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
                                -i-

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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
                                 -ii-

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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
                               -iii-

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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.
                                        -2-

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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
                                                                                              i
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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).
                                        -5-

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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
                                        -7-

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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).
                                        -13-


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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
                                        -16-

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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.
                                       -18-

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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.
                                        -19-

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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-

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 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-

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 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-

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 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  •
                                       -38-

-------
'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
                                        -39r-

-------
 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

                                        -40-


-------
 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).
                                         -41-

-------
               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%).
                                       -42-

-------
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.
                                      -43-


-------
      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).
                                       -44-

-------
           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.
                                         -46-

-------
                      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.)
                                        -47-

-------
 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.
                                        -48-
                                                                                               .{

-------
      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
                                         -49-

-------
 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
                                        -50-

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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)
                                       -52-

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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
                                        -53-

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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.
                                        -54-
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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)
                                       -55-

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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
                                       -56-

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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*.
                                         -57-

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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
                                        -59-

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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.
                                        -€0-

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                               V.   REFERENCES


Abrahamsen,  L.  H. ,  and  Jerkofsky,  M.A.  198P.  Interactions of hunan varicella-
zostsr virus with  Savin in cultures of  hunan cells [abstract! .  Unpublished
report from  University  of  Maine at Orono.

Ahmed, F.E. , Hart,  R.W. , and  Lewis, .N.J.  19T7a.   Pesticide induced DMA damage
and  its  repair  in cultured hunan cells.  Mutat.  Res.  42:151-174.

Ahmed, F.E. , lewis, N.J. ,  and Hart, R.W. 19~?7b.   Pesticide induced ouabain
resistant mutants  in Chinese  hamster V79 cells.   Chem.  Biol.  Interact.   19:369-
374.

Aly,  C.M.. , and  El-Dib,  M.A. 1971.   Studies on the persistence of some csrbamate
insecticides in the aquatic environment..  I. Hydrolysis of Sevin,  Baygon,
Pyrolan  and  Diinetilan in waters.   Water Res. 5:1191-12C5.

Mer, S. 1935.   Cytological effects of pesticides* I-  Mitotic effects of  N-
methyl-1-naphthyl carbamste (Sevin). Cytologia  30:175-181.

»mer, S.M. and  Earah, O.R. 1968.   Cytological effects  of  pesticides.   H!..
Meiotic  effects of  N-methyl-1-naphthyl carbamate (Sevin) .   Cytologia 33:324-337.

?ner, 3.M. ,  Hanraouda, M.A.., and Farah, O.R.   1971. Cytological and
morphological effects of the  insecticide N-methyl-l-naphthyl-carbamate (Sevin) .
Flora  (Jena)  ISC: 433-439.

Andrianova,  M.M.,. and Alekseev, I.V.  197B.   Cn  the carcinogenic properties of-
the pesticides  Sevin, Maneb,  Ciram, and Cinbe. ^p. Pitan. 29:71-74.
Ashvjooc-Smith , M.J. , Trevino, J. , and Ring, R.   1972.  • Mutagenicity of
dichlorvos. Nature  2^!!?: 418-420*

Saron, R. , Sphon, J. /Chen, J. ,  Lustig,  E. , Eoherty, J. ,  tensen,  E. , and Kolby,
S. 19^9.  Confirmatory isolation and identification of a metabolite of carbaryl
in urine and milk.  J. Agr. Ebod Chem. 17:883-257.

Benson, B.W., Scott, W. J. , and Beliles,  R.P. 1967.  Sevin — Evaluation  by
Tsra.to logical Study in the Nbuse.  Unpublished  report  from tee 'Xbodard
Research Corp.  (Proprietary.)

Bionetics Laboratory.  1958 .  Unpublished Report from  the Bionetics Research
Laboratories, Inc.  (Proprietary.)

Blsvins, R.D. , Lse, M., and Regan, J.D.  1977.  Mutagenicity screening  of five
methyl carbamate insecticides and their  nitroso  derivatives using mutants of
Salmonella typhimurium LT2»  Mutat. Res. 56:1-^.

Sollag, J. , and Liu, S. 1971a.  Fungal transformation of the insecticide
carbsryl r abstract] .  Bacteriol. Proc. 71:] 5.

Bollag, J. *nd Liu, S. 1971b.  Degrada'tion of Sevin by soil microorganisms.
Soil Biol. Bioeham. .1:337-345.
                                       -61-

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 Brankovan, 0.  1972.  The meiotic effect of Sevin 50 after treatment of com in
 the embryonic and generative phases of development.  Arhiv za Poljoprivredne
 Nauke 25:125-132.

 Br*eskii, V.  1972.  The study of the mutagenic properties of an insecticide
 from the carbamate group (Sevin).  Sov. Genet. 8:798-800.

 CAG. 1977.  The Carcinogen Assessment Group's Preliminary Report on Carbaryl,
 dated September 28, 1977.  toy E. Albert, M.D., Chairman.

 GAG (Anderson).  1980a.  Memorandum:  Evaluation of Carcinogenicity Studies on
 Carbaryl.  From Larry Anderson, lexicologist, Carcinogen Assessment Group, to
 Bipin Gandhi, Project Manager, Special Pesticide Review Division.

 CAG (Anderson).  1980b.  Summary of Carcinogenicity Studies on Carbaryl and N-
 Nitrosocarbaryl.  Presentation delivered before the F1FRA Scientific Advisory
 Panel, July 23, 1980.

 Carpenter, C.P., Weil, C.S.,  Palm, P.E., Wbodside, N.W., Nair, J.H., and
 Smyth, ELF. 1961.  Mammalian  toxicity of l-napthyl-*Hnethyl carbamate (Sevin
 insecticide).  J. Agricx. Ebod Chem. 9:30-39.

 Chaisson,  Christine 1980.  Memorandum:  Carbaryl:   Evaluation of Studies on
 Mutagenicity annd Epidemiology (Spermatogenic Effects), dated May 27, 1980.
 From Christine  F. Chaisson, Acting Deputy Branch Chief, Toxicology Branch, BED,
 to  Robert Brown, Branch Chief, SPRD.  Attachments (2):   reports by Clement
 Associates, Inc..   ,              -

 Cbernoff,  Neil.  1976 [updated 1980].  Memorandum:  Review of data bearing upon
 the potential of the  pesticide carbaryl to induce  effects on the reproduction
 and perinatal development of  mammals.  From Neil Chernoff, Ph.D., Neurobiology
 Branch,  Experimental  Biology  Division,  HERL/RTP, to James Benslcin, Ph.D.,
 Office of  Special Pesticide Reviews.  Includes Appendix:  "Summary Critique of
 Available  References."

 Chin,  B.,  ESdridge, J.,  and Sullivan, L.   1974.  Metabolism of carbaryl  by
 selected human  tissues using  an organ-maintenance  technique,   din.  Tbxicol.
 7:37-56.

 Christie,  A.  1969.  Effects of insecticides on algae.   Water  Sewage Works
 116:172-176.

 Collins, T.F.X.,  Bansen,  W.H.,  and  Keeler,  H.V.  1971.   The effect of carbaryl
 (Sevin) on  reproduction of  the  rat  and  the gerbil.   Tbxicol.  Appl.  Pharamacol.
 19:202-216.

 Cook, W.L., Crow,  S.A., and Bourquin, A.W.  1977.   Inhibitory effect of
 pesticides  and polychlorinated  compounds on representative surface slick
 bacteria [abstract].   Annu. Mtg. Am.  Soc. Microbiol.  77:243.

 Cook, W.L., 1980.  File note of telephone conversation with Dr. W.L.  Cook,
Georgia State University, and Vicki Dellarco,  Reproductive Effects Assessment
Group, EPA.  July 3, 1980.
                                       -62-

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Crosby,  D.G. ,  Lsitis,  E. ,  snd  Winterlir. ,  W.L.  l^S.   Photo-decomposition of
     nste  insecticides.  J.  Agric.  Ebcd Chan.  13(3) :2C4-2P7.
Declune, C. , and  Bernard,  P.   197*7.   Ebetal  accumulation of [14C]  carbaryl in
rats and mice  autoradiogrsphic study.  Tbxicology 8:95-1^5.

D2Giovanni-Connelly,  R., KOlbye,  SJ1. ,  and Greeves,  P.D.  1D5S.   The effects of
IPC, CTPC,  Sevin  and  Zecrran  on Sacillus subtilis.   Experientia 24:8C-P1.

Degraev*:, N. ,  Msutschen-Dahnen, M. ,  Hsubrechts,  N. ,  and Collzzi,. A. 1975.   The
hazards of  the insecticide carbaryl  used alone and in association with
nitrites.   Bull Sac.  R. Sciliege  45:45-57.

DeLorsnzo,  F. , Stsiano, N. , Silengo, L. , and Cortese, R. 1978.   Mutsgenicity of
Dtallats, Sulfulate,  and Triallste and  relationship betveen structure and
mutsoenic effects of  carbanates used widely  in agriculturs..  Cancer Res. 38:13-
15.

Deparrment of  Hsaltii, BSucstion,  and Welfare. 1977...  Approaches  to determining
the mutagenic  properties of chemicals:   Risk to  future generations^  Report of
the SuIxrjiiBuitte on. Environmental  .Mutagenesis to  th?  DHEW Cbrami-ttee to
Coordinata Toxicology and  Related Programs.   Apr- 1.,  1977..

Dikshith, T.S., Gupta, P.K.,  Gaur, J»S.f Eetts,  K.K. , and Msthur,  A.K.  1975.
Ninety day tcxicity of carbaryl in male rats- ESrviron. Res- l^l'il-lTfJ.

Darough, H., and  Casida, J. 1954. Nature of certain metabolites of the
ins«?cticide Sevin. J.  Agric.  PDod Chen. 12:294-304.

Dorough, H.W.  1979.   Mstsbolism of Carbsmats Insecticides.  EPA  Grant No.
RSC51430-2, Progress  Report,  March 25,  1979.

Coughsrty, W. J. ,  Goldberg, L., and Coulston,  F.  1971.  The effect  of csrbsryl
or. reproduction in the monkey  (Macscca mulatta) .    Tbxicol.  Appl.  Pharascol.
13:355.

Dougherty, W.J. 1974.  Tsratogenic evaluation of csrbaryl  in  the rhesus monkey
(.M£C5cca mulgtta) .    Institute of Comparative and  Rman Tbxicology Report.
(Proprietary . )

Earl, F.L. , filler, E. , 2nd Van Eoon, E.J.   1973.  Reproductive, te-ratogenic,
2nd neonatal effects of some  pesticides and  related compounds in beagle dogs
and ministure  swine.   Pesticides  and  the Environment:   A continuing
controversy.   Inter-taerican  Conference in Tbx.  snd  Ccc.  I«ed., Symposia
Specialist, ed. William Deichnan. M.  Miami,  Florida.

Edwards, W.7.  1?2S.   "tenor and urn:  Lack  of Lndication  of Delayed  Meurotoxicity
Potential for  C?rbsryl, dated tey 9,  1SPC.   rron K?.  Thomss Edwards,
Pharmacologist, TE/HEE, to  Bipin  Gandhi,  Project ^ten^ger  for Crrbsryl,  SPRD.
a.trachnents :  validation reports.

Egert, G. , nnd Grein,  H. 197").  Formation of mutsgsnic  N-nitroso compounds  from
ths pesticides prcmetryne, dcdinc, =nd  carbaryl  in ths-  presence of nitrite  at
pH 1.  Mutat.  Res. 37:17?-1P1.

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 Eisenbrand,  G.;  Ungerer,  0., and Preussnsnn, R. 1975.  Ihe reaction of nitrite
 with pesticides.  II.   Formation.  Chemicsl properties and carcinogenic
 activity of  the  N-nitroso derivative of N-raethyi-1-naphthyl carbsmate
 (Carberyl) .   Pood Cosset. Tbxicol. 13:355-35^.

 Elespuru, R. , Lijinsky, W. , and Setlow, J.K. 1974.  Nitroso-carbaryl as a
 potent nutagen of environmental significance.  Nature 247:385-287.

 Environmental Protection  Agency. 1980.  Mutagenicity Risk Assesanents:
 Proposed Guidelines.   Fed.  Reg.  45(221):  79184-79488 (November 13, 1982).

 Epstein, S. , Arnold, E. ,  Andrea, J. , Bass, W. , and Bishop,  Y. 1972.  Detection
 of chemical  mutagens by the dominant lethal essay in the mouse.  Tbxicol.
 Appl.  Pharmecol. 23;283-325.

 Srcegovich,  C.D. , and  Rashid, K.A- 1977.  Mutagenesis induced in mutant strains
 of Salmonella typhimurium in pesticides. Abstr. No. 43.  174th ACS National
 Meeting..  Aug". 29 - Sept. 2, 1977.

 Fahrig, R.   1974. Comparative mutagenicity studies with pesticides.  IARC   *
  10:161-101.

 Fiscor, G. ,  and  Nil Lo Piccolo, G.M. 1972.  Survey of pesticides for
 mutagenicity by  the bacterial plete assay method.   EMS Mewsl. 5:6-8.

 Gaines, T.B. 1959.  Acute toxicity of pesticides.   Taxied. Appl.. Pharmaccl.
 14:515-534.

 Greim,  H. , .Bimboes* D. , Egert,. G. , Coggelmann, W. , and Kramer, M.. 1977.
 Mutsgenicity and chromosomal aberrations as an analytic tool for in yi±ro
 detection of mammalian enzyme mediated formation of reactive metabolites.
 Arch. Tbxicol. 39: 159-
Guthrie,  F.E. , Monroe, J.R.,  and Abernathy,  C.O.  1971.  Response of the
laboratory mouse to selection for resistance to insecticides.  Ttoxicol. Appl.
Ph.-raacol.   18:92-1C1.

Halpin, G. J. , M.D. 198B.  Report on Cape  tey County.  Epidemiological  study
conducted by the New Jersey State Health  Dept., Staff of  Parental and  Child
Health Services.

Hart, E.R. 1972.  Teratology  Study - Sevin,  vitamin A, aspirin, and  malathicn -
Final Report.  Bionetics Litton.  (Proprietary.)

Inning, R.J. , Shafer, B., and Wbodard, C. 19^9.   Sevin.   Safety evaluation  of
feeding to female beagles from day one of gestation through weaning  of
offspring.  Unpubl. Rpt. of the Vbodard Research  Corp. (Proprietary.)

Lines, J.R.M. , inland, B.M. ,  Vslerio, M.G. ,  Petrucelli, L. , Fishbein,  L. ,
H=rt, E.R. , Pallotta, A.J. , Batss, R.R. , Falk, H.L. , Cart, J. J. , Klein, M.,
Mitchell,  I., and Peters, J.  1959.  Bioassay of pesticides =nd industrial
chsmicsls for tunorigenicity  in mic«>:  A preliminary note.  J. Nat.  Csncer
Inst. 42:11CT-1114.

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 International Agency for Research on Cancer. 1976. Carbaryl.  IARC Monographs
 on the Evaluation of Carcinogenesis Risk of Chemicals to Man 12:37-53-  WHO,
 Lyon.

 Isbidate,  M.,  and Odashima,  S. 1977.  Chromosome tests with 134 compounds on
 Chinese hamster cells in vitro:    A screening for chemical carcinogens.
 Mutat.  Res.  48:337-354.

 Ishidate,  M. 1980.  Letter from Motoi Isidate Jr., M.D., National Institute of
 Hygienic Sciences, Division  of Mutagenesis,  Tokyo, Japan, to Dr. Donna Kuroda,
 Reproductive Effects Assessment Groups, EPA.  Dated June 25, 1980.

 Johnson,  D., and Stansbury,  H.  1965.  Adaption of Sevin insecticide (carbaryl)
 residue method to various crops.  J. Agr. Food Chem. 13:235-238.

 Kazarnovskaya,  M.L.,  and Vasilos,  A.F. 1977.  The effect of Sevin on the
 chromosomal  apparatus of cells in vitro.   Zdravaookhranenie 204:14-16.

 Khera,  K.S.  1976.  Significance  of metabolic patterns in teratogenic testing
 food safety, din. Toxic. 9:773-790..

 Kitagawa,  K.,  Wakakura,  M.,  and  Ishikawa, S. 1977.  Light microscopic study
 of endocrine organs of rats  treated by carbamate pesticide. J.  Tox.  Sci. 2:53-
 60-

 Knaak,  J.B., Tallant,  M., Kbzbelt, S-, and Sullivan, L. 1968.   Hie metabolism
 of carbaryl  in man, monkey,  pig,  and sheep.  J.  Agr. Food Chem.  16:465-470.

 Knaak,  J.B., Tallant,  M., Hartley, W., and Sullivan, L. 1965.   The metabolism
 of carbaryl  in rat, guinea pig and man. J. Agr.  Food Cbem. 13:537-543.

 Knaak,  J.B., and Sullivan, L.J.  1967.  Metabolism of carbaryl in the dog.  J.
 Agr. Food. Chem.,  13:537-539-

 Krishna, J., and Casida,  J.  1966.   Fate in rats  of the radiocarbon from ten
 variously  labeled methyl- and  dimethyl- carbamate-C  insecticide chemicals
 and their  hydrolysis products.   J. Agr.  Food Chem.   14:98-105.

 Krylova, J.V.,  and Denisova, A.V.  1973.   Action  of Sevin on the Mongolian  Tree
 Creeper.   SLol.  Nauki  16:25-30.

 Suhr, R.,  and  Casida,  J.  1967.   Persistent glycosides of metabolites of methyl
 carbamate  insecticide  chemicals  formed by hydroxylation in bean plants.  J.
 Agr. Food  Chem.  15:814-824.
                                                                                              I
£uhr, R. 1970.   Metabolism of  carbamate insecticide chemicals in plants and                  [
 insects.   J. Agr.  Food Chem. 18:1023-1030.                                                    |

 LaFleur, K.  1976.   Movement of carbaryl through  Cpngaree soil into ground
water.  J. Environ. Qual.  5:91-92.

Lijinsky,  W. and Taylor,  H.W.  1977.   Transplacental chronic toxicity test  of
 carbaryl with nitrite  in  rats.  Fd.  Cosmet.  Toxicol.  15:229-232.

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 Lijinsky,  '.«;.,  and Taylor,  H.W.  1975.  Csrcir.ogenesis in Sprague-Dawley rats of
 N-nitroso-N-slkylcsrbanats esters.  Cancer Lett. 1:275-279.

 Makovskaia,  E.I., Rappoport,  M.B., and Pintshuk, V.G. 1955. On the possibility
 of carcinogenic effect of some  insecticides belonging to the carbanate group.
 Vaprosy Eksperimentalnoi  Qikologii 1:67-74.

 Marshall,  c.,  Borough, H.W.,  and Swim, H.E. 1976.  Screening of pesticides for
 mutagenic  potential  using  Salmonella typhimurium mutrnts. J. Agric. Food Chem.
 24:55P-553.

 Matsumura, F., and Ward,  C. 1955.   Degradation of insecticides by the human and
 the rat liver.  Arch.  Environ.  Health 13:257-251..

 McCann, T.,  Choi, E.,  Tfiamasaki, E., and &nes, B.N.  1975.  Detection of
 carcinogens  as mutagens in the  Salmonella/Microsane test: assay of 3CJ0
 chemicals.  Proc. Nat. Acad.  Set.  72:5135-5139.

 McCann, J. 19S0.  Letter  from Dr.  Joyce McCann, university of California,
 Lawrence Berkely Laboratory,  to Vicki Dellarco, Reproductive Effects
 .Assessment Group, EPA. Dated June 3, 1980.

 Mellon  Institute Special Rsport f26-53. June 11, 1953.  (Proprietary.)

 Mellon  Institute Special Report 125-122, skin painting  study.  December 26,
 1TS2.  (Proprietary.)

 Mensie, C.M. 1974.  Metabolism  of  Pesticides — An  update.  USDI.   Fish and
 Wildlife Ser.  Epl. Sci. Rpt.  — Wildlife No. 134.  Washington,  D.C. p. 73-78.

 Menzie, C.M. 1959.  Metabolism  of  Pesticides.  USDI Bur. Sport  Fish and
 Wildlife.  Spl  Sci. Rpt. Wildlife No.  127:72-75.  Washington,  D.C.

 Metcalf, R.L.  1977.  validation Report on Gaines (1969)  studyi  "Acute  toxicity
 of  pesticides."

 Murray,  F.J.,  Staples, R.E.,  and' Schwetr,  B.A.  1979.  Tteratogenic  potential of
 carbaryl given  to rabbits  and mice by gavage or by  dietary inclusion.
 Tbxico.  Appl.  Pharmacal. 51:81-*?9.

 Nagy, A.,  Mile,  I.,  and Antoni,  F.  1975.   The nutagenic  effect  of  pesticides on
 Escharichia  coli   VJP2  try  -.  Acta. Microbiol.  Acad.  Scie.  Kung.   22:3H9-314.

 Orlovs, N.V., and Zhalbe,  E.P.  1958.   Maximum pemissible  amounts  of Sfvin
 in  food products. Vbp. Pit?n. 27(5):49-55.   Chem. Abstr. 70:454221.(Original
 in Russian.)

 Fanciers,  R.J.   19<>7.    Determination of  teratogenic  properties of orally
 =cainistered ,l-naphthyl-*Hnethylcarbaaate  (Savin) in sheep, June 21, 19-37.

 PL^IS. 198fT.  Pesticids Incident Monitoring System Report No.  285.  Summary  of
 Reported Pesticide Incidents Involving Carbaryl, dated January, 19SH.   Compiled
by Esslth  Effects  Branch,  Hazard Evaluation  Division, Office  of Pesticide
 Programs,  Environmental Protection Agency.

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 Prsussnan,  R.,  Eisenbrsnd,  G.,  and Schnahl,  D.  1976.  Carcinogenicity testing
 of low doses of N-Nitrosopymolidine and of N-Nitrosobenzthiazuran and N-
 Nitrosccarbaryl in rats.   Institute of Toxicology and Chemotheragy, German
 Cancer Research Center.

 Cuarles,  J.M.,  and Tennant, R.W.  1S75.  Effects of nitroso-carberyl on EALB/3T3
 calls. Cancer  Res. 35:2537-2645.

 Rashid, K.A. 1578.  The  relationships between mutagenic and DNA damaging
 activity  of pesticides and  their  potential  carcinogenesis..  Perm St. Univ.,
 Ph.D.  dissertation.

 REAG.  1982.  The Reproductive Effects Assessment Group's Preliminary Report on
 Carbaryl.

 Regan, J.D., Setlow,  R.B.,  Francis, A.A., and Lijinsky, W.  197G.
 Mitrosocarbaryl:  its effect on hunan DNA.   Mut. Res. 38:293-302.

 Regan, j.,  Setlow, R,, Etancis, A., and Lijinsky, W. 1975.  Nitrosocarbaryl:
 its effects on  hunan DMA.   Mutat. Res. 38:293-301..

 Rickard,.  K.W. 1979.   Chemical properties and potential toxicological
 significance of N-nitrosocarbaraates.  ttiiv.  Kentucky Ph.D. dissertation.

 Robens, J.F. 1959.  Tteratologic studies of carbaryl, diazinon,  norea,
 disulfiram,  and thiram in snail laboratory animals.   Ttoxicol. Appl.  Pharmacol.
 15:152-153.

 Shaffer,  B.C.,  and Levy, A.C. 1958.   Evaluation of the teretogenic  potential of
 Sevin  in  rabbits fed  the compound from day 9 to day IS of the gestation
 period.   Unpublished  report from  the  Wbodard Research Corp. (Proprietary.)

 Shimkin,  M.E.,  Wieder, R.,  McDanough,  M., Fishbein,  L., and Swern,  D.  1969.
 Lung tixnor  response in strain A mice  as a quantitative bioassay of  carcinogenic
 activity  of  some carbamates and aziridines.   Cancer  Res.  29:218/!-2190.

 Shirasu,  Y., Moriya,  M., Kato,  K., Puruhashi, A., and Kada, T.  197S.
 Mutagenicity screening of pesticides  in the  microbial system.   Mutat.  Res.
 4C:19-3C.

 Shpirt, M.E. 1975.  Tbxicologicsl  evaluation of dichlorodipheny trichloroethane
 (DET), hexcchlorocyclohexane (BZCH),  tetramethyl  thiuramdisulf ide  (T?-TTD),  Sevin
 and Zinsb vhen  acting  on a  hunan  cell  structure.   Gig.  Tr.  Prof. Zabol.   17:32-
.34.

Shtenberg, A.J.,  and Rybakova, M.N. 1958.  Effects of carbaryl  on the
neuroendocrine system of rats.  Food  Cbsnet. Ttoxicol.  6:461-4-57.

Shtenberg, A.I.,  and Ozhovan, M.v.  1971.  The effects of low Sevin doses  on  the
 reproductive function of animals  in a  number of generations.  Vaprosy  Pitaniia
 3":42-42.    (Original  in Russian,  translation supplied by the Office  of Special
 Pesticide RevievvS.)

Siebert,  D., and  Eisenbrsnd, G. 19*74.   Induction  of  mitotic gene conversion  in
Saccharonycss csrevisise by N-nitrosated pesticides.  Mutat. Res. 22:121-125.

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 Snalley, n.E.,  O'Har?, P.J., Bridges, C.H., and Radeleff, R.D. 1959.  The
 affects of chronic carbaryl administration on the new muscular system of
 swine.  Toxicol. Appl. Pharmacol. 14:429-419.

 Snalley, H.E.,  Curtes, J.M., and Earl, F.L. 1958.  Teratogenic action of
 carbaryl in beagle dogs.  Tbxicol. Appl. Pharaacol. 13:392-423.

 Strother, A. 1972.  In vitro metabolism of methylcarbanate insecticides by
 human and rat liverfraction.  Tbxicol. Appl. Pharmacol. 21:112-129.

 Sullivan, L., Chin, B., and Carpenter, C. 1972a.   In Vitro  versus in vivo
 chrccnatcgrsphic profiles of carbaryl anionic metabolites in man and To"wer
 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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