CARBARYL
DECISION DOCUMENT
DECEMBER 1980
OFFICE -OF PESTICIDES AND TOXIC SUBSTANCES
Environnentsl Protection Agency
421 M Street, S.W.
Washington, D.C. 20450
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TABLE OF CONTENTS
Page
I. INTRODUCTION 1
II. GENERAL INFORMATION 2
A. Chemical Identity 2
B. Registered Products, Uses,
and Tolerances 3
1. Products and Production 3
2. Uses 3
3. Tolerances 4
C. Regulatory History 6
D. Environmental Fate 6
1. Hydrolysis 6
2. Photoreactivity 6
3. Fate of Carbaryl in
Soil — Persistence Studies 7
E. Biological Fate —Metabolism 7
F. Pesticide Incident Monitoring
System (PIMS) Reports 12
III. CARBARYL AS A POTENTIAL RPAR CANDIDATE 14
A. Introduction 14
B. Teratogenic and Fetotoxic Effects 16
1. Laboratory Data 18
a. Rodents 18
b. Other Mammalian Species 22
2. New Jersey Epidemiological Studies 29
to Assess Carbaryl-Related Birth
Defects
C. Mutagenicity . 31
1. Evidence Concerning Point 32
(Gene) Mutations
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TABLE OF CONTENTS (CONT'D)
(a) Bacteria 32
(b) Mammalian Cells in Culture 38
(c) Drosophila 38
2. Evidence Concerning Primary DNA
Damage 39
3. Evidence Concerning Chromosome
Effects 4C
4. Evidence Concerning Whether Carbaryl
Reaches the Germinal Tissue 42
a. . Epideniological Data 42
b. Rodent Data 45
D. Oncogenic Effects 47
S. Neurctoxicity 53
F. Viral Enhancement 56
IV. CONCLUSIONS AND RECOMMENDATIONS 55
A. Summary of Conclusions 58
1. Teratogenic and Fetotcxic Effects 53
2. Mutagenic Effects 58
3. Oncogenic Effects 59
4. Neurotoxicity 59
5. Viral Enhancement 59
5. Overview — Determining Considerations 59
B. Recommendations - 50
V. REFERENCES 51
APPENDIX A — Review of Experimental Evidence on
the Mutagenicity of N-Nitrosocarbc.ryl A-l
APPENDIX B — Review of Experimental Evidence on
on the Carcinogenicity of N-Nitroso- B-l
carbaryl
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LIST OF FIGURES AND TABLES
Figure 1. Primary Carbaryl Metabolites in Mammals 9
and Routes of Formation (Hydrolysis or
Hydroxylation)
Table 1. Point Mutations in Bacteria 33
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CAFBM&L: DECISION DOCUMENT
I.
Cartaaryl is a widely used pesticide which is manufactured in the United
States by the Onion Carbide Corporation under the trade name "Sevin." Carbaryl
.is a member of the carbamate family of pesticides, and like other family
members, derives its efficacy from its ability to inhibit the enzyme cholin- '
esterase. The inhibition of this enzjme renders the nervous system inoperable,
thereby killing the target pest. In general, N-methyl carbamates have been
found to be more efficacious than N-dimethyl carbamates . Carbaryl belongs
to the N-methyl carbamate class of insecticide » Two distinct characteristics
have ™*3«* carbaryl the most popular carbamate the world over. These
— low """""» ^ "j»" nra^ and ^oT^nal tBxicity, and a
spectrum of- insect control — have led to its widespread use in a wide range I
t
Carbaryl use was first called into question in 1968, after a study by
Smalley et al. (1968) found carbaryl to be a potent teratogen when administered
in low doses to pregnant beagle dogs. A 1969 study of beagles (Inning et al.,
1969) r-nmn-jce -inroad by the Onion Carbide Corporation also showed positive
results. Since then, carbaryl has been the subject of numerous studies of its
effects on a number of mammalian species.
This position document summarizes the results of the Agency's review of
currently available toxicological evidence on carbaryl as it pertains to the
potential human health hazards of the pesticide. The document is i^jn^"1 IP^ of
five sections. Section I is this introduction. Section II discusses general
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products, uses, production, and tolerances. Section HI ac
the primary
purpose of the review: it compares data on potential adverse effects of
carbaryl to humans with the Agency's criteria for a Hebuttable Presumption
Against Registration. Section IV summarizes the conclusions of **"« review of
carbaryl and recommends several actions to be taken in response to these
conclusions. Section V is a MM•*'T'-Thi*-8!! listing of works cited.
EL. GENERAL INPORMATICN
A. Chemical Identity
The chemical name for carbaryl is 1-Naphthyl methylcarbamate or 1—
Naphthyl N-nethylcarbamate. The P*»"i^al Abstracts Service (CAS) has assigned
to carbaryl the registry number 63—25—2. The chemical f''1"".^* is
C12HUIC2 ^^ a nolecular weight of 201. The structural formula of
carbaryl is shown below:
0-C-NHCH.
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The pure form of carbaryl is a white crystalline solid. The technical
product is pink, lavender/ or tan in color. It is essentially odorless and
melts at 142°C. The vapor pressure is less than 0.005 mm Hg at 26°C.
Carbaryl is slightly soluble (40 pom) in water at 30°C. It is fairly soluble
in certain polar organic solvents, including acetone/ cyclohexanone and
dimethyl formamide. It is slightly soluble in hexane, benzene, and methanol.
B. Registered Products/ Uses, and Tolerances
1. Products and Production. Carbaryl has been registered for pestic.irtal
use since 1959. Carbaryl is used in 1,518 federally registered products, made
by 295 registrants. Production and consumption in the United States in 1972.
were estimated at 53 and 25 m-m im pounds, respectively, an estimated 28
pounds having been exported.. In fcpn-mg of general use categories, the
pattern of 1972 domestic consumption was roughly as follows (approximations
expressed in millions of pounds): agriculture - 19, home and garden - 3.5,
government - 1.5, industrial - 1.0 (Van Ranker et al., 1974) . Production for
domestic consumption in 1979 has been estimated to be between 15 million to 25
million pounds .
2. Uses. Carbaryl is registered as an insecticide/acaricide, as well as
a plant regulator used in thinning apples. Its major uses include applications
to cotton, peanuts, soybeans, field and sweet corn, ornamentals and turf,
forest and shade trees, deciduous tree fruits, many other fruit, vegetable, and
nut crops, poultry and pets (Von Rumker et al., 1974) . The technical product
is at least 99% pure and is formulated for use in wettable powders, dusts,
granular" products, flowable suspensions and baits (Von Rumker et al., 1974) .
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Products formulated for use range from 0.5% (aerosol) to 80% (wettable powder)
concentrations of carbaryl.
3. Tolerances. Dnder Section 408 of the Federal Food, Drug, and Cosmetic
Act, the Environmental Protection Agency determines legally permissible tole-
rances for pesticidal chemical residues in or on raw agricultural products*
tolerances for carbaryl and its nyat-ahni-i •t-o^ r including 1—naphthol,
are li^tpd in 40 CFR 180.169, and are summarized below:
i« 100 parts per mi 1 -ion (pom); alfalfa, ai/fgifa hay,, barley
(green fodder and straw), bean forage, bean bay, clover, clover
hay, cDiii fodder, CUJLTI forage, cotton forage., cowpea forage, cowpea
hay, grass, grass hay, oats (green fodder and straw), peanut hay,
pea vines, rice straw, rye (green foddtyr and straw) , yrghm" forage,
soybean forage, soybean hay, sugarbeet tops, wheat, (green, fodder
and straw) .
ii. 40 parts per mi 111 on; almond hulls.
iii. 12 parts per miliionr blackberries, boysenberries, collards,
dandelions, dewberries, garden beets (tops), kale, loganberries,
mustard greens, parsley, raspberries, spinach, Swiss chard, turnips
(tops).
iv. 10 parts per million; apples, apricots, asparagus, bananas,
beans, blueberries, broccoli, brussels sprouts, cabbage, carrots,
cauliflower, celery, cherries, Chinese cabbage, citrus fruits,
cranberries, cucumbers, eggplants, endive (escarole), grapes,
kohlrabi, lettuce, melons, nectarines, okra, olives, peaches,
pears, peas (with pods), peppers, plums (fresh prunes), pumpkins,
salsify (tops), sorghum grain, strawberries, summer squash,
tomatoes, winter squash. j
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v. 5 parts per million; corn (kernels and kernels plus cob,
determined after removing husks present when marketed), cottonseed,
cowpeas, garden beets (roots), horseradish, meat and fat of
poultry, parsnips, peanuts, radishes, rice, rutabagas, salsify
(roots), soybeans, farrn-rpg (roots)•
vi. 1 pai-t psr nn THon; aiirmnrig,. chestnuts, filberts (v»a?»i nuts),
pecans, and walnuts.
vii. 0.5 part per million; maple syrup.^
viii. 0.2 part per million (negligible residue): potatoes.
ix. Zero; grains of barley, oats, rye, and wheat.
. In addition, an interim tolerance of 0.5 ppm has been set for residues of
carbaryl in eggs (40 CFR 180.319).
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C. Regulatory History
Carfaaryl was first registered for use in 1959 as an insecticide on
cotton. With subsequent registrations, the total number of proved sites of
application has risen to more than 80. The first tolerances were- granted in
1959, and the number of tolerances granted now numbers 101. The most recent
tolerance (0.2 ppm on potatoes) was granted in 1975.
In November 1968, carbaryl was classified as highly +""rir to bees subject
either to direct exposure or to indirect exposure via residues. As a result,
carbaryl products intended for foliar applications to crops and trees, and for
f were r^^j'JT^"^ to ^O^T* «3nH<*»n»ty statements/warnings
against use in the vicinity of bees.
Fate
1. Hydrolysis. Carbaryl is fairly stable to hydrolysis in acidic and
neutral mpdlums. In an alkaline medium at ph 9 to 10, carbaryl decomposes to 1-
naphthol and N-methyl carbamic acid> N-aethyl carbamic acid is unstable, and
it further decomposes to ».whe»» dioxide and methyl amine (Christie, 1969;
Dkeles, 1962).
2. Photoreactivity. Pure carbaryl is stable in daylight and in ultra-
violet light. When it is irradiated in ethanol solution, its products include
1-naphthol and other, unidentified products which have anticholinesterase
action (Crosby et al., 1965). 1-Naphthol has also been identified as a
f
product which ojcurs when carbaryl is irradiated in aqueous solution (Aly,
1971) .
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3. Fate of Carbaryl in Soil—Persistence Studies. Carbaryl is moderately
persistent in soil. LaFleur (1976) applied carbaryl to a Congaree sandy loam
field plot at 25.4 kg/ha. After four months, carbaryl was not detected in the
upper 20 cm of soil. After 16 months, 6% of the applied carbaryl was found in
the upper one meter of soil. LaFleur (1976) also demonstrated that retention
of carbaryl varies widely depending on' the soil. Using columns of six
different South Carolina soils, he found that those with high organic content
were able to absorb several times as much carbaryl as sandy soils. Johnson and
Stansbury (1965) applied carbaryl in three different concentrations and found
it to have a "half life* of approximately eight days under normal conditions.
After 40 days, Johnson and Stansbury did not detect any carbaryl.
Carbaryl is mo«-ahiii-i*ii7t> carbaryl either by hydrolysis to
form 1-naphthol or by any of four hydroxylation steps affecting the ring or the
N-methyl group (Menzie, 1974). Whether carbaryl is initially broken down by
hydrolysis or by hydroxylation, carbaryl metabolites are ultimately conjugated
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by sulfaticn or glucuronidaticn, and the conjugates are eliminated in the urine
and/or feces. In some cases, some of the hydrolysis and hydroxylation products
have been detected in the urine at low levels in the free form (Menzie, 1974).
For most Tramnals, hydroxylation and glucuronidation are the preferred
2/
mechanisms. The primary metabolites of <
formation are shown in Figure 1 as follows:
2/
mechanisms. ' The primary metabolites of carbaryl and their routes of
2/ Although most animals metabolize at least a portion of administered j
carbaryl through hydrolysis, this does not seem to be a significant mechanism i
in monkeys and pigs (Menzie, 1974). {
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FIGURE 1—PRIMARY CARBARYL METABOLITES IN MAMMALS AND ROUTES OP FORMATION (HYDROLYSIS OR HYDROXYLATION)
0 - 6LUCURONIDE
0-C-N
0-SUtWE
6LUCURONIDE
n
0-C-NIICH2OII
l.-Naphthol
Hydrolysis
I
0-C-NIICIh
11
0-C-NIICII,
Hydroxylatlon
1-Naphthyl-N-hydroxymethyl
oarbaryl
0
II
0-C-NIICH3
Hydroxy- HO*
lation u1
Gatbaryl . n||Q ||
5f6-Dlhydrb-S,6-dihydroxy carbary1
Hydroxylation
-C-NHCH3
S-Bydroxyoarba^yl
4-llydroxycarbaryl
I \\
\ o-c-niicii,
0-GLUCURON1DE
(E)
SUlfME
0-6LUCURONIDE
(6)
8
0-C-I1IICII3
SULFATE
(II)
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Various studies have been performed to elucidate the identity and quantity
of carbaryl metabolites in various mammals, including man, the dog, and the
rat. Die studies to date indicate that the metabolic patterns observed in
mammalian species are qualitatively identical, with few exceptions. The nature
of the metabolites produced is fairly consistent, although there are specie
variations in quantity and order of distribution, with sane species producing
metabolites not found in others (Ruhr et al., 1970; Baron et al., 1969; Borough
et al., 1964; Knaak et al., 1965; Krishna et al., 1966).
In man, the primary nvyrhar1^*81" of carbaryl
hydrolysis. As identified in non-radioactive studies by Knaak, the major
urinary metabolites are 1-oaphthyl glucuronide and 1-naphthyl sulfate. In
addition, 4-hydroxy-carbaryl glucuronide has also been identified in human
urine, indicating that humans mo*-aK-ii * •» carbaryl through the hydroxylatian as
well as the hydrolytic pathways.
The metabolic profile of the dog appears to be qualitatively similar to
that of man and displays minor quantitative differences. In a 1967 study
performed by Knaak and Sullivan in three dogs using radioactive carbaryl,
carbaryl metabolites were found to be eliminated by both the urinary and fecal
routes. The only identified urinary metabolite was the glucuronide of 5,6-
dihydro-5,6^ihydroxy-carbaryl, a hydroxylation product. A second major
metabolite, chromatographed as 1-naphthyl glucuronide, did not exhibit the
fluorescent properties expected of this compound. The neutral metabolites
appeared to include an unspecified naphthalenediol; other tentatively
identified compounds were the 4-hydroxycarbaryl glucuronide and the 4-
nydroxycarbaryl sulfate.
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In a subsequent radio-labeling study performed by Sullivan et al. (1972a),
metabolites identified in the dog and dog liver preparations included neutral
catDounds, which although not specifically identified, were believed to include
carbaryl, 1-naphthol, and 4 and s-} iy'^r''vyr?rtv''ryl compounds. Other compounds
identified were S,6-dihydro-5,6-dihyUroxy-carbaryl, hydrcotycarbaryl
glucuronide, hydroxycarbaryl sulfate, and a non-fluorescent compound
tentatively identified as 1-naphtfayl glucuronide. The absence of fluorescence
for the compound believed to be 1-naphthyl glucuronide may be due to the
presence of an unknown metabolite with fluorescent-quenching properties cc—
chramatographed with the 1-naphthyl species. TJae identity of metabolites
reted in the feces was not investigated.
Studies in the rat have demonstrated that carbaryl. is metabolized by both
the oxidative and hydrolytic routes in the species. In a 1964 study performed
by Dorough and Casida, carbaryl was incubated with rat liver mi<-rr*Mnai
» the mo^ahri^ jt-oc idoir^ •Pi aA included 1— naphthol, 4— faydroxy—
carbaryl, 5-hydroxycarbaryl and
study detected, but did not identify, four other major metabolites, two of
which contained intact carbamate groups. A radio-labeling study performed in
1965 by Knaak et al. in male rats identified the sulfate and glucuronide
conjugates of naphthol, 4-hydroxycarbaryl and 5,6-dihydro-5,6-dihydroxycarbaryl
glucuronide as the major urinary metabolites. In later studies of carbaryl
(Matsumura and Ward, 1966; Strother, 1972), the metabolism in the intact rat
liver confirmed the findings of the Dorough and Casida rat liver mieroscme
study: the metabolites which were identified were 1-naphthyl, 4- and 5-hydroxy-
carbaryl, and 1-naphthyl-N-nvdrcocymethylcarbamate. Another study of carbaryl
jnoj-ai-oi i «am in the rat identified the presence of tduoether conjugates in rat
f
bile and urine.
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A recent preliminary study by Dorough (1979) regarding the comparative
chemical nature of carbaryl metabolites in the rat and the dog indicated that
there is no appreciable difference in the manner and rate at which carbaryl
equivalents are voided from these two species.
In sumnary, although the comDarative carbaryl metabolism in such species
as man, dogs, and rats has not been fully detailed, there is evidence that all
these species metabolize carbaryl by g
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No clear trend in the number of carfaaryl-related incidents reported over
the years emerges from PIMS records. Fewer than 10 incidents were recorded : j
each year from 1966 through 1971 and again in 1979. Fewer than 50 incidents Ij
were reported in 1972 and in 1974. Between 50 and 100 incidents were recorded \
in 1973 and- in 1975. One hundred or more incidents were recorded in 1976 and •
in 1978.
PIMS records indicate a wide range of sites and circumstances associated
with carfaaryl-related incidents. Tbe largest category/ however, comprised
incidents in and around the home, and the second largest category comprised
agricultural incidents.
Among home incidents/ ingestion of the pesticide was tbe most penmen type :
of episodes involving children. Other types of hone incidents included
accidental splashing or spilling, contamination while spraying (home garden
use), and contamination via contact with residues on hone garden crops.
Agricultural incidents mast ccnnonly involved field workers laboring for a
number of days in fields which h** been sprayed or dustffd with carbaryl or a
combination of pesticides including carbaryl.
Two points gty*1!^ be emphasized in connection with the overview given
above: 1) Recorded incidents include only those which have actually been
reported to PIMS. Because other, unreported incidents have in all likelihood
occurred, the number of incidents cited here should be considered a minimum.
2) Not all incidents recorded for the period 1966 to January 1980 have been
formally confirmed as results of pesticide exposure (PIMS, 1980).
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III. CARBARYIi AS A KJi'hKTIAL HEAR CaNDIEftlE
A. Introduction
Sec. 3(a) of FIERA requires all pesticide products to be registered by
the Admin is UaLut of EEA before they nay be sold or distributed. Sec. 6(b) ;
of FIFRA authorizes the Administrator to issue a notice of intent to cancel the •
registration of a pesticide or to change its ffia«g-i-Hff*H
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of "Rebuttable Presumption Against Registration." The 40 CFR 162.11
regulations require that an opportunity then be provided for registrants,
applicants, and interested persons to submit evidence to rebut the presumption,
or evidence relating to the economic, social, and environmental benefits of any
use of the pesticide. If the presumptions of risk are not rebutted, the
evidence on the benefits of the pesticide is evaluated and considered along
with the infornation on the risks from the pesticide. The Agency then analyzes
various methods of reducing the amount of risk from the pesticide together with
their costs and determines whether the pesticide can be regulated so that the
benefits of continued use outweigh the competing risks. If measures short of
cancellation cannot reduce the risks associated with any given use of the
t
pesticide to a level which is outweighed by benefits, the use in question must
be cancelled.
Carbaryl was referred to the EPA as an active ingredient suspected of
meeting 40 CFR 162.11 risk criteria warranting a rebuttable presumption.
Teratogenicity as a potential hazard of the pesticide was the primary basis of
concern, but carbaryl has also been subject to examination for other possible
adverse effects. Hence the Agency initiated an intensive review of the
presently available toxicological evidence. The following effects are at issue
in this review: teratogenicity and fetotoxicity, mutagenicity, oncogenicity,
neurotoxicity, and viral enhancement. The conclusions of this decision
document constitute the Agency's determination as to whether a rebuttable
presumption against pesticide products containing carbaryl is warranted at this
time.
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B. Teratogenic and Fetotoxic Effects
40 CFR 162.11 (a)(3)(ii}(B) provides that a rebuttable presumption against
a pesticide's continued registration shall arise if that pesticide's ingre-
dient (s) "produces any ... chronic or delayed toxic effect [other than an
oncogenic or mutagenic effect] in test animals at any dosage up to a level, as
determined by the Administrator, which, is substantially higher than that to
which humans can reasonably be anticipated to be exposed, tricing into account
an ample margin of safety." To determine whether or not carbaryl might pose a
teratogenic or fetotoxic hazard warranting a rebuttable presumption, the Agency
has contliicfegd a through review and evaluation of tte currently available
literature.
By way of introduction to the following discussion of teratogenicity and
fetotoxicity as possible adverse effects of exposure to carbaryl, it will be
^gtyfuT tO xAAwtus gnmta basic •iggiya*? of Aa^ifii'Hnn- Generally, tfrte for?"
"teratogenic* is defined as the tendency to produce physical and/or functional
defects in offspring in utero. Ihe trm "fetotoxic" has traditionally been
used to describe a wide variety of embryonic and/or fetal divergences from the
normal which cannot be classified as gross terata (birth defects) — or which
are of unknown or doubtful significance. Types of effects which fall under the
very broad category of fetotoxic effects are death, reductions in fetal weight,
enlarged renal pelvis edema, and increased incidence of supernumary ribs. It
should be emphasized, however, that the phenomena of terata and fetal toxicity
as currently defined are not separable into precise categories. Rather, the
rtrum of adverse embryonic/fetal effects is continuous, and all deviations
the normal must be considered as examoles of dg^»l<^pngn*"a1 toxicity.
Gross morphological terata represent but one aspect of this spectrum, and while
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the significance of such structural changes is more readily evaluated, such
effects are not necessarily more serious than certain effects which are
ordinarily classified as fetotoxic—fetal death being the most obvious example.
In view of the spectrum of effects at issue, the Agency suggests that it
might be useful to consider developmental toxicity in terms of three basic
subcategories. The first subcategory would be embryo or fetal lethality, "frig
is, of course, an irreversible effect and may occur with or without the
rrence of gross terata. The second subcategory would be teratogenesis and
would encompass those changes (structural and/or functional) which are induced j
prenatally, and which are irreversible. Teratogenesis includes structural \
defects apparent in the fetus, functional deficits which may become apparent :
only after birth, and any other long-term effects (such as carcinogenicity) ''.
which are attributable to in utero exposure. The third category would be j
^^" --^• • j
embryo or fetal toxicity as comprised of those effects which are potentially ;
reversible. iTijg subcategory would therefore irvTlu^ such effects as weight
reductions, rgftuc*"]ffn in the dg^Li'iJ of skeletal osslfi<^»fi""r and delays in !
oiyaii maturation* j
i
Two major problems with a definitional scheme of this nature must be |
pointed out, however. The first is that the reversibility of any phenomenon is j
i
extremely difficult to prove. An organ such as the kidney, for example, may be j
delayed in development and then appear to "catch up." Unless a series of i
I
specific kidney function tests are performed on the neonate, however, no [.
conclusion may be drawn concerning permanent organ function changes. This same
uncertainty as to possible long-lasting gftgr effects from developmental
deviations is true for all examples of fetotoxicity. The second problem is
that the reversible nature of an embryonic/fetal effect in one species might,
under a given agent, react in another species in a more serious and
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irreversible manner. The Agency must therefore consider all such deviations
frcsn nomal developnent in its risk assessment process, regardless of any
appearance of reversibility.
1. Labor?tory Data. Data concerning the potential of carbsryl to induce
adverse prenatal effects in mammalian species are extensive, more extensive
than has been the cass for other pesticides vhich have come under Agency
review. Numerous studies have been conducted on diverse mammalian species
including the mouse, rat, gerbil, hamster, guinea pig, rabbit, swine, sheep,
monkey, and dog. All of the studies are not of equal utility, however, for
purposes of assessing the potential of carbaryl to act as a perinatal toxicant
in the environment. A nunber of the studies were done with inappropriate
protocols, and some of the older American studies, as well as many of the
foreign papers, sre seriously flawed by inadequate presentation of data. The
Agency has therefore chosen to base its conclusions concerning the potential,
teratogenic and fetotoxic risk from carbaryl exposure primarily on those
studies which are valid and interpretable (Oiernoff, 1975 [updated 19SS1).
Because msny of the studies discussed below are seriously £L?wed, detailed
statistical analyses end "p" (pool) values are generally not included, since
such detail would suggest a false statistical rigor. In the following
discussion, an overall weight of evidence approach is applied in an analysis of
currently available data.
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a. Rodents. A number of studies have been conducted on the teratogenic
potential of carbaryl in various species of rodents. In the mouse, Murray et
al. (1979) showed that administration of carbaryl during gestation by oral
gavage (as much as 150 mg/kg/day) or in the diet (5660 ppm) did not result in
increased incidences of terata (birth defects) even at doses producing maternal
toxicity: Some reduced fstal weight and skeletal ossification were noted in
the dietary experiment, but significantly reduced maternal weight gain was also
noted at this dose level.-/
The potential of carbaryl to affect the development of the rat has been
studied in a number of experiments. Weil et al. (1972) administered carbaryl
in the diet and observed fetal toxicity only at the highest dose (500
mg/kg/day), which also produced significant reductions in maternal weight
gain. At dose levels where no maternal effects were noted (100 mg/kg/day),
ii/
there were no fetal effects. No terata were seen at any dose level.
3 A nunber of other studies, have been done on the mouse, but are of less
importance for a variety of reasons. A study by Qithrie et al. (1971)
attempted to determine the ability of mice to adapt to pesticide poisoning over
a series of generations. The protocol was not comparable to any known
teratological study, and therefore the results (no terata seen) are impossible
to interpret.
The experiments done by Benson et al. (1967) used very small nunbers of animals
and low doses (30 mg/kg/day), and this study (which showed no effects) is
therefore of little value in determining the fetotoxic potential of carbaryl.
A series of studies was done at the Bionetics Laboratory (1968), but the
inappropriate route of administration (subcutaneous) and a failure to replicate
effects that were initially noted also render this set of studies of little
regulatory value.
A study by Hart et al. (1972) also used the rat to determine the
teratogenic potential of carbaryl. The dose administered was low (37.5
mg/kg/day), and no effects were noted. There have also been a nunber of
studies done by scientists in Russia. These lack critical details (number of
animals tested, nunber of litters examined, source and purity of carbaryl) and
ars therefore extremely difficult or impossible to interpret.
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Likewise the potential of carbaryl to affect the. actual process of
reproduction has also been studied in the rat. Weil et al. (1973) administered
the compound either by gastric intubation, or in the diet. Maternal toxicity
was evident as animals showed cholinesterase inhibition, decreased body weight,
and increased mortality. A depression of the reproduction index occurred in
the first generation of anjmaig receiving 100 mg/kg/day by oral intubation.
This depression did not occur in subsequent generations, however, and may have
been a random occurrence since the chances of selecting a.resistant sub-strain
in one generation are minimal - Embryo or fetal lethality was seen in litters
of this dose group by a decrease in viable fetuses in the F2a - F2b generations
and a decrease in the number of live pups born in the F1a - F2a and F2a - F3a
generations. The interpretation of these effects should take into account the
significant maternal mortality in treated groups at all breeding periods. No
reproductive or fetal effects were seen at lower dose levels (25 mg/kg/day and
less). Carbaryl-induced effects were far less severe in am™!" receiving the
pesticide in the diet (200 mg/kg/day). Decreased body weight was noted in
fln-ima^g during the initial portion of the experiment, and an increase in the
average number of days from the first mating to the birth of the litter was
seen only in the F1a - F2a generation. No fetal effects were noted in this
experiment.
Collins et al. (1971) glso performed a 3-generation study in t|ie rat.
Lack of information concerning the status of the mothers and, in some cases,
incorrect methods of calculating the results render this study difficult to
interpret. One example of these flawed calculations is in the calculation of
the average litter size. Average litter size was calculated by dividing the
total number of progeny by the number of females exposed to mating. Since
animals which did not produce litters were not examined for pregnancy, the
presumption of 100J pregnancy inherent in this calculation is unjustified. An
error is therefore introduced into these calculations since an animal which
failed to conceive would carry the same weight in the calculation as an animals
whose entire litter died in utero . These are not the same phenomena, of
Bourse, and a sterile or "unwilling" animal might skew the results. The
authors did not report significant neonatal effects at the lowest dose group
(123 mg/kg /day).
-20-
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Effects of carfaaryl en the developing guinea pig were studied by Weil et
al. (1973). Carbaryl was administered either by oral gavage, or in the
diet. The high (200 mg/kg/day) oral gavage dose resulted in a significant
reduction of maternal weight gain. (Recorded differences in maternal weight
gain were not adjusted, however, to take differences in litter size into
account.) There was no reduction in fetal weight and no increase in fetal
anomalies observed. The results of dietary administration of carbaryl (as high
as 300 mgAg/day) were also completely negative in terms of fetal effects. The
carbaryl administered in the diet appeared to induce less severe maternal toxic
effects ^hTi ^1^ carbaryl arimjr" gfoyrgd by oral gavage. Weil et al. concluded
that the dietary administration of carfaaryl also had no effect on guinea pig
t
development. '
6 / '>
1 A study by Bobens (1968) showed that carfaaryl is teratogenic when •
administered to guinea pigs at doses which are lethal to a significant portion \
of the maternal animals. With doses of 300 mg/kg/day given on. multiple days \
during gestation (precise days not specified in paper), there were
ai'n'H-irinai -i *"\es present in 11 of the 46 fetuses, i^ig dosing regimen resulted in i
40% mortality in the treated dams. The lack of significant protocol ;
information in the paper (precise days dosed, number of litters with affected \
fetuses) and the high degree of maternal mortality render this study extremely j
difficult to interpret in terms of the developmental toxic potential of [
carbaryl. i
There have been two other rodent species in which the potential of carbaryl to j
induce adverse developmental effects has been studied. Collins et al. (1971) t
studied the effects of dietary administration of carbaryl for three generations ;
in the gerfail. The g*™* problems of interpretation aiiirfpd to in connection '<
with the work of Collins et al. (1971) with the rat apply to their work with :
the gerfail. (Refer to note 5 above.) It does appear that at the doses |
administered in thig experiment, carbaryl resulted in gif*? perinatal toxicity I
as reflected in smaller litter sizes and poorer survival to day 4 in many of I
the treated groups. The lowest dose level administered was 200 mg/kg/day. j
i
Robens (1968) also studied the effects of carbaryl in the hamster. All doses j
resulted in significant maternal toxicity. No fetal effects were noted in 125 •
rag/kg dose group, although considerable maternal toxicity was noted (diarrhea, [
salivation, and uncoordination). The graii number of litters examined in **ri«
study (maxlman of 8), coupled with the maternal toxicity, make the
interpretation of these results difficult, although it does not appear that the
fetal hamster is sensitive to carbaryl.
-21-
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b. other Map™]j*n Species. Carbaryl's potential to adversely effect
mamnalian development has also been studied in a variety of non-rodent
species. The rabbit has been studied by Murray et al. (1979), who found that
carbaryl was teratogenic at doses which induced maternal toxicity.
Craphaloceles (fissures in the ventral body wall) were noted in litters of dams
given 200 ng/kg/day by oral gavage in oil (this dose aicn resulted in reduced
maternal weight gain and diarrhea)• A single case of <^nphairv»oio vras «ig" noted
at the 150 mg/kg/day dose group, a dose which aicn produced seme maternal
toxicity (diarrhea). The rabbit has alsn been studied by Kobens (1968), who
administered 200 mgAg/day in a gelatin capsule and observed neither maternal
nor fetal toxicity. '
Two series of experiments have investigated the potential effects of
carbaryl on the development of the Rhesus monkey. Dougherty et al. (1971)
administered doses of carbaryl to monkeys beginning on the day sperm were found
in the vaginal tract and continuing throughout gestation. Pregnancy was
verified by a hioassay for monkey chorionic gonadotrcpin (MCG) using female
mice. The control group comprised 5 pregnant females. The 2 mg/kg/day and 20
rag/kg/day groups consisted of 2 and 6 pregnant females, respectively. An
increase in abortions was seen in the treated groups. Specifically, 2
abortions were reported in the 2 mgAg/day group, 3 in the 20 mg/kg/day group,
/ Shaffer and Levy (1968) administered dietary doses of 10 and 30
mgAg/day to the rabbit during gestation. These are low doses, and no effects
in either the maternal or fetal animals were seen.
i !
-22-
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and 1 in the control group. Of the five abortions noted in treated animals,
all but one occurred early in pregnancy* Since fetuses were not recovered in
these early abortions, and since early abortion in the Rhesus monkey is
difficult to detect and may be confused with placenta! bleeding, the crucial
factor in this first study would be the validity of the pregnancy test used.
No terata were noted in any of the recovered fetuses or neonates. In a second
study (Dougherty et al., 1974) , Rhesus monkeys were given carbaryl on days 20-
38 of gestation after verification of pregnancy. Pregnancy was determined by
for MCG, a different method Ctw that used in the previous
study (Dougherty et al., 1971). Obese experiments did not indicate any
carbaryl-related abortifacient (abortion-inducing) effect. No terata were seen
in any of the groups. Two infants in the 20 mg group had low birth weights,
one of which subsequently died at 10 days of age. Ohe growth of infants of
carbaryl-txeated mothers was not different from the growth of control infants.
Obese two studies (Dougherty et al. , 1971, and Dougherty et al. , 1974) are
not comparable in two crucial respects. The first is the validity of the
active pregnancy tests used in these studies since the key finding in the
first study involved early abortions, which are difficult to detect. The
second, and more important, is that the dosage regimen differed markedly in the
two studies, the first being throughout gestation, while the second was during
the period of major organogenesis. Thus animals in the first study received
carbaryl during the first part of pregnancy when early abortions might be more
readily induced, while in the second study the compound was administered only
during the period of organogenesis, when pregnancy is further advanced. Also,
the cumulative dosage differed, and at day 38 of pregnancy, animals in the
first study had received approximately 27 doses (5 fjm^g per week), while
animals in the second had received 19 doses. The second study therefore does
-23-
-------
not negate the first, which way have indicated an early abortifacient potential
for carfaaryl. The very snail number of animals used in the first study (a
total of 13 comprised controls and two treatment groups)/ coupled with the
difficulty in accurately identifying early abortion by the method employed,
allows no definite conclusions to be made* An arH-i^-irmai study of the
potential of carbaryl to induce early abortion in the monkey would be necessary
to answer this question raised by the first study (Dougherty et al., 1971). It
should be noted that neither study in the monkey indicated any teratogenic
potential of carbaryl in that species.
Two studies have attempted to determine the teratogenic potential of
carbaryl in the beagle dog. Smalley et al. (1968) administered the pesticide
in the diet throughout gestation at doses ranging from 3.125 mg/kg/day to 50
mg/kg/day. Birth defects were found in dose groups of 6.3 mg/kg/day and
above. The defects were of a broad spectrum and included lack of tail,
agenesis of gJcEgrr>8|i genitalia, •failtiro of fehg p'th-ie and jgrfiivim to develop,
openings in the ventral body wall, and the visceral agenesis* Teratogenic
effects were seen in 11.6% of the pups born to dams receiving carbaryl as
compared to 0% in the controls. "Difficult births" (dystccia) were seen in all
treatment groups and were absent in the controls.
Imming et al. (1969) attempted to repeat the Smalley study in the dog.
They administered carbaryl throughout gestation at doses ranging from 2.0 to
12.5 mg/kg/day in tine diet. They found a gn»n increase in still births in the
12.5 mg/kg/day and 5.0 mg/kg/day groups, as well a slight reduction in survival
until weaning at the highest dose level. Defects were seen in the 5.0 and 12.5
mg/kg/day dose groups and included umbilical hernia, cleft palate, and
gastrointestinal anomalies. No defects were seen in the low dose (2.0
mg/kg/day) or control groups. The authors also reported that some of the
bitches had difficulty during parturition. It would therefore appear that both
-24-
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the pregnant female and fetal beagle are sensitive to carbaryl and that this
compound is teratogenic in this species at doses of 5.0 mg/kg/day or greater.
The Agency feels that the dog studies should be repeated, with special
attention paid to sufficient numbers of animals in the dose groups, the
condition of the bitches throughout the period of dosing, and possibly
8/
maternal and fetal blood levels of the compound.
After reviewing all tine above data, two general conclusions may be made
concerning the potential of carbaryl to affect mammal-tan development. The
first is that the administration of carbaryl to pregnant animals (at
sufficiently high dose levels and/or sufficient duration of treatment) may
result in adverse effects to the embryo or fetus. Of those studies from which
definite conclusions may be drawn, carbaryl has been shown to produce terata in
the guinea pig, rabbit, and dog; and fetotoxicity in the mouse, rat, and
gerbil. The second conclusion which may be arrived at is that these effects
8/
Two additional non-rodent species have been used in carbaryl studies.
Earl et al. (1973) reported on experiments in which carbaryl was administered
in the diet to swine throughout gestation. Fetal resorptions and stillborn
were 22% in the 8 mg dose, 21% in the 16 mg dose, and 3% in controls in the
first of two experiments. In the second experiment, resorptions were not
noted, but the rate of stillborns was not dose-related, being 2% in the ;
controls, 11% in the 16 mg dose, and 2% in. the 32 mg dose. There was no <
evidence of any dose-related fetal anomalies in either study. No information j
is given concerning the effects of carbaryl on the dams. The swine studies are I
perhaps suggestive of seme fetal toxicity, but there is not sufficient |
information at present to make any definite conclusions concerning th^ effects j
of carbaryl on this species. i
Panciera (1967) studied the effects of carbaryl en pregnant sheep. Two cardiac
anomalies were found in the treated groups, but the gmaii numbers of pregnant
animals, together with no knowledge of the nwmai incidence of such anomalies,
make it unoossible to draw definite conclusions concerning tjtis study.
-25-
-------
have generally occurred only at dose levels which are grossly toxic to the
maternal animal. Adverse developmental effects have been seen at levels which
resulted in maternal death in the guinea pig, cholinergic toxicity in the
rabbit, and weight loss in the rat and mouse (the health status of the maternal
gerbils was not given in the published study concerning that species). The dog
appears to be the only exception to this conclusion, and in this species the
treated females had difficulty giving birth, a possible sign of carbaryl-
induced maternal toxicity.
The Agency fully acknowledges that the evaluation of these studies in
terms of their applicability to the human population must be done with great
care. In assessing the totality of the experiments, the Agency recognizes that
carbaryl has been tested in an extremely wide variety of species and has been
found to be teratogenic only in three species (guinea'pig, rabbit, and dog), of
which defects were found in only one species (the dog) at doses below those
causing maternal toxicity. In view of the circumstance that there are adequate
prenatal studies in eight species, it would appear that carbaryl is not a
potent teratogen. This same close dose relationship exists between maternal
toxicity and forms of adverse fetal effects other than teratogenicity.
In judging the relevance of embryonic fetal effects which are "confounded"
by gross maternal toxicity, several factors must be kept in mind. In an
experiment where significant differences are seen between control and treated
groups, these differences are by definition attributable to treatment. If
adverse perinatal effects are found in carbaryl-treated litters, the cause of
-26-
-------
these effects is understood to be carbaryl. Even if these effects are seen
only in litters of mothers who were themselves adversely affected by the
carbaryl treatment, we must still conclude that carbaryl is the cause of the
developmental effects.
When the Agency begins to consider the relevance of such studies to the
human population, however, other factors must be considered. Foremost are
possible mechanisms by which the effect in the "developing organism was
obtained. If, for instance, a compound is administered which affects the
maternal animal's desire to eat, the mother may lose weight and be unable to
provide sufficient nourishment to either the fetal or neonatal animal. A study
such as this raises the question, however, whether any* type of food restriction
would have resulted in the same effect. If this is the case, the primary
effect would be the food restriction, and the mechanism for producing it would
become almost incidental. In .the study by Robens (1968), for'example, carbaryl
produced terata in guinea pigs at doses which resulted in 40? maternal
mortality. While carbaryl was by definition the teratogenic agent, it is very
possible that the actual cause of the terata lies in some part of the general
•
debilitation of the surviving animals. Without a control group which is
equally sick it is impossible, however, to distinguish between the unique
effects of carbaryl and the effects of the severe maternal toxicity. In all
the species tested, with the exception of the dog, adverse fetal effects were
not seen when there was no maternal toxicity. Ihere is a valid question,
therefore, as to whether carbaryl itself has any properties which render it
fetotjoxic or whether carbaryl, like most pesticides, can cause severe maternal
toxicity when administered at sufficiently high doses, and some aspect(s) of
this toxicity in turn results in adverse developmental effects.
-27-
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Another aspect of the relationship of maternal toxicity to adverse fetal
effects is the relationship between the effect doses of both types of
effects. It is generally felt that a compound which is fetotoxic at much lower
dose levels than it is maternotoxic has the potential to be a greater human
hazard than one in which the effect levels are similar. The reason for this
is that the former case is far more insidious environmentally, since a slight
rise in terata or other fetal toxicity is extremely difficult to identify by
epidemiological means. Acute health problems in the adult population are
easier to identify, and corrective actions may -be swiftly taken. The
relationship between maternal and fetal toxic dose levels clearly approaches
equality in most species tested with carbaryl. Even in the case of the dog,
some signs of maternal toxicity occurred at doses where terata were noted,
indicating that in *•*** species, too, adverse developmental effects were not
seen at doses below those which elicited maternal toxicity.
In view of the overall weight of evidence of studies which are valid and
interpretable, the Agency has concluded that currently available data on
carbaryl do not indicate that a rebuttable presumption on the basis of
teratogenic and fetotoxic effects is warranted at this time. In the Agency's
judgment, the extremely high doses of carbaryl used to elicit effects in the
developing organism, coupled with the positive correlation of maternal and
fetal toxicity in the multiple species tested (the dog being a possible
exception), indicate that carbaryl would not constitute a potential human
teratogenic or reproductive hazard under proper environmental usage. As
articulated above, however, the Agency believes that the dog study should be
repeated, with special attention paid to sufficient numbers of animals in the
dose groups, the condition of the bitches throughout the period of dosing, and
possibly maternal and fetal blood levels of the compound (Qiernoff, 1977
[updated 1980]).
-28-
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2. New Jersey Esidemiologle°l Studies to Assess Carbarvl-Related Birth
Defects. The State of New Jersey, Department of Health, carried out
epidemiologies! studies for adverse reproductive effects and birth defects due
to carbaryl spraying for gypsy moths (Halpin, 1980). Three counties were
selected for the studies: Cape May, Morris, and Monmouth. These counties were
selected because residents and the press alleged that human and animal
abortions and birth defects occurred because of carbaryl spray exposure.
The survey was conducted by the staff of Parental and Child Health
Services of the New Jersey State Health Department to investigate several cases
of birth defects that occurred in Cape May County. Five cases of birth defects
were reported from births during the period from December, 197-9, to January,
1980. The coincidence of these cases with the spraying of carbaryl in the
spring of 1979 led to speculation that carbaryl was the cause of the defects.
The study was carried out to explore the possibility of a correlation
between use to control gypsy moths and the occurrence of the birth defects.
In particular, the study was initiated to answer two Questions: 1) Is there
an increased rate of birth defects in municipalities where carbaryl is used in
aerial spraying for gypsy moths? and 2) Is there a temporal correlation
between the occurrence of birth defects and this spraying?
Live births (3^,355) from January 1, 1977, through March 31, 1980, were
surveyed in Morris, Manmouth, and Cape May counties, which were chosen on the
basis of their level of spraying and their common suburban/rural
characteristics.
The records of all ten hospitals with maternity services in these counties
were reviewed by the Health Department. Three physicians studied and
classified the possible cases, scoring only major birth defects (Down's
Syndrome, major organ system defects, cranio-facial defects, defects of nervous
-------
system development, and absence of any limb or portion of a limb). Minor birth
defects (webbing between fingers, extra digits, minor abnormalities of the
external genitalia, and maldevelopment of the feet) were not included in the
statistical analysis, nor were birth marks/discolorations, hip joint
dislocation, malposition of the feet, hernias, and undescended testicles.
The rate of birth defects per 10,000 live births was calculated for each
municipality. These rates were then compared between those municipalities that
sprayed carbaryl for gypsy moths and those that did not. Analysis of the
possible temporal relationship between the spraying and the defects was based
on a calculated two-week interval of probable conception.
For sprayed municipalities the average rate of birth defects was
44.36/10,000 live births, with a standard deviation of 6U.44. The average rate
for non-sprayed municipalities was 68.55/10,000 live births, with a standard
dsviation of 219.02. The State Health Department concluded that there is no
difference between the rates of birth defects in sprayed muni ^-i patj tig* and in
non-sprayed municipalities.
Analysis of the time of conception and the spraying of carbaryl (no
statistics given) also led to the conclusion that there is no difference
between the sprayed and non-sprayed municipalities (Halpin, 198C).
.-.3O-
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C. Mutrgenicity
4C CFR 152.11 (a) (3) (ii) (A) provides that "a rebuttable prescription shall
sris!? if a pesticide's ing red lent (s), metsbolite(s) , or degradation product(s)
... induces mutagenic effects, as determined by multitest evidence." 4P CFR
1^2.2(y) defines mutagenicity as "the property of a substance or mixture of
substances to induce changes in the genetic complanent of either somatic or
germinal tissue in subsequent generations." .""utagenic chemicals are recognized
as posing a potential risk to human health because of their ability to cause
heritable changes in genes and chromosomes. Such germline changes can, for
rscsmple, lead to birth defects or to the accumulation of deleterious mutations
in the human gene "pool. In addition, somatic mutations may be involved in the
etiology of cancer.-
. Tb determine whether or not carbaryl might pose a mutagenic risk*
the Agency has reviewed the currently available deta and conducted an
sssessment of the mutagenic potential of carbaryl (KEAG, 19*£). Specifically,
the primary objective of a mutagenicity risk assessment is to determine the
potential of a chemical to cause heritable germline effects in men, as the
Agency has articulated in its Etoposed Guidelines for Mutagsnicity Risk
Assessments, (Fed. Reg. 45(221): 74954-74955 [November 13, 198!!]).
•——™—•*^__
Only those data which are pertinent to mutsgenicity as a possible
acverss effect of carbaryl per se ere evaluated here. The possibility of a
nutsger.ic risk posed by formation of >J-r.itrosoccrbaryl in stomach physiology
will be addressed in a se??rat* Agency review of nitroso compounds. (See
Appendix A for a sinmary of current information concerning the mutsgenicity of
N-r.itrosocr.rbsryl.)
-------
In broad sumrry, carbaryl has been reported to produce gene nutations in
bffcteri?, Drosophila, and msnmalian cells in culture. Itowsver, th«re are
several inadequacies in these studies, m addition, the results of cytogenetic
t?sts imply that carbaryl may induce chromosomal effects in rasmmalian cells in
culture, in whole mammals, and in plants, and carbaryl has been shown to cause
primary I3JA damage in cultured hunan cells. Collectively, all of these factors
strongly suggest that carbaryl may act as a mutagen. Tb cause heritable
effects in man, however, s chemical with intrinsic mutagenicity must reach the
germinal tissue. Evidence that carbaryl and/or its active metabolites reaches
the gonads is only suggestive. Averse gonadal effects — e.g., abnormal sperm
morphology, reduction in the number of spermatogonia and spermatozoa in the
seminiferous tubules, and reduced sperm motility — have been observed in
rodents exposed to carbaryl (Degraeve et al., 1975; Shtenberg and Rybakava,
1955; Kitagawa et al., 1977). in addition, abnormal sperm head morphology has
been reported in workers with known exposure to carbaryl. Therefore, given the
weight of evidence, carbaryl may have the potential to act as a germ cell
mutagen. It should be emphasized, however, that carbaryl is not intrinsically
a potent mutagen in the reported studies, and probably acts as a weak mutagen
only. A review of the most pertinent evidence follows.
1. Evidence Concerning Point (Gene) Mutations. Paint (gene) mutations
are alterations which affect single genes. They are defined as intralocus
changes (base-psir substitutions, frameshift mutations, small deletions, and
additions).
s. S?cteri£. Studies concsrning point mutations in bacteria ere
summarized in Table 1 as follows:
-------
Reference Test System
Asliwood-Smlth Reversion
et al., 1972 to tryptophan
prototrophy In £. colli
spot test
Blevlns et al., Salmonella testi
1977 plate Incorporation •
and spot test
<•*
* Cook et al. Salmonella testi 1
(abstract, 1977) plate Incorporation
DeOlovannl-Donnelly Back mutation at
et al., 1968 Iratole locus
of Bacillus
subflTH
1
Detorenzo Salmonella
et al., 1978 testi plate
Incorporation
'
i .1
•J
i ;
\
Strains
W2
TA 1535
TA 1537
TA 1538
TA 98
TA 100
A 100
1681-
W 1535
TA 1537
TA 1538
TA 98
TA 100
TABU 1 — POINT MUTATIONS IN BACTERIA
Activation Concentration
None 10% solution
In phosphate buffer
saline (Img/dlsk)
Nona 50 nanomole
or 11.5 ug/plate
dissolved In ethanol
)
Nona . 0.2, 2, 20,
' 400 ug/plate
Nona 0.07% solution
Phenobarbltal 10 ug to 1500
Induced rats, ug/plate,
B-9 liver dissolved In
preparation EMSO
(96-991 purity)
• •
'
•
Results
Negative
Negative
Weakly
nutagenlc
at 400 ug/
plate
Negative
•
Negative
with or
without
metabolic
activation
Comments
1. Data not presented.
2. Concentration of carbaryl
Is above solubility In water
(fl.fllll Carpenter et al., 1976).
1. Data not presented.
2. Spontaneous frequencies not given.
3. No positive controls for tester
strains.
1. Abstract without data.
2. Data obtained by personal
communication (Cook, 1900).
1. Article was brief on
methodology.
2. No positive controls.
Data not presented.
•
•• -. * ••*•>
II
1
-------
Reference
Test System
IftBLE 1 — POINT MUTATIONS IN BACTERIA (COMT'D)
JJtralna . Activation Concentration
Results
Comments
< Egert and
Grelm, 1976
•' Egert and
Grelm, 1976
!> Elespuru
t et al., 1974
..; Fahrlg,
:.} 1974
';.]
1.
I
Hit at Ion to K12_ House-liver
prototrophy In gal" . mlcrosomeg
E. col It spot test nad~ . end NAOMI
"'91
M1B
Salmonella testi TA 1538 Mouse-liver 100 uH
IlquT3 suspension mlcrosomes
and NADFII
j
forward mutation HI Id- typo None j| 0.1 MH
to novoblocln t ,
resistance In }
llaetnophllus Influenzae
* . '
Gpot testi back
nutation to prototrophy
In two auxotrophlo strains
of Be r rat la marcescenat
to galacfcose prototrophy
In JU coll
Negative
with or
without
activation
Increase In
nutagenlclty
after
activation
Negative
Negative
1.
2.
3.
1.
2.
No positive controls.
Insufficient Information on
procedure employed.
Quantification of results not
presented.
The positive results were not
confirmed by repeating tin
experiment.
Dose related response not
determined.
Old not examine a range of
concentrations.
1.
2.
Report was a review of published
and unpublished data.
Method and quantification of
results not given.
\
i
-------
Reference
Fahrlg,
1974
Flcsor aid Nil lo
Piccolo, 1972
McCann at al.,
1975
f
Harsliall et al..
1976
TABLE 1 ~ POINT MUTATIONS IN BACTERIA (OOHT'D)
Test System Strains Activation Concentration Results
Liquid holding testi
forward mutation to
streptomycin resistance
In £. coll
Reversion to I96.
prototrophy In leu"
E^ colli spot test cys
Salmonella testi TA 1535
plate Incorporation TA 1516
TA 10B
TA 98
Salmonella testi TA 1535
plate Incorporation TA 1536
TA 1537
TA 1538
None
Aroclor 1254
Induced rats,
8-9 liver
preparation
Rienobarbltal
Induced rats,
S-9 liver
preparation
Commercial spray
with 51 carbaryl
10 ug up to
2000 ug/plate
fl
I
90 ug to
1000 ug/plate,
dissolved In CMSO
Negative
Negative
Negative
with or
without
activation
Negative
with or
without
metabol Ic
activation
Comments
1. Report was a review of published
and unpublished data.
2. Method and quantification of
results not given.
Method and quantification of
results not presented.
1. Mass screening of 300 chemicals.
2. Data presented only for 2000
ug/plate.
1. Data presented only for 1000
ug/plate or where a 2-fold
Increase above background was
observed.
2. Positive control data not
presented.
-------
Reference
Nagy et al.,
1975
1
Rashld, 1976
^ Slilrasu
-------
Originally, MrCann et si. (1975) classified csrbsryl es nonmutagenic in
the Salmonslla/microsome assay using four histidine-requiring strains.
Metabolic activation did not augment the nutation frequency. Later, however,
McCam and associates (personal communication, 193B) re-examined their data and
conducted cdditional experiments at different concentrations of carbaryl to
detect reversion at the histidins locus of Selnonella typhimurium TA 1535 (base-
pair substitution sensitive strain). In the absence of metabolic activation
they found that carbaryl appears to be weakly mutagenic. In one experiment,
sight doses were examined (0 to 1CC0 ug/plate), and carbaryl exhibited a dose-
response effect. At the carbaryl concentration 500 ug/plate, which yielded
the maximum mutagenic response, the mutation frequency was increased to about
2-fold over control values.
The mutager.icity of carbaryl was also evaluated by Rashid (1578),
employing five strains of Salmonella typhimurium. Rashid found that
carbaryl produced a weak response -in only strain TA 1525 (missense) at 125
ug/plste (.1.5-fold increase above the spontaneous mutation frequency) and at
i
525 ug/plate (1.9-fold increase above the spontaneous mutation frequency) in
the absence of metabolic activation. The presence of rat liver (S-9)
microsoraal enzymes did not increase the reversion frequency. Cook (abstract
published in 1977) observed weak mutagenic activity (2-fold increase) on
another base-pair substitution sensitive strain TA ISC at 4JJC ug/plate in the
absence of metabolic activation (Cook, personal communication, 19Sf!). Egert
and Grsim (1975) investigated the mutagenic activity of c?rbaryl using the
Salmonella typhimurium strain sensitive to frameshift mutagens, TA 1538. After
prsincubstion in mouse-liver microsomes ?.nd NADPH, 3S revertants/lfr-
Q
survivors were observed st ICC uM, whereas "<1" revertant/l£T survivors were
found in the nontrcatsd control.
-37-
-------
In contrast, several investigators have reported negative responses with
carbaryl using the Salmonella/Ames test. Other bacterial tests in which
carbaryl has been reported as negative include back mutations in Bacillus
subtilis. forward mutations in Haemonhilus influenzas . and back and forward
mutations in Eaehgriehia coll (Table 1 ) . It should be acknowledged that
these negative results might be due to the relative sensitivity of the tests,
solubility problems, or cytotoxicity.
b. Mammalian Cells in Culture. Ahmed et al. (1977) found approximately a
9-fold increase in the nunber of anaha -s n-reg i atant (OUA7 mutants over
spontaneous mutants after V79 Chinese hamster ^iis were treated in monolayer
o
with TO uM of carbaryl (66? cell survival) . These CUA mutants were
-«
reported to be phenotypically stable . The apparent ability of carbaryl to
generate ouabain-resistant mutants would be consistent with, its activity as a
base-pair substitution mutagen. Although carbaryl was found to be weakly
mutagenic in this gene mutation assay, several inadequacies are apparent in
tihjg report which reduce the weight of the positive result: e.g., Da
concentration-related increase in mutation frequency was not demonstrated, 2)
concurrent positive controls were omitted, and 3) data were not presented to
support the statement of phenotypic stability of the ouabain-resistant
phenotype .
C. Prosophila; Sex-linked Reegssive Lethal Test. Brezheskii (1972)
exposed Drosophila melanogaster males to a 1% suspension of "Sevin" (355
carbaryl) in dilute sugar for 24 hours (505 survival). Brezheskii found a
small increase (piO.01) in the percentage of complete (heritable mutations) and
partial (not all mutations transmitted to progeny) recessive lethals (0.025) as
compared to control values (0.05 experimental control; 0.05 + 0.025 in •
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'historical controls). If only the complete lethals are considered, the
frequency observed (0.091?) is not significantly different from the spontaneous
value. However, the Agsncy points out that a larger sample size would have to
be examined to classify carbaryl either as weakly positive or as negative.
2. Evidence Concerning Primary DNA Damage. DMA repair tests do not
measure mutation per se, but DNA damage as induced by chemical treatment of a
cell. Microbial test systems measure this damage as cell killing. Mammmalian
cell systems — both in vitro and Jn vivo — measure the damage to DNA either
directly, or as it is being repaired (DHEW, 1977).
Mammalian cells synthesize DNA during only one stage of the cell cycle.
This Is referred to as "scheduled DNA synthesis." However, when the cellular
DNA is damaged, repair synthesis can occur when scheduled DNA synthesis is not
taking place. This "unscheduled DNA synthesis" can be measured in
synchronously growing cells as uptake of radioactive thymidine in the cell's
DNA. It can be measured in cells in culture, or in meiotic and post-meiotic
mouse spermatoevtes in vitro CDHEtf. 1977).
Ahmed et al. (1977b) have shown that exposure of virally transformed hunan
cells (VA-4) in culture to carbaryl initiates unscheduled DNA synthesis at
exposure as low as 1 uM as determined 1) by autoradiography and 2) by
photolysis of brcmodeoxyuridine (BrdUrd), which is incorporated into DNA during
DNA repair synthesis. Metabolic activation by liver microsomes did not enhance
carbaryl's ability to induce unscheduled DNA synthesis. The cytotoxicity of
the carbaryl doses used was not given in this report.
Regan et al. (1976) treated a culture of human skin cells with 100 uM of
carbaryl for 1 hour and found no evidence of DNA damage. The technique
employed, however, was not the same as that used by Ahmed et al. (1977b).
Regan et al. (1976) determined the sedimentation profiles in alkaline sucrose
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gradients of cellular DNA treated with carbaryl as a detection method for DNA
damage. This method of detection, however, may not be as sensitive as the
(ErdUrd) photolysis method.
Siebert and Eisenbrand (197*0 used a diploid strain of Saegharomvees
eerevisiae D4 heteroallelic at the gene loci ade-2 and try-1; to assay for
the ability of carbaryl to induce mitotic gene conversion in these loci. This
is another assay that detects damage to DNA. In this organism, genetic
activity (genetic damage) was not produced by a 16-hour carbaryl (1000 ppm)
treatment. Yeast cells cultured in this solution showed only a 225 lethality.
3. Evidence Concerning Chromosome Effects. Mutagenesis involves not
only point mutations but also the gain, loss, or rearrangement of portions of
chromosomes as well as gain or loss of intact chromosomes.
Several cytogenetic studies have shown that carbaryl can cause chromosome
abnormalities (eolchieine mitosis, chromosome lagging, chromosome
fragmentation, multipolar anaphases, anaphase bridges, multinucleated cells) in
both meiotic and mitotic chromosomes of plants (Wuu and Grant, 1966; Amer,
1965; Amer et al., 1971; Amer and Farah, 1968; Brankovan, 1972). Although
carbaryl is capable of breaking chromosomes in plants, predominantly it causes
mitotic disturbances by interfering with the spindle mechanism. This may
result in chromosome loss and/or gain. Russian studies provide suggestive
evidence that carbaryl may also act as an antimitotic agent in human cells in*
culture and in rats (Shpirt, 1975; Kazarnovskaya and Vasilos, 1977; Vasilos et
al., 1972, 1975).
In addition, Ishidate and Odashima (1977) studied the effects of carbaryl
on chromosomes of cultured Chinese hamster fibroblasts. Three different doses
(0.0075, 0.015, 0.03 mg/ml) were added to cell cultures (Ishidate, personal
communication, 1980). At the maximum effective dose, 0.03 mg/ml (50? growth
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inhibition dose), several types of chromosome aberrations (35% aberrant cells)
were reported 48 hours after treatment. Specifically, chromatid gaps and
breaks, chromosome breaks, translocation, ring formation, and fragmentation
were observed with a higher frequency than in non-treated control cultures (1%
aberrant cells). Although the authors stated the "gaps" were the predominant
chromosomal effect, the frequency of occurrence for each particular type of
aberration and the frequency of aberrations within a cell were not given. At
lower doses, 0.015 mg/ml resulted in 24% aberrant cells, and 0.0075 did not
appreciably affect chromosome structure (1% aberrant cells). The authors did
not give the toxicity of these doses.
The dominant lethal assay in rodents, which detects chromosome damage in
germ cells, was used both by Epstein et al. (1972) and by Mail et al. (1973).
Using male mice, Epstein et al. (1972) administered 1000 mg/fcg and 50 mg/kg
(subtoxic doses) of carbaryl by gavage in daily portions over five consecutive
days. Reportedly, this dosage schedule did not produce significant, early fetal
deaths or preimplementation losses. However, data were not presented in this
report to support this statement. Weil et al. (1973) looked for dominant
•
lethality in rats using a 3-generation study and found no significant lethal
effects. The authors do not state if the carbaryl dosage level was the maximum
tolerated dose. Furthermore, the number of males treated, the number of virgin
untreated females mated with each treated male, and the number of implant and
fetal deaths per female of test or control groups are not given in this
report. It should be noted that in general the dominant lethal assay is
recognized as an insensitive test for purposes of detecting weak mutagens.
This is because of the small number of animals used in such a study and because
of the high background of fetal wastage observed in control animals. In
addition, chromosomal effects are usually observed at higher chemical doses
than are point (gene) mutations (REAG, 1980).
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4. Evidence Concerning Whether Carbaryl Reaches the Germinal
Tissue. In order for any mutagen to cause genetic alterations that may be
inherited by future generations, it must reach the gonads. As the Agency
articulated in its Proposed Guidelines for Mutagenicity Risk Assessments,
"S/idence that a chemical reaches or affects the germinal tissue, as provided
by such sources as data demonstrating the alkylation of DMA or other cellular
macromolecules, unscheduled ENA synthesis, sister chromatid exchange, or
chromosome aberrations, in germinal cells; and non-specific accumulation of
radioactive label in the gonads following administration of the labeled
chemical [is considered a factor which contributes insight into the rautagenic
t
activity of a chemical substance]. Other relevant evidence includes adverse
gonadal effects following acute, subchronic or chronic toxicity testing; and
adverse reproductive effects, such as decreased fertilization index, r^uced
spsrm count, or abnormal sperm morphology T (Fed. Reg- 4S f22] :74987) »
Numerous inadequacies are apparent in the available rspcrts • concerning the
adverse gonadal effects which might be associated with exposure to carbaryl,
and the evidence reviewed below is considered suggestive rather than conclusive
(REAC, 10SC; Chaisson, 198!!).
3. Epidemiological Data. The sperm-abnormality assay is ah indicator
that a chemical agent may be damaging the germ cells (Wyrobek and Bruce,
197S). Wyrcbek et al. (1?9C) analyzed semen samples fron 50 carbsryl
production workers who hsd spent at least one year on the job. Thirty-four new
hires provided semen for control purposes. Carbaryl-exposed males had sperm
counts or sperm with fluorescent bodies (thought to be caused by raeiotic
nor-disjunctioni similar to control values. There wes, however, r significant
elsvstion (p<0.£C5) of sperm abnormalitias (abnormal head morphology) in
currently exposed workers (52.£ ^ 2.'>%) ?s compared to controls (41.9 ± 2.1%).
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Previously exposed workers (an average of 5.5 years since last carbaryl
•sxposurs) did not exhibit a significant elevation of spem sbnormalitiss from
control values. Because of the snail sample size, hox>«ver/ it cannot be
established if carbaryl effacts are reversible. When current workers were
classified as low (e.g., supervisors, foremen, maintenance personnel) and high
(baggers, operators) exposure groups, both groups wers shown to have
significant differences in sperm abnormalities from the control group..
However, there were not appreciable differences between ths high and low
exposure groups.. In addition, there was a negative correlation between the
nunber of years exposed to carbaryl and the percentage of abnormal sperm in
current workers. Wyrobek and coworkers speculated on several mechanisms to.
explain this odd relationship 1) the likelihood that men working longer may be
exposed less because of seniority, 2) biological or pharmacological adaption
to exposure, 3} selection for non-affected males. Although this study
demonstrates a correlation between working in a carbaryl-exposed area and an
alteration of human spermatozoa, this is suggestive evidence only that these
effects are due to carbaryl and/or its metabolites. It must be established
that these defects of sperm morphology are not the result of other factors,
such as exposure to chemicals other than carbaryl.
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IT. an earlier epidemiologicsl study performed with workers at the sane
plant studied by 'Ayrobek et al. (19SO), Whorton and Milby (ig^S) exanined semen
samples provided by 47 workers who volunteered to participate in the
Tf» /
experiment."'' Thirty-six of these workers also provided blood samples for
hormone assays. The mean age of the 47 volunteers was not given. Itowever,
fchorton and filby's cohort of 99 workers (selected from employment records),
which included the 47 contributors, was described as follows: 53 baggers,
(me?n age = 41.1 years) , 23 operators (mean age - 34.8 years'! , and 23 others
(mean age 44.8 years). The experiment included no onsite controls. Hswever,
the results of the semen counts were compared to semen counts obtained from 93
members of a composite control population (chemical plcnt workers not exposed
to any known infertility-producing agents), the data used for control purposes
having been provided by Environnental Health Associates, Inc. (EHA).. A higher
percentage of the carbaryl-exposed workers (14.9%) showed depressed sperm
counts (less than 20 million per ml) than did the control group (5.5%), but the
difference is not considered statistically significant (p = S^CSSSK
Reproductive hormone levels in the carbaryl-exposed workers were "unremarkable"
(normal).
Of 49 origins! volunteers, 47 provided technically satisfactory srmen
samsles. These same 49 volunteers later participated in the study conducted by
%yrcbek et al. (19BC).
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b. Rodent Data. In another study performed in Russia, Krylova and
^^^^^^^^^^^^^^ t
i
Denisova (1972) examined tha process of spsrmatogenesis in the Mongolian tree {
creeper, a small rodent. Animals were trapped which inhabit an area that was j
[
sprayed with 85% Sevin wettable powdsr. The authors reported a reduction in !
I
r
the nunber of spermatozoa, spermatids, and sperraatocytes statistically
significant (p
-------
effect or. melp fertility was dependent on the carbaryl concentration and
duration of exposure.
In contrast, some investigators have reported no significant gonscal
effects attributable to carbaryl. Dikshith et al. (1975) administered carbaryl
(2CTC rag/kg for 3 days a week) orally to male rats for a period of SB days. No
histological changes were observed in the testes and epididymis. Also, these
authors found no effects on the fertility of male rats. Weil et. al. (1?73)
also observed no significant effects of carbaryl (ID ncj/kg/day) en fertility
in s 3-gensration rat study.
Taking the full weight of evidence into account, the Agency has concluded
that currently available data on carbaryl do not indicate that a rebuttable
presumption on the basis of mutagenic effects is warranted at this time. Due
to the weak mutagenic responses which have been measured, and due to the
suggestive rather than conclusive nature of the evidence available as to the
potential of carbaryl to reach the mammalian germinal tissue, the Ajer.cy
believes that general exposure reduction methods typical of those already on
many of the labels are appropriate and will be pursued prior to any further
REAR review.
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D. Qneogenia Effects
40 CFR l62.11(a)3)(i)(A) provides that a rebuttable presumption snail
arise if a pesticide's ingredient(s), metabolite(s), or degradation product(s)
induce(s) "oncogenic effects in experimental mama "Man species or in man as a
result of oral, inhalation or dermal exposure." The term "oncogenic" is
defined in 40 CFR I62.3(bb) as "the property of a substance or mixture of
substances to produce benign or malignant-, tumor formation in living animals."
To determine whether or not carbaryl might pose an oncogenic hazard warranting
a rebuttable presumption, the Agency has conducted a review of the available
literature (GAG, 1977). A summary of the Agency's findings follows.
Innes et al. (1969) administered 4.64 mg/kg carbaryl in 0.52 gelatin daily
by stomach tube to mice, strains X(c5-BL/6xC,H/Anf) and YCCcyBL/-
6xAKR)F1, beginning at 7 days of age and continuing until mice were 6 weeks
old. (Carbaryl dosage was not adjusted according- to weight gain during
the 6-week period.) Vehicle groups and untreated control groups consisted of
18 animals each. Subsequently, the vehicle groups were fed a diet containing
14 ppm carbaryl, and the experiment was terminated when the mice were
approximately 78 weeks of age. Treated mice showed no significant increase in
the incidence of tumors over controls. It should be pointed out, however, that
the 14 ppm dietary dosage of carbaryl was probably too low to be a firm
Only those data which are pertinent to oncogenicity as a possible adverse
effect of carbaryl per se are evaluated here. The possibility of an onco-
genic risk posed by formation of H-nitrosocarbaryl in the stomach will be
addressed in a separate Agency review of nitroso compounds. (See Appendix B
for a summary of current information concerning the carcinogenicity of N-
nitroso-carbaryl.)
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indicator of no oncogenic potential. The Agency points this out in view of 1)
the circumstance that many of the carbaryl tolerances on food are as high as 12
ppm (Fed. Reg., special ed. [July 1, 1976]: 180.169), and 2) the fact that
in a 1963 study conducted by the Mellon Institute (reviewed below) mice were
fed 400 ppm carbaryl in the diet for 2 years and did not show any marked
toxicity.
In a parallel experiment, limes et al. (1969) administered a single
subcutaneous dose of 100 mg carbaryl/fcg in UEO to weanling mice. Tumor
incidence in treated animals was not slgrP'f'irrarit'Ty increased*
In a study conducted by the- Mellon Institute (1963), groups of 48 male and
48 female CD-I mice were fed diets containing 0.04% (400 ppm), 0*01% (100 ppm),
and 0% carbaryl. Seventy-two of the 288 anynal« included in the study were
given gross and higtspa'^hol'?g^«»1 examination* Of thg other 216 animals, those
which died during the initial 80-week period (50%) were subjected to gross
pKami nation, and tissues suspected of having pathology were examined
histologically. Of the 65 animals allowed to live for 2 years, 9 males and 21
females were sacrificed at the end of the period, and no information was given
on the remaining animals. No significant difference in tumor incidence between
treated and untreated animals was observed in those mice sacrificed after 80
weeks, those which died during the 80-week period, or those sacrificed at the
end of 2 years. Because this study is very seriously flawed, however, it
cannot stand as a basis for any determination for or against the carcinogenic
potential of carbaryl (GAG, 1977). Among numerous drawbacks, the most serious
problems are 1) that no information is available concerning 93 of the 288
animals with which the experiment began, and 2) that for animals dying during
the study, histopathology was carried out only on animals suspected of tumor
growth and not on all animals.
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In another experiment conducted by the Mellon Institute (1962), a mixture
of 40 parts Sevin 85 sprayable powder (85% carbaryl) and 60 parts water was
applied (schedule of application unspecified) on the skin of 36 mice for 30
months. None of the animals developed tumors. In the same experiment,
metfaylcholantfarene (0.2 % solution in acetone) was applied to a group of 32
animals. All animals in this group developed turers by the end of 12 months.
Details concerning pathology and experimental procedure were not available for
review, however.
Matovskaia et al. (1965) administered weekly doses of carbaryl (60 mg/kg
in a 2% solution in sunflower oil) intraperitoneally to groups of line A and
c~HA mice. The experiment included 400 carbaryl-treated animals, 150 animals
t
treated with urethane (200 to 1000 mg/kg intraperitoneally) as a positive
control group and 100 animals in the untreated control group. Animals were
sacrificed at 1, 3, 5, 9, 12, 15, 18, and 24 months after the test began, and
several organs (lungs, kidneys, liver, heart, spleen, pancreas, thyroid, and
adrenals) were reportedly examined histopathologically. Makovskaia et al.
reported that "carbaryl treatment did not produce any new growth in lungs or
liver," but their report failed to provide experimental details necessary for
evaluation (CAG, 1977). Kb conclusion concerning the carcinogenic potential of
carbaryl can be drawn from this study.
Carpenter et al. (1961) maintained groups of male and female CP-N rats on
a mixture of Purina Chow and carbaryl (in concentrations of 0.04%, 0.02%,
0.01%, 0.005%, and 0.00%) for two years. Hats were 60 days old when the
experiment began, and 20 rats were included in each group. Sixty-one of the
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102 animals which died during the study, and 98 animals sacrificed at the end
of the study, were examined histopathologically. Although female rats were
found to have more pituitary tumors relative to males, no significant increase
in tumors was found in treated groups relative to controls. Hie validity of
Carpenter et al. is 'A»«^':"'l»**^f however, in that the study began not with
newborn or weanling rats but with 60-day-old animals. Because they are known
to be more sensitive to ctigft'i"*'1 carcinogen^sis than are adults, newborn or
weanling animals are preferred for purposes of cancer bioassays (GAG, 1977).
For purposes of their experiments with rats, Andrianova and Alekseev
(1970) obtained carbaryl (reportedly 97.65% pure) from the Schelkovsky Chemical
Plant. A group of 60 adult male rats (mongrels) was given 30 mg/kg carbaryl
orally (as a water suspension) twice weekly by gavage for 22 months. This dose
was approximately one-twentieth of the IE^Q dose. Tumors, as well as organs
suspected of having tumors, were- examined histopathologically. Out of 12 rats
alive in the treated group at the end of the experiment, four animals were
found to have ™*i ^nr^**1^ tumors. All tumors were sarcomas (2 fi">^'>e^>' *.* *nagf 1
polymorphocellular sarccma, and one osteosarcoma with various metastases). In
•
another experiment, Andrianova and Alekseev (1970) introduced 20 mg of carbaryl
(enclosed in a purified paraffin capsule weighing 250 mg) hypodennically in a
group of 48 male rats. Tumors were discovered in 2 out of 10 rats that
survived 22 months. In both animals, ^imrirg. (diagnosed as f ^ i-»rr?gau"i !?;ina )
were observed under the skin near the back, not near the site of the implant.
Whether or not controls were similarly implanted with gelatin capsules without
the chemical was unclear.
Andrianova and Alekseev used a group of 48 untreated rats as controls for
both these experiments. Among controls, one fibrosarcoma was discovered after
11 months, at which time 46 of the original controls remained alive. The
number of controls surviving the full 22 months of the experiment was not
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given. The Agency points out that if all 46 controls survived to 22 months,
the positive results of the feeding experiment would be significant.
Andrianova and Alekseev state, however, that "the results concerning the
average life span ... show that the pesticide doses selected were sufficiently
well endured by the animals" (translation), and survival in experimental and
control groups way have been comparable (GAG, 1977). Agency efforts to obtain
further liifonpgt: ion necessary to evaluate tng adequacy and significance of this
Russian study have not been gffim»gg^»7.
Two additional studies of t&e carcinogenic potential of carbaryl which
were not included in the original review by EPA's Carcinogen Assessment Group
(CAG, 1977) have since been evaluated by the Agency (GAG [Anderson] 1980a,
•
1980b). In a study performed by Shimkin et al. (1969), the carcinogenic
potential of carbaryl was tested with the pulmonary tumor response in male A/Be
mice, 7 to 9 weeks old. Carbaryl was prepared by reacting its chloroformate
with ammonia. The dosing vehicle was 99.9% pure tricaprylin. Intraperitoneal
injections of 20 mg carbaryl/kg were made 3 times weekly for 4 weeks. One
mice were sacrificed at 20 weeks after the final injection. Necropsies were
performed, and lungs were examined microscopically. Untreated and vehicle-
treated control animals were similarly majnfjHn»d and evaluated.
Fifteen of an original 16 carbaryl-treated animals survived to 20 weeks.
Upon sacrifice and examination, one lung tumor was found in each of 4 animals,
and 2 lung tumors were found in each of 2 animals. Twenty-eight of an original
32 vehicle control animals survived to be sacrificed. Open examination, 1 lung
tttprr was found in each of 6 animals, and 1 animal had 2 tumors. Thirty-one of
an original 32 untreated control animals survived to be sacrificed. Upon
examination, 1 lung tumor was found in each of 2 animals. The number of lung
tuiioL's per mouse in the carbaryl-treated, vehicle control, and untreated
control groups was, respectively, 0.7, 0.3, and 0.1.
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The tumor incidence observed by Shimkin et al. in carbaryl-treated mice
(40%) was slightly higher than in the vehicle control group (25%). However,
this difference is not statistically significant: p> 0.05, by the Qii-squared
test. Moreover, Shimkin et al. in fact state that a slightly higher tumor
incidence in vehicle-control mice (used as the standard comparison group in the
above calculation) over untreated control mice is ccomcnly observed in this
strain.. In the Agency's judgment, no conclusion about the potential human
carcinogenicity of carbaryl can be made from this test (GAG [Anderson] 1980a).
This particular bioassay is a short-term test with a mouse strain highly
susceptible to lung tumor formation, and findings in this strain cannot be
considered equivalent to a lifetime mouse bioassay. The essay is a"|g» flawed
by other limitations; 1) the actual purity of the carbaryl test sample is not
described, 2) the sample size of the groups is small, and 3) since the bedding
was heat-treated absorbent cedar cubed wood, it is possible that terpenes or
other substances in this material r"^* have altered the rate of- carbaryl
metabolism in the mice.
A study has been reported by A-J. Triolo (1978) in a TT.S. EEA document,
which attempted to evaluate the ability of carbaryl to enhance the incidence of
forestomach tumors in Ha/ICR female mice and lung tumors in A/J female mice
treated with benzo(a)pyrene (BP). Dietary levels of carbaryl up to 2000 ppm
did not increase the incidence of forestomach tumors in mice treated with 300
ppm benzo(a)pyrene in the diet for 12 weeks. In A/J mice given 3 mg
benzo(a)pyrene par as on days 7 and 21, 1000 ppn carbaryl in the diet produced
a statistically significant (P<0.05) increase in lung tumor incidence (IB/IB)
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as compared to the BP-treated control (15/17). This increase is slight,
however, and in a repeat experiment the incidence in the carbaryl-BP group
(16/3*0 was similar to that in the BP group (16/31). The A/J mice were on
study for 20 and 16 weeks, respectively, in these experiments. The Agency's
conclusion with respect to this study is- that a co-carcinogenic effect has not
been conclusively demonstrated (GAG [Anderson], 1980b).
Based on the weight of evidence of available studies on carbaryl the
Agency has concluded that a rebuttable presumption on the basis of oncogenic
effects is not warranted at tfrte time.
E. Neurotmgieitv
As stipclated in 40 CRF l62.11(a)(3)(ii)(B), "a rebuttable presumption
shall arise if a pesticide's ingredient (s), metabolite(s), or degradation
product (s) ... produces any other chronic or delayed toxic effect in test
at any dosage up to a level, as determined by the. Administrator, which
is substantially higher than that to which hiring can reasonably be anticipated
to be exposed, taking into account ample margins of safety."
Three available studies concerning the neurotoxicity of carbaryl have been
reviewed by the Agency. To test the neuromuscular degenerative potential of
carbaryl as compared with that of triorthocresyl phosphate (TOCP) , Carpenter et
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al. (1961) administered single subcutaneous injections to 2-year-old maul ting .
Rhode Island Bed hens. ' These chemicals were mixed or suspended in lard
melting at or below 37°C and administered at 0.25, 0.50, 1.0, 2.0f and 3.0
gram/kg dosage levels at concentrations of 25% to 40%. Thirteen hens were
administered carbaryl, and 10 were administered TOCP thus diluted. In
addition, undiluted TOCP was give: to one chicken, and undiluted lard was given
to two others. Five control chickens received no injections.
No adverse effects were observed at 1.0 g/kg or lower doses of carbaryl.
Chickens administered 2.0 g/kg carbaryl showed leg weakness on the first or
second day following dosing, and in only one case was the chicken nonambulant
for 3 days. A nephrotoxic action was »ig" observed in fowl which received 2.0
g/kg or larger injections of carbaryl.. (Nephrotoxic action was apparent as a
deposition of fine fat droplets within the epithelial cells lining the
proximal tubules.) Necrosis was not present. In hens which received 3.0 g/kg
carbaryl, lesions (focal loss of striation and fatty infiltration of
' The subcutaneous route was used because no satisfactory vehicle was
then known by which to administer carbaryl intravenously.
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K
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gastrocneinius muscles) were observed. Such lesions wars found at all levels in
hsns examinee after injection with TOCP, with the anomalous exception of the
2.0 g/kg dosage. Likewise at all dosage levels tested, animals injected with
TOCP showed fatty deposition of a similar though more diffuse nature to that
observed in fowl administered 2.3 g/kg or larger doses of carbaryl. At 3.C
g/kg, TOCP proved lethal in 3 to 1C days, with leg weaknesses observed in 3 of
4 cases only on the day of death. At all lower dosage levels, weakness was not
apparent until the thirteenth or fourteenth day. Upon histopathological
examination, slight evidence of demyelination was observed in three of the 13
TOCP-treated hens, whereas carbaryl-treeted hens showed no signs of
demyelination.
•
In screening tests for the production of paralytic effect in chicken hens,
Gaines (1959) tested 30 organic phosphorous pesticides an 9 csrbamate
pesticides including carbaryl. Ihe vork of Gaines has been reviewed and
validated for the Agency (Melcalf, 1977).. Gaines (1959) administered carbaryl
subcutsnsously to hens at single doses of 8C0 and 1*530 rag/kg. Prior to
csrbaryl treatment, the hens were treated with 15 mg/kg of atropine orally.
All animals were observed for 24 days. The hens treated with 15CS mg/kg dose
of carbaryl showed leg weakness within 24 hours, but all chickens recovered by
day 24. Ihe TOCP-treated animals developed paralysis after 14 days, and the
paralysis continued until death.
anslley et si. (1959) administered carbaryl at a dose of 150 mg daily for
72 and S3 days to one male pig and one female pig, respectively. In a second
experiment, one female and tvo males were given 152 mg/kg of carbaryl for 28
dr.ys, followed by 3DZ mg/kg/day for either 18 (males) or 57 (females)
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sedition.-! days. Ataxis was observed in both experiments. Microscopic
examination of skeletal muscle showed myodegeneration. The author reported
that hydrochlorothitzide, a diuretic, reversed the signs of toxicity of
carbaryl adainistrstion in chronic testing of pigs.
Eased on currently available evidence as susnarired above (Edwards, 1983},
the Agency has concluded that carbaryl does not pose a neurotoxic hazard and
that a rebuttable presumption on the basis of neurotoxicity is not warranted at
this time.
F. Viral Enhancement
A recent study (Abrahamsen and Jerkofsky, 1980) which has come to the
Agency's attention concerns viral enhancement as a possible adverse effect of
hunr.n exposure to carbaryl. Abrahamsen and Jerkofsky investigated the effect
of Sevin 4 oil on the replication of the human herpes virus varicella-zoster
(VZ) in primary human embryonic lung (HEL) and HEP-2 cell cultures. Complete
Sevin 4 oil, its active ingredient (analytic grade carbaryl) and its "base oil
plus inert ingredients" in sub-toxic concentrations, were tested for the
ability to enhance the growth of VZ virus as measured by an infectious center
assay. A 12- to 15 -fold increase in virus production by cells pretreated with
Sevin 4 oil and carbaryl was observed. No enhancement was observed in base oil-
treated cultures. Similar results were obtained when HEP-2 cells were used.
Viral enhancement appeared to be concentration-related in that decreasing
concentrations of csrbaryl brought decreases in viral enhancement. Experiments
with herpes simplex virus type 1, however, showed no viral enhancement by Sevin
4 oil of any of its components. Abrahamsen ?.nd Jerkofsky suggest that the
results of their work may be pertinent to studies of Rsye's synrlrome^, an
encephalopsthy with fatty infiltration of the viscera "since published
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epidemiological evidence has suggested a possible relationship between
pesticide spraying, certain viral diseases including chickenpox or varicella ,
and the subsequent occurrence of Reye's syndrcne" (Abrahamsen and Jertofsky,
1980 [abstract]).
With regard to viral enhancement as a p^gslbl^ adverse effect of exposur
to carbaryl, the Agency's determination at **•»« juncture is that the work of
Abrahamsen and Jerkofsky is preliminary in nature and that current data do not
constitute a basis on which to conclude that earbaryl poses a human hazard in
terms of viral enhancement. Ike Agency has therefore concluded that a
rebuttable PL^SM"^*. ^ "n is. not warranted at frh^g time*.
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V. CONCLUSIONS AND RECOMMENDATICMS
A. Summary of Conclusions
1. Terategenic end FeTotoxic Effects. Based on the weight of evidence of
currently available studies which are valid and interpretable, the .Agency has
concluded that a rebuttable presumption on the basis of carbaryl-related
terstogenic and fetotoxic effects is not warranted at this time. In the
Agency's judgment, the extremely high doses of csrbsryl used to elicit effects
in the developing organism, coupled with the positive correlation of maternal
and fetsl toxicity in the multiple species tested (the dog being a possible
exception), do not indicate that the pesticide carbaryl constitutes a potential
human teratogenic or reproductive hazard under proper environmental usage.
However, the Agency believes thet another study in dogs should be conducted,
with special attention paid to sufficient numbers of animals in the dose
groups, the condition of the bitches throughout the period of dosing, snd
matemrl and fetal blood levels of the compound.
2. Mutagenic Effects. Based on the weight of extensive existing evi-
dence, the Agenry has determined that the current data base does not support a
conclusion that carbaryl poses a mutsgenic hazard to humans. Due to the weak
mutager.ic responses which have been measured, and due to the suggestive rsther
thsn conclusive ncturc of ths evidence available as to the potential of
cerbaryl to recch the mormalian germinal tissue, the Agency believes that
general exposure-reduction measures typical of those alre?dy on many of the
labels, ar«* appropriate end will be pursued prior to any further RPAR review.
A rcbu*tcbls presumption or. the b?sis of csrbaryl-releted mutsgenic effects is
therefore not w?rr3nt3d at this time.
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3. Qneogenig Effects. Based on the weight of existing evidence, the
Agency has concluded that the current data base does not indicate that carbaryl
poses an oncogenic hazard to humans. A rebuttable presumption on the basis of
carbaryl-related oncogenic effects is therefore not warranted at this time.
3. Neurotereleitv. Based on available evidence, the Agency has concluded
that carbaryl does not pose a human health hazard in terms of neurotoxic
effects. A rebuttable presumption on the basis of neurotoxicity is therefore
not warranted at this time.
5. Viral Enhancement. The Agency's determination at fcM« juncture is
that research into viral enhancement as a possible adverse effect of exposure
to carbaryl is preliminary in nature and that current information does not
constitute a basis on which to conclude that carbaryl poses a human hazard in
this area. A rebuttable presumption on the basis of viral enhancement is
therefore not warranted at this time.
6. Overview—Determining Considerations. Recognizing that the data base
on any chemical is necessarily a continuum, the Agency's determination not to
proceed with an RPAE action against carbaryl at this time takes into account a
number of considerations in connection with the present toxicological picture
of the pesticide. As has been pointed out, the current data base under review
^
is extensive, more extensive than has ordinarily been the case for pesticides
which have come under Agency review. This is particularly true for terato-
genicity/fetotoxicity and mutagenicity, which are the toxicological areas of
primary concern, and it is unlikely that resource-intensive RPAE procedures
would surface data not already in the Agency's possession via other channels.
Although the current data base is extensive, risk data are not
unequivocal, and study results, again in the areas of teratogenicity/feto-
toxicity and mutagenicity, have been inconsistent. The current toxicological
picture of carbaryl thus reflects a degree of uncertainty. It is in the face
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of such uncertainty that the Agency must determine whether or not to proceed
with an RPAR action and the detailed risk/benefit analysis the RPAR process is
intended to implement. In the case of carbaryl, consideration of the overall
weight of current evidence leads the Agency to conclude that the responsible
call is not to initiate RPAR proceedings at this Juncture but rather to address
the concerns at issue via the recommendations made below. Should further
review of data indicate that current use patterns of the pesticide pose
unreasonable adverse effects to human health or the environment, however, the
Agency will re-open the case of carbaryl as an RPAR candidate.
8. Bflsuinfflttntl st ions
Because the Agency has concluded that a rebuttable presumption against
registration and continued registration of pesticide products containing
carbaryl is not warranted at tMs time, the Agency's recommendation is that
carbaryl be returned to the registration process. Ibis recommendation is made
with the following stipulations: 1) that a FXFRA sec. 3(c)(2)(B) action be
initiated requiring that an additional study of the teratogenic and fetotoxic
effects of carbaryl in dogs be conducted and that any other necessary data be
generated as will be specified in a forthcoming 3(c)(2)(B) action; 2) that
appropriate label changes be implemented according to forthcoming negotiations
between the Agency and registrants to ensure that exposure is minimized.
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sr.inals. Toxicol. Appl. Pharmacol. 22:151-174.
Sullivan, L., Eldridge, J., Knaak, j.., and Tallant, M. 1972b. 5,S-dihydro-5,5
dihydroxycarbaryl glucuronide as a significant metabolite of carbaryl in"the
rat. J. Agr. food Chem. 2Q:98C-985.
Thomas, J.A., Dieringer, C.S., and Schein, L. 1974. Effects of carbaryl on
mouse organs of reproduction. Tbxicol. Appl. Pharaacol. 28:142-145.
Uchiyaraa, M., Tskeda, M., Suzuki, T., and Yoshikawa, K. 1975. Mutagenicity of
nitrosoderivatives of N-raethylcarbaraate insecticides in microbiological
method. Bull. Environ. Cbntam. Tbxicol. 14:373-394.
Ukeles, R. 1962. Growth of pure cultures of marine phytoplankton in the
presence of toxicants. Applied Microbiology 10:532-537.
Vashakidze, V.I* 1967. Mechanism of action of pesticides (Gronosan, Sevin,
Dinoc) or. the reproductive cycle of experimental animals. Soobshch. Akad, Nauk
Gruz, SSR. 48:219-24. Chem. Abstr. SS:28759flx, 1968. (Original in Russian.)
Vashakidze, V.I., Shavladze, M.S., and Gvineriya, I.S. 19^-3. Harmful effects
of nitrogen-containing pesticides (Dinoc and Sevin) on the sexual function of
male rats. Sb. Tr., Nauch.-Issled, Inst. Gig. If. Profzabol, Tiflis 12:205-258.
Chem. Abstr. 58:113583m, 19S8. (Original in Russian.)
Vasilos, A.F., Enitriento, V.D., and Shroyt, I.G. 1972. Colchicine-like
action of Sevin on human embryonic fibroblasts in vitro . Bull. Eskp. Biol.
Med. 73:91-93.
Vasilos, A.F., Qnitriento, V.D., and Shroyt, I.G. 1975a. Disruption of the
raitotic system following acute Sevin poisoning. Izv. Akad. Nauk Mold. SSR Ser.
Biol. Kiim. Nauk. 3:64-57.
Von Ruraker, R., lawless, S.W., and Meiners, A.F. 1974. Production,
distribution, use and environmental impact potential of selected pesticides.
Case Study >b. 2 Carbaryl. pp. 133-145. Contract study EPA 54n/l-71-f!?l for
Office of Pesticide Programs and Council on Environmental Quality.
Weil, C.S., and Carpenter, C.P. 1955. Evaluation of the teratogenic potential
of insecticide Sevin in rats. Mellon Institute Special Report, Report 24-2D
Corrected. (Proprietary.)
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'.veil, C.S., Vtoodside, M.D., Carpenter, C.P., and Snyth, H.F., Jr. 1972. |
Current status of tests of carbaryl for reproductive and teratogenic effect. j
Tbxicol. Appl. Pharraacol. 21:39^-404. !
Weil, C.S., and Carpenter, C.P. 1965. Results of a three generation j
reproduction study on rats fed Sevin in their diets. Mellon Institute Report, j ;
Report 28-53. (Proprietary.) • i j
Vfe.il, C.S., Waodside, w.D., Bernard, B., Condra, N.I., and Carpenter, C.P.
1973. Studies on rat reproduction and guinea pig teratology of carbaryl fed
either in the diet or by stomach. Tbxicol. Appl. Pharaacol. 25:521-538.
Weil, C.S., Wbodside, M.D., Bernard, J.B., Condra, N.I., King, J.M., and
Carpenter, C.P. 1973. Comparative effect of carbaryl on rat reproduction and
guinea pig teratology when fed either in the diet or by stomach intubation.
Ibxicol. Appl. Pharmacol. 25:621-638.
Whorton, D., and Milby, T. 1978. Testicular Function among Carbaryl Exposed
Employees at the Union Carbide Institute Plant. A report to Union Carbide
Corporation from Environmental Health Associates, Die.. (Confidential;
Proprietary.) Reviewed for the Agency by Clement Associates, Washirgton, D.C.
'vuu, K.D., and Grant, W.F. 19SS. Morphological and somatic chromosomal.
aberrations induced by pesticides in barley (Hordeum vulgare). Can. J.
Genat. Cytol^ 8:4S1-5C1. -
'.Vyrobek, A«J., Watchmaker, G., Gordon, L., Wang, R., and .Msore, D. 1980. Sperm
abnormalities in carbaryl exposed employees* Submitted for publication.
V.yrobek, A.J., and Bruce, W.R. 1973. Ihe induction of sperm-shape
abnormalities in mice and hunans. Chem. Mutagsns 5:257-22C.
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APPENDIX A — REVIEW OF EXPERIMENTAL EVIDENCE ON THE
MUTAGENICITY OF N-NITROSOCARBARYL
Carbaryl has been shown in vitro to react with sodium
nitrite under acidic conditions (pH 1) to form N-^nitrosocarbaryl
(Etsenbrand et al., 1974). Because nitrite is present in human
saliva and food products, the formation of nitrosocarbaryl in
stomach physiology is possible/ in view of the widespread use of
carbaryl. Rickard (1979) demonstrated the in vitro formation of
nitrosocarbaryl in the stomach of rats and guinea pigs. When
guinea pigs were given either simultaneous intubation of
carbaryl (1 umol) and sodium nitrite (1160 umol), or when these
components were mixed with feed, approximately a 1.5% yield of
t
nitrosocarbaryl was detected. The formation of this nitroso
derivative was dependent on the amount of nitrite and the pH,
and not particularly by the amount of carbaryl present.
Increasing the amount of carbaryl from 0.025 to 2.5 umol did not
increase the yield of the nitroso compound. In rats, the
stomach pH (3.5-5.5) is higher than in guinea pigs (pH 1.5), and
in that species a very low yield of nitrosocarbaryl was found
(0.02%) at the same concentrations of nitrite and carbaryl.
A-l
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Nitrosocarbaryl has been shown to be strongly mutagenic in
bacteria. Blevins et al. (1977) found that the base-pair
substitution sensitive Salmonella strains TA 100 and TA 1535
were reverted by this agent without metabolic activation. The
reversion frequency in TA 100 was increased by approximately 1.6-
fold at 1.15 ug/plate and 6-fold at 11.5 ug/plate., and TA 1535
by about 3-fold at 1.15 ug/plate and 76-fold at 11.5 ug/plate.
Nitrosocarbaryl was not as active on the frameshift sensitive
strains TA 98, TA 1537, and TA 1538. Marshall et al. (1976)
found that nitrosocarbaryl increased the number of histidine-
t
independent colonies of TA 1535 by approximately 6-fold at 0.5
ug/plate and by 367-fold at 50 ug/plate without metabolic
activation. Marshall et .al. also found nitrosocarbaryl .to be
slightly active (above 6-fold over background values) on the
frameshift sensitive strains TA 1537 and TA 1538 at 50
ug/plate. Both Blevins et al. (1977) and Marshall et al. (1976)
found that the mutagenic activity of nitrosocarbaryl was dose-
related.
Elespuru and coworkers (1974) measured the induction to
novobiocin resistance in Haemophilias influenzae. These
authors found that nitrosocarbaryl was approximately an order of
magnitude more potent than the mutagen N-methyl-N1-
nitrosoguanidine (MNNG). In Escfaerichia coli nitrosocarbaryl
was also more potent in the induction to arginine prototrophy
than MNNG (Elespura et al., 1974). Uchiyama et al. (1975) found
mutagenic activity as tested by the ability to cause reversion
at the tryptophan locus in Escfaericfaia coli (data not
guantitated).
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Generally, metabolic activation was not required for the
mutagenic response of nitrosocarbaryl. For example/ when
Marshall et al. (1977) incorporated the S-9 fraction in the
Salmonella assay, a decrease in mutagenic activity was
observed. Greim et al. (1977), however, found an increase in
mutagenicity after metabolic activation by mouse-liver
microsomes.
Siebert and Eisenbrand (1974) reported that nitrosocarbaryl
was active in causing mitotic gene conversion in Saccfaaromyces
cerevisiae. Incubation for 2 hours on 1 ppm of nitrosocarbaryl
increased the relative conversion frequency 3-fold for the
ade-2 locus and 5-fold for the trp-5 locus, and at 30 ppm
increases were 139-fold for the ade-2 locus and 885-fold for
the trp-5 locus. In this study a dose-related effect was
shown using 5 concentrations of nitrosocarbaryl. Regan et al.
(1976) demonstrated that nitrosocarbaryl was able to induce DNA
damage in culture human cells as measured by unscheduled DNA
14
synthesis. In addition, by using methyl labeled [ C] and
ring labeled [ H] nitrosocarbaryl, Regan et al. (1976) found
that the 14C label was associated with cellular DMA, whereas
the H label was not. Because nitrosocarbaryl has been
observed to cause reversion of base-pair substitution sensitive
strains (TA 100, TA 1535), these results suggest that the
nitrosocarbaryl molecule was split and the resultant methyl
group could alkylate DNA and cause base-pair substitution type
mutations.
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Ishidate and Odashima (1977) reported several chromosome
aberrations (80% aberrant cells) in Chinese hamster cells 24
hours after exposure to nitrosocarbaryl (0.015 mg/ml). The
toxicity of nitrosocarbaryl was not reported.
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APPENDIX B — REVIEW OF EXPERIMENTAL EVIDENCE ON
THE CARCINOGENICITY OF N-NITROSOCARBARYL
As stated in Appendix A in connection with the mutagenicity
of nitrosocarbaryl, carbaryl has been shown to react in vitro
with sodium nitrite under acidic conditions to form
nitrosocarbaryl, and formation of nitrosocarbaryl in stomach
physiology is possible, given the presence of nitrite in human
saliva and food products. N-nitrosocarbaryl is clearly a
carcinogen in experimental animals, and this relates to the
known carcinogenic properties of N-nitrosamines as a group.
Preussman et al. (1976) administered 130 mg/kg N-
nitrosocarbaryl to "equal numbers * of 100-day-old male and
female Sprague-Dawley rats, 200 g, twice weekly by gavage until
death. Animals were given gross and histopathologic
examinations* Twenty male and 20 female control animals were
also on study. Hyperkeratoses, papillomas, and squamous cell
carcinomas were found in the forestomach in 17 of 32 treatment
rats. Mean induction time was 167 days. These anomalies were
not present in control animals, although other types of tumors
were found in 4 of the controls. The Agency notes that this
study is flawed in that only one dose was used, but agrees that
the results were clearly positive.
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In a study performed by Lijinsky and Taylor (1976), twelve
female Sprague-Dawley rats, 8 to 10 weeks old, were given 0.2 ml
of a 0.11 M solution of nitrosocarbaryl in olive oil once weekly
for 10 weeks to yield a total dose of 50 mg. Fifteen male
Sprague-Dawley rats, 8 to 10 weeks old, were similarly treated
with a 0.16 M solution twice weekly for 20 weeks to produce a
total dose of 300 mg. Control animals were included in this
study. Animals were examined for .tumors with follow-up
histopathologic evaluation on spontaneous death. In another
experiment, a 19 umol solution of nitrosocarbaryl in acetone was
applied twice weekly onto the backs of mice.
Females survived up to 110 weeks and males up to 90
weeks. Carcinoma in the forestomach was found in 9 females and
7 males, and papilloma in the esophagus or trachea was found in
1 male and 1 female. None of these tumor types was observed in
control animals. Tumor metastases were found in treatment
rats. Results were negative in the skin treatment experiment.
The Agency notes that small numbers of rats were used,
which were dosed for a short period of time. However, this test
was clearly positive. A lack of experimental details precludes
a definite conclusion on the strengths and weaknesses of the
skin treatment study.
In another study performed by Lijinsky and Taylor (1977),
recrystallized technical grade carbaryl was administered to rats
by gavage, either as a 30 mg/ml suspension in water or in 4.0%
sodium nitrite (NaN02) solution. Three groups of 8 Sprague-
Dawley female rats, 12 to 14 weeks old, were housed with males
for mating during a weekend. One group was given 30 mg carbaryl
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in water daily for 10 days during gestation days 4 to 18
(weekends omitted). One rat died during treatement, and 3 of
those remaining gave birth to 9 female and 13 male pups. The
second group was given 30 mg carbaryl in 4.0% NAN02 solution
on gestation days 4, 5, and 6. Six animals die.dr and one rat
gave birth to 7 female and 3 male offspring. The third group
was given 18 mg carbaryl in 4.0% NAN02 on gestation days 14 to
18. None of these animals died, and 6 dams gave birth, to. 32
male and 32 female pups. In addition, a fourth group of 12
female rats was given the same treatment as the third group for
5 successive days. Whether or not this fourth group was mated
was not specified, however, nor (if they were mated) is an
evaluation of offspring shown. All adults and offspring were
allowed to live until natural death: except that moribund- animals
were sacrified. Necropsies were performed, and lesions and
tumors examined histologically.
At least 50% of the dams and offspring reportedly survived
to 100 weeks, and some survived 150 weeks. A statistically
significant (p< 0.05) difference in tumor incidence between rats
treated with carbaryl and those treated with carbaryl and
nitrite was not evident. Data concerning all dams dosed with
carbaryl and nitrite are summed together, however, and this
makes a comparison with dams treated with carbaryl alone
difficult. Nonetheless, adrenal, liver, and uterine tumors —
which were not evident in dams treated with carbaryl alone —
were detected in 2/22, 3/22, and 2/22 dams, respectively, given
carbaryl plus nitrite. Among male offspring exposed during
gestation days 14 to 18, breast tumors were diagnosed in 0/13
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pups in the carbaryl group and in 4/32 pups in the carbaryl plus
nitrite group; pancretic tumors were observed in 1/13 pups in
the carbaryl group and in 6/32 pups in the carbaryl plus nitrite
group.
The Agency points out that the lack of concurrent control
groups severely limits the usefulness of this study as an
evaluation of the carcinogenic potential of carbaryl, and of
carbaryl plus nitrite, under the experimental conditions
described. With respect to statistical sensitivity, the 'sample
sizes used in this study were rather small. The results of
this study do not reflect a lifetime exposure of the
experimental animals to carbaryl. Moreover, the effect which
the administration of a suspension has on the actual absorption
of carbaryl from the gastrointestinal tract needs to be
considered and evaluated (CAG [Anderson], 1980).
In a study performed by Eisenbrand et al. (1975), a single
subcutaneous injection of 1000 mg N-nitrosocarbaryl/kg in oil
suspension was given to 8 male and 8 female Wistar rats.
Vehicle controls were also evaluated. Another group of 37 male
and 37 female Wistar rats (90 days old) received single gavage
dosages of N-nitrosocarbaryl ranging from 200 to 1500 mg/kg.
Untreated animals served as controls.
Fourteen rats treated subcutaneously died by day 450. All
of these animals developed local tumors at the site of
injection, which.were diagnosed as polymorphic-cell sarcomas.
Orally treated rats did not develop tumors by 21 months. Tumors
were not found in control animals.
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The Agency notes that only single doses of N-
nitrosocarbaryl were administered to evaluate carcinogenic
potential, presumably with respect to the lifetime of the
animals. Subcutaneous administration did not produce tumors
beyond the site of administration, which makes these results
difficult to interpret in relation to possible carcinogenic
effects from other routes of administration^
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