976. Screening of
pesticides for mutagenic potential using Salmonella typhimurium
mutants. J. Agr. Food Chem. 24(3): 560-63.
12. Kuhr, R. J., and H. W. Dorough. 1976. Carbamate Insecticides:
Chemistry, Biochemistry and Toxicology. CRC Press, Cleveland.
301 pp.
13. Dorough, H. W. 1976. "Biological activity of pesticide conjugates",
in Bound arid Conjugated Pesticide Residues. American Chemical
Society Symposium Series No. 29. pp. 11-34. Eds. D. D. Kaufman,
G. G. Still, G. D. Paulson and S. K. Bandal.
234
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14. Huhtanen, K., and H. W. Dorough. 1976. Isomerization and Beckmann
rearrangement reactions in the metabolism of methomyl in rats.
Pest. Biochem. and Physiol. (In Press).
15. Rodriguez, L. D., and H. W. Dorough. 1976. Degradation of carba-
ryl by soil microorganisms. Arch. Environ. Contarn, and Toxicol.
(In Press).
16. Dorough, H. W., and D. E. Nye. 1977. "Fate of Croneton (2-ethyl-
thiomethylphenyl N-methylcarbamate) in large animals" in Fate of
Pesticides in Large Animals, Eds. G. W. Ivie and H. W. Dorough,
Academic Press, N. Y. (In Press).
17. Hurst, H. E., and H. W. Dorough. 1977. A convenient method of
synthesis of 14C-carbonyl methylcarbamates. J. Labelled Compounds
and Radiopharmaceuticals. (In Press).
Thesis and Dissertations
1. El-Shourbagy, N. A. Glycoside Conjugation in Insects and Mammals,
Masters Thesis. 1972.
2. Lin, T-H. Influence of Selected Biologically Active Chemicals on
the Mammalian Metabolism of Carbamate Insecticides, Ph.D. Disserta-
tion. 1972.
3. Rodriguez, L. D. Studies in Degradation of Carbaryl by Soil Micro-
organisms, Ph.D. Dissertation. 1973.
4. Hurst, H. E. Chemical, Metabolic and Toxicological Considerations
of the Insecticide Croneton (2-Ethylthiomethylphenyl Methylcarba-
mate), Masters Thesis. 1976.
5. Marshall, T. C. Potential Toxicological Significance of Conjugate
and Bound Residues of Carbamate Insecticides Formed by Plants,
Masters Thesis. 1976.
6. Chen, K. C. Role of Glutathione Conjugation in the Biochemical
Degradation of Pesticides, Ph.D. Dissertation. 1976.
235
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VI SUMMARY
The current research on "Metabolism of Carbamate Insecticides" was con-
ducted under a continuation grant (R-802005) sponsored by EPA for the
period covering February 1, 1974 through January 31, 1977. A detailed
progress report covering the prior project period was submitted in
September 1972 and is identified as Report Number EPA-650/1-74-002. The
present summary progress report consists of abstracts of published works
and summaries of unpublished data resulting from research funded by EPA
Grant Number R-802005 since 1973.
Isomerication and Beckmann Rearrangement Reactions in the Metabolism of
Methomyl in Rats - Methomyl {S-methyl-N-[(methylcarbamoyl)-oxy]thioacet-
imidate}, also known as Lannate, may exist in two geometric configura-
tions and the more stable syn [ll*C=N] methomyl [CH3S (CH3)C=NOC(O)NHCH3]
was metabolized to respiratory ltfCC>2 and CH314C=N in a ratio of about 2
to 1. Studies with the anti isomer showed that it was metabolized pre-
dominantly to CH311+CSN. These and other data are presented supporting
the contention that syn methomyl is partially isomerized to the anti
isomer in the animal prior to the hydrolysis of the ester linkage. Af-
ter hydrolysis, the syn oxime [CH3S (CH3) 1I+C=NOH] is further metabolized
to lt+CO2 while the anti oxime is metabolized to CH31'tC=N. Proposed im-
mediate precursors to the carbon dioxide and acetonitrile, formed by
Beckmann rearrangement of the syn and anti oximes, are CH3S1I+C (O)NHCH3
and [CH314+C=NSCH3]x~, respectively.
Fate of Croneton (2-Ethylthiomethylphenyl N-Methylcarbamate) in Rats -
The fate of Croneton, 2-ethylthiomethylphenyl N-methylcarbamate, was
determined in rats following both single oral or dietary exposure to the
llfC-carbonyl- and 1LfC-r ing-labeled insecticide. Greater than 95% of the
[lltC]-Croneton equivalents was excreted in the urine or as a combination
of 14CC>2 (47%) and urinary products (41%) 72 hr after a single oral dose.
The feces contained 2-7% of the dose. A similar excretion pattern was
observed during a 7-day feeding period. The principal urinary metabo-
lites were Croneton sulfoxide (23-28% of the dose), phenol sulfoxide
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(20-23%) , phenol sulfone (9-25%), and Croneton sulfone (3-11%) after a
single oral dose and similar in the long term study. The carbamates
were excreted primarily as free metabolites while the phenolic constit-
uents were eliminated as acid labile conjugates. The 24-hr acute oral
LD5g values of Croneton, Croneton sulfoxide, and Croneton sulfone to
mice were 71, 59, and 282 mg/kg, respectively.
Fate of Croneton in Large Animals - A lactating Holstein cow and a male
Yorkshire pig treated with a single oral dose, 0.5 mg/kg, of ring [1LtC]-
Croneton excreted 97.8% and 90.0% of the dose via the urine after 24 hr.
Residues were not detected in swine tissues sampled 24 hr after treat-
ment, and of the bovine tissues, only the kidney, liver and skin con-
tained detectable radiocarbon (0.016, 0.017, and 0.05 ppm 11+C-Croneton
equivalents, respectively). Milk collected 6 hr after treatment con-
tained 128 ppb [1LfC]-residues; 60% was as the free carbamate metabolites,
Croneton sulfoxide and sulfone. White Leghorn hens given ring [ll+C]-
Croneton as a single oral dose (0.5 mg/kg) or as daily treatments for 7
days (0.5 mg/kg twice daily at 12-hr intervals) exhibited patterns of
excretion and metabolism similar to that observed in the cow and pig.
Birds sacrificed 4 hr after the last of the daily doses contained f1 C]-
Croneton equivalents ranging from 0.019 ppm in the fat to 0.324 ppm in
the kidney. By 24 hr, only the liver and kidney (0.444 and 0.022 ppm)
contained residues in excess of 0.01 ppm, and these had declined to 0.01
ppm or less by 4 days. Residues in eggs were on the order of 0.03-0.04
ppm [ C]-Croneton equivalents after 2 days of treatment and reached a
maximum of 0.06-0.07 ppm after 7 days. They declined rapidly when
treatment was terminated and were below detectable levels after 3 days.
About 75% of the radiocarbon in the eggs was as free phenol sulfoxide
and sulfone, 10% as free Croneton sulfoxide and Croneton sulfone, 5% as
water soluble metabolites and 5% as unextracted residues. The remaining
5% was unknown metabolites in the organoextractable fraction.
Fate of Insecticides Administered Endotracheally to Rats - Administra-
tion of carbaryl, leptophos, parathion and chlordane as aerosols to rats
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via the trachea resulted in quantitative retention of the inhaled insec-
ticides. None of the radioactivity was detected in the exhaled air ex-
cept 2.5% of the C-carbonyl carbaryl dose as carbon dioxide. Rapid
ester hydrolysis of carbaryl, leptophos and parathion was evidenced by
the different levels of radiocarbon in the blood following treatment
with the same chemical, but with the radioactive carbon on the opposite
sides of the ester linkage. The three compounds were eliminated in the
urine in amounts greater than 90% of the inhaled doses by three days.
With chlordane, excretion was primarily via the feces: 52% of the dose
was eliminated after 6 days. It was concluded that the ultimate fate
of these insecticides when inhaled was about the same as when ingested.
Insecticide Residues in Cigarette Smoke. Transfer and Fate in Rats -
A quantitative smoking system was devised for mainstream smoke collec-
tion or for delivery to the lungs of rats via the trachea. Percentage
transfer of 1I+C-labeled insecticide equivalents to the mainstream smoke
of cigarettes impregnated with five different ^C-labeled insecticides
(carbaryl, carbofuran, leptophos, DDT, and mirex) was approximately the
same per milliliter of smoke, 0.2-0.3% of the ltfC-labeled insecticide
contained in the burned tobacco, with puff volumes of 35 and 5 ml.
Thirty-five milliliter puffs represent the average puff volume of man
while a 5-ml puff was the volume administered to rats. Total recovery
(ash, butt, sidestream smoke, and mainstream smoke) ranged from 88 to
102% of the ring-^C-labeled insecticides added to that tobacco subse-
quently burned during the smoking process. Radioactive carbaryl, carbo-
furan, parathion, leptophos, and DDT were added to cigarettes and the
mainstream smoke was directed to the lungs of rats via the trachea.
Total radiocarbon transfer to the lungs ranged from 0 to 15% of that in
the tobacco burned during a smoking process involving eight 5-ml puffs.
Exhalation of ^C-residues during this time was 24 to 30% of that in-
haled with all insecticides except carbofuran, of which 42% of the resi-
dues was exhaled. After 5 hr, total exhalation of the consumed radio-
carbon was 35% for parathion, 65% for carbofuran, and approximately 50%
for the other products. The nature of the lltC-residues inhaled, their
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urinary and fecal excretion, and their deposition in and dissipation
from various organs and tissues are presented.
Chemical and Metabolic Characteristics of 1-Naphthyl g-D-Glucoside -
1-Naphthyl g-D-glucoside was stable under a variety of conditions en-
countered in metabolism studies. When 1-naphthyl-11+C glucoside was
given orally to rats, 67% of the radiocarbon was eliminated in urine af-
ter 24 hr. Of this, 28% was the administered compound, 35% 1-naphthyl
glucuronide, 15% 1-naphthyl sulfate, and 14% was 1-naphthol. Cleavage
of the glucoside linkage was possibly the sole initial step in the
metabolism of the compound. l-Naphthol-^C given as a single oral dose
was eliminated from rats more rapidly, 90% of the dose in the 0-24-hr
urine, than was its glucoside conjugate. 1-Naphthyl glucuronide con-
stituted 81% of the radiocarbon while 17% was 1-naphthyl sulfate and
only 1.6% was free 1-naphthol.
Glycoside Conjugative Activity in Different Insect and Vertebrate Spe-
cies - The occurrence and comparative activity of glycoside conjugating
enzymes were investigated in insect and vertebrate species. Although
some quantitative differences were noted, all species readily conjugated
the substrate, 1-naphthol. Glucosyltransferease activity was essential-
ly the same in whole body homogenates and in selected tissues of the
adult housefly, Musca domestica L., and the American cockroach, Peri-
planeta americana (L.), and larvae of the alfalfa weevil, Hyper postica
(Gyllenhal), tobacco hornworm, Manduca sexta (L.), and Indian meal moth,
Plodia interpunctella (Hiibner). In the rat, the comparable enzyme,
glucuronyltransferase, was more active in the liver, kidney, and lungs
than in the intestine, stomach, heart, fat, and brain. Glycoside con-
jugating enzymes were stable when sotred in the intact tissue or in
various subcellular fractions at -20° and 0°C for 90 days. In vitro
glycosylation of 1-naphthol by houseflies was not influenced by develop-
mental state, age, sex or insecticide resistance.
Glucosylation by Housefly Microsomes and Effect of Monoamine Oxidase
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Inhibitors - Glucosylation by housefly microsomes was studied using 1-
11+C-l-naphthol as the substrate. With Tris-HCl buffer the optimum pH
for the formation of naphthyl-8-D-glucoside was 8.5. Magnesium was the
most effective divalent ion for activity when compared with cobalt,
nickel and manganese. The apparent K for UDPG was 2 x IQ~k M and for
m
1-naphthol 5-7 x 10~5 M. p-Nitrophenol was a competitive inhibitor for
1-naphthol glucosylation. The monoamine oxidase inhibitors, harmaline
and tranylcypromine, non-competitively inhibited the conjugation of 1--
naphthol.
Role of Glutathione Conjugation in Carbaryl Metabolism - In vitro and
in vivo glutathione (GSH) conjugation in rats was investigated using 1-
naphthyl-l-^C carbaryl and naphthalene-l-ll*C. Maximum in vitro conju-
gation (72%) occurred after incubation of naphthalene-l-ll+C with the
15,000 g supernatant of a rat liver homogenate fortified with NADPH and
GSH. The amount of conjugates formed was dependent on the amount of
glutathione added. Microsomal enzymes were required for naphthalene to
form GSH conjugates, demonstrating that oxidative metabolism preceded
conjugate formation. Glutathione conjugates isolated by tic included S-
(l,2-dihydro-2-hydroxy 1-naphthy1)glutathione and l,2-dihydro-2-hydroxy
1-naphthyl glucuronic acid. By comparing the nature of the glutathione
conjugates with those obtained by the metabolism of carbaryl, it was
concluded that glutathione conjugation did not occur with this carbamate
insecticide. The same conclusion was drawn from studies using 1-naph-
thol. When naphthalene- l-^C and 1-naph thy l-l-^C carbaryl were injected
into rats, 50 and 80% of the radioactivity was excreted in the urine
within 72 hr, respectively. With naphthalene, 3 major metabolites were
detected in the urine, with the mercapturic acid derivative accounting
for 32% of the dose. In contrast, mercapturic acid metabolites were not
evident in the urine of carbaryl-treated animals. Glucuronic acid and
sulfate conjugates were the predominant types of metabolites found in
the urine of rats dosed with carbaryl.
Bioavailability of Bound and Conjugated Carbamate Insecticide Residues -
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Bound and conjugated forms of pesticides and/or their metabolites may
occur as major terminal residues in plants destined for human consump-
tion. The potential bioavailability of such residues in mammals was in-
vestigated by administering bound (radiocarbon remaining in the plant
matrix after thorough solvent extraction) or conjugated (water soluble)
plant metabolites of four C-labeled methylcarbamate insecticides
(carbaryl, carbofuran, aldicarb and Croneton) orally to female Sprague-
Dawley rats. After two days 8.0 and 1.3% of the carbofuran and carbaryl
bound-residue dose were eliminated in the urine, while 78.8 and 99.5%
was voided in the feces. Only 1.4 and 0.4% of the respective bound in-
secticide treatment was found in the bile of biliary fistulated rats.
When Croneton bound residue from sorghum was fed to rats, 16.4% of the
dose was excreted in the urine and 90.8% in the feces after 48 hr, while
only 2.5% was recovered in the bile of a bile duct-cannulated rat.
Within 36 hr, the urinary excretion of the water-soluble metabolites of
carbaryl, aldicarb, Croneton, and carbofuran was 87.7, 87.5, 81.2, and
64.8% of the dose. Radiocarbon equivalents in the feces were 9.7, 1.0,
3.7, and 8.3%, respectively. These data demonstrate that bound residues
of the test compounds were very poorly absorbed from the gastrointesti-
nal tract of rats, while the conjugated ones were readily absorbed.
Influence of Insecticide Exposure on the In Vivo and In Vitro Metabolic
Activity of Rats - Insecticides were administered to rats as single
doses, oral and IP, at approximately 1/10 their acute oral LDso levels,
or in the diet at concentrations where the daily intake was equivalent
to the single doses. Carbofuran decreased the rate of urinary elimina-
tion of carbaryl-^C from rats whereas Ruelene tended to increase the
rates. However, the changes were minor, 12% or less, and the nature of
the carbaryl metabolites was not altered. Carbaryl and carbofuran
caused a reduction in liver protein of rats injected with the carbamates
for five days, but had no affect when fed in the diet for 40 days. DDT
increased the liver protein when injected but not when fed in the diet.
When administered in combination, by injection or in the diet, DDT and
carbaryl did not modify the protein content of the liver. Liver and
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kidney microsomes of rats fed DDT in the diet, alone or with carbaryl,
resulted in increased oxidase activity. Neither of the compounds af-
fected microsomal glucuronidation of 1-naphthol. In a four-week feeding
study, the rate of weight gain was 15% less in animals fed carbaryl plus
Ruelene or coumaphos plus Ruelene than in the control rats. No signifi-
cant difference from the controls was observed with carbaryl or carbaryl
plus carbofuran. Feeding these compounds did not affect urine pH; but
the urea content was increased by all treatments and the glucose content
increased by all except treatment with coumaphos plus Ruelene.
Influence of Dose on Carbamate Hydrolysis - An examination was undertak-
en of the relative roles played by the urinary and hydrolysis-dependent
respiratory routes of elimination following various doses of C-carbon-
yl Croneton. Hydrolysis inferred from 14CC>2 expiration from rats ex-
pressed in terms of the administered dose showed that relative hydroly-
sis was slowed and decreased with increasing dose. When rate of hydro-
lysis was examined graphically with time, biphasically exponential elim-
ination of 11+CC>2 was observed with a relatively fast a-phase lasting for
about 12 hr followed by a generally slower 3-phase. Half-times of the
a-phase were nearly constant at low doses (about 4 hr at 0.2 or 2 mg/kg)
but increase dramatically in good correlation with doses producing
severe toxicity (14.5 hr at 100 gm/kg). Urinary excretion expressed as
a percentage of the administered compound also declines at high doses.
Administration of a moderate dose of atropine prior to Croneton did not
alter hydrolysis, but increased urinary excretion.
Screening of Pesticides for Mutagenic Potential Using Salmonella
typhimurium Mutants - The mutagenic activity of several pesticides and
related analogues was examined with a set of four strains of Salmonella
typhimurium (Ames Assay, TA1535 series; deep rough strains without ex-
cision repair). Nitrosocarbaryl, a derivative of the insecticide carba-
ryl, proved to be a potent base-pair substitution mutagen showing activ-
ity at 0.5 yg/plate, as well as demonstrating a relatively mild frame-
shift activity. Captan at 25 ug/plate showed both frameshift and base-
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pair substitution mutagenesis. The mutagenic properties of these two
compounds decreased when exposed to rat liver homogenate. DDT, DDE,
heptachlor, heptachlor epoxide, dieldrin, carbaryl, linuron, and diazi-
non were not mutagenic in this system. The Salmonella typhimurium
strain TA1535 mutants had no significant effect upon the metabolism of
carbaryl. Rapid conversion of carbaryl to water soluble metabolites
indicated that the S-9 rat liver homogenate was a very efficient in
vitro preparation. Detection of hydroxylated carbaryl intermediates
common to known pathways suggested that the metabolism within the Ames
test was representative of the in vivo mammalian metabolism. Addition
of S-9 enzymes to the system resulted, therefore, in the mutagenic
potential of the carbaryl metabolites being analyzed rather than just
that of the parent compound as was the case in the absence of the liver
preparation.
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TECHNICAL REPORT DATA
(Please read InXructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-77-012
3. RECIPIENT'S ACCESSIOC+NO.
4. TITLE AND SUBTITLE
Metabolism of Carbamate Insecticides
5. REPORT DATE
February 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
H. Wyman Dorough
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Entomology
University of Kentucky
Lexington, Kentucky 40506
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
R-802005
12. 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
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Results of studies conducted to determine the metabolic fate of carbamate insecti-
cides and its toxicological significance are presented. Methomyl metabolism in
rats was investigated in detail as was Croneton in the rat, cow, pig and chicken.
Carbaryl and carbofuran were administered to rats endotracheally either as aero-
sols or as components of tobacco smoke and their fate determined. Carbaryl and
nitrosocarbaryl were among a series of pesticides assayed for mutagenic/carcino-
genic activity using the Ames assay system. In addition, a study was conducted
to determine if the in vitro metabolism of carbaryl under conditions of the Ames
assay was representative of that which occurs in vivo. Respiratory l^C-carbon
dioxide from carbonyl-labeled carbamates was evaluated as a technique for deter-
mining the effects of various factors on the metabolism of carbamate insecticides.
Carbaryl and certain of its analogs were used in studies designed to define the
role of glutathione conjugation in carbamate metabolism in rats. Conjugate and
boud residues of carbaryl, carbofuran, Croneton and aldicarb formed by plants
were administered orally to rats and their bioavailability ascertained. Biliary
excretion and enterohepatic circulation were considered in addition to urinary
and fecal elimination.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. cos AT I Field/Group
Insecticides
Carbamates
Toxicity
Metabolism
In Vivo
In Vitro
Pesticides
Chemical analysis
Carbamate Inseciticides
Conjugates
07 C
06 F, T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
257
20 SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
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