UPfl.
               S-
                  Chlorinated Hydrocarbons
                  Insecticide Versus  Carcinogenic Action
                  Ohio State Univ.
                  Columbus
                                       Repository Material
                                     Permanent Collection
                  Prepared for

                  Health Effects  Research Lab-
                  Research Triangle  Park, NC
                  Mar 83
   a
   CVJ
   f r>
                                       i.ri Avs
                                       ''; 2CC04
' EJBD
 ARCHIVE
' EPA
, 600-
 1-
 83-
 003
U.S. Department of Commerce  '
National Technical Information Service

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EPA-600/1-S3-003
». ••~'.s —N^ sk-v.£
CHLORINATED HYDROCARBONS: I
ACTION
:•
NSECTICIDE VERSUS CARCINOGENIC
'"^srV^D'Ambrosio, N. J. Lewis, R. w. Hart, W. J. Collins
9. 'S3PCSMING CXCA.NIZADCN NAMB Af
The Ohio State University
Colun:bus, Ohio 43210
'•I. SPCNSC"ING ACeNCv NAMK ANO ACC
Health Effects Research Labi
Office o^ Research and Deve
U.S. Environmental Protectii
Research Iriangle Park, NC
10 Aoo«asa
;«ess
Dratory
lopment
an Agency
27711 '
3. HBC:PIENI~S ACCESSIONXSO.
r$1 3 181578
9. AEPOHT OATH
March 1983'
0. P«HCOHMINQ ORGANIZATION COCB

1O. ^AQCRAM 6U&M&NT NO.
CCAE1A
11. CSNTBA«iT< GRANT NO.
Grant No. R805008
13. TYF« OF REPORT ANO CfiHICO CSVER
Final
1A. SPONSORING AGENCY COOS
EPA 600/11
13. SUP"V.aV»e.NTA«Y .NOTES
Project Officer: Stephen Nesnow
     The purpose of this grant was tho determination of those structural characteristics
responsible for the deleterious vs beneficial effects of chlorinated pesticides.  These
studies have led to tl-e development of a model system for the rational design, synthesis
and evaluation of insecticidal compounds with reduced genetic hazard.

     Various halonenated hydrocarbons and their analogs were designed and synthesized for
this study.  The test systems employed for evaluation of the mammalian and insect effects
were:  a) normal (CI and" 153) and SV-40 transformed (VA-4) human fibroblasts;-b)-D:iA repair
by UDS ana BUdR photoTysis; c) measurement of association of   C-labeled synthetic analogs
with genetic material; d) cytotoxicity; e) metabolic activation studies using liver
homogenates; and f) topical and oral toxicities of standards and test compounds in house
flies (Muse a domes tica) and mosquito larvae fAedcs aegypti).
     These studies indicated that modification of the 6,7-double bond of aldrin (and the
6,7-eooxide of dieldrin could lead to potent insecticidal agents with reduced cytotoxicity,
DNA repair and DMA association.  These studies included the synthesis and evaluation of
three distinct series of pesticide analogs including two series of halogenated cyclodienes
with modified n-electron character and a series of aromatic pyrethrin-related agents.
                              a/C'oao
  Pel ease to public
                                                unclassified
                                                                          III NO. OF.-AOcS
                                                unclassified  .

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                                               EPA-600/1-83-003
                                               March  1983
                CIILORINATKD HYDROCARBONS:
          INSHCTICIUK VERSUS CAKCINOGENIC ACTION
                            by
 S.M.  D'Ambrosio, N..1.  Lewis,  <<.W.  Hart and W.J.  Collins
Chemical  Bloraedical Environmental Research Croup (CBERG)
                The Ohio  State University
                   Columbus, Ohio 43210
                    Cr.«:»t No.  R805008
                     I'roject Officer

                      Stephen Noenow
                   Biochemistry  Branch
           MD68 EPA Mo.ilth  Effects laboratory
          Rc::c.ircli Tri.ingle P.irk, N.C.   27711
           ilo.ilth r.ffect*  Research  Laboratory
           Office of RL"?e.irch .ind  Dcvolopnent
          U.S.  KnvlroiiiiLMiL.i I Protection Agency
          Reso.irch Trl.n«le Pcirk,  N.C.   27711
                 • •P'Ol'l i  ;.
                  NATIONAL  TECHNICAL
                 INFORMATION SERVICE
                     ij   -•m.'ii c: C'""i"Cl
                        • s.l'n3 >«  ' J

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                                   f.'OTlCE
nPC,.ss,nly  reffoc  the v 'I  I ,'  .t?.tP" rcvl{1-' flnd therefore  does not
no inrorreJ.                   °' tPA and n° ofMci«l enrtcirsewnt should

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                               FOREWORD
     The many benefits of our niodc-rn, developing, industrial society  are
accompanied b> certain hazards.  Careful assessment of the relative risk
of cxi stilt; and new r.-.an-made onviionmcntal hazards  is necessary  for the
establishment of sound regulatory policy.  These- regulations serve to
cnhanco the quality of our environment in order to  promote the public
health and uclfare ar.d the productive capacity of our Nation's population.

     The, complexities of environmental problems originate in the deep
interdependent relationships between the various physical and biological
segments of nan's natural .nnd social world.  Solutions to those,  environ-
mental problens require an integrated program of research and ucvelop-
pient using input fron a number of disciplines.  The Health Effects
Research Laboratory conducts a coordinated environmental health  research
progran in inhalation to-iicology, genetic toxicology, neurotoxicology,
developmental and experimental biology, and clinical studies, using
human volunteer subjects.  These studies address problems in air pollu-
tion, water pollution, non-ionizing radiation, environmental carcinogetiesi b
and the tcxicology ol pesticides and other chemical pollutants.  The
Labor.itory participates in and provides data for the development and
revision of criteria do'-uments on pollutants for which national  ambient
air quality and water quality standards exist or ar<_- proposed, pro\ides
the d.ita for resist r:ition of new pLslicic'es or proposed suspension of
those already in use, conducts research on hazardous and-toxic materials	
and is primarily icsponsible for providing the he.ilth basis for  non-
loniiing radiation standards.  Direct support to the regulatory  function
of the Agency is provided in the form of expert testimony and preparation
of affidavits a" well as expert advice to the Administrator to assure the1
atfr-quacy of er.vi ronmcntal regulatory 'decisions involving tno protection
of the health and welfare of all U.S. inhabitants.

     Identi-y those' structural features of pesticides (halogenated hydro-
carbon class) that  arc- responsible  for the species  specific effects.
                                   F. Cordon llueter, Ph.D.
                                   Director
                                   Health Effects Research Laboratory
                               • 111

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                                   PKHFACE


     Population growth  and  an Increase  In groups which  do  not  directly
participate  In  food  production liuve Increased the demand  for  more efficient
agrlciilLur.il productivity.  This demand has hucn satisfied by  both higher crop
yields  and by  eliminating  nan's  natar.il competitors  with pesticides.
Pesticides have  thus  become  some  of the mos,t  widely used env I r onne n ta 1
chemicals today.   Sono  of these chemicals although not  showing .in Inured late
effect  in vl vo at  concentrations  normally used  In  agriculture, may  pose a
significant long  torm hazard  to man.  The ideal pesticide  should selectively
affect a desired species  for a  specified  period  of  time  and then  disappear
without  .my tr*ce.  There Is no such  pesticide  and  the  present policy of
pesticide use is a compromise between deslr.ible and undesirable effects.   It
was  the  purpose  of  this study to develop an approach  to effectively evaluate
the relationships th.it  exist between  insecclctd.il agents used In agriculture
and the Induction of genetic damage  In nviram.ilIan systems.

     This study was directed towar-ls  Identifying those  structural features  of
the halogenated  hydrocarbons class of pesticides that  ire responsible for the
species  specific  effects  observed.  We  found  that  It  was possible  to
selectively reduce  the deleterious genetic  effects of many  of these agents In
ma-imalian systems while rointainlng  a  high  level  of Insect toxlcity.   This
research suggests  that nore effective and envlronnentally safe  pesticides niy
be attainable through  in  1 nterd Isc 1 pi I IIH ry approach combining  chemical,
cellular, molecular  and entomological studies.
                                     iv

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                                    MJSTKACr


      The purpose  of  this -rant  w.s ihe de te r.nl ni t Ion  of Chose  str.ictural
 .'-hanccerlsclcs  responsible  for Che deleterious  vs beneficial  effecCs of
 chlorlnitud pesticides.   These studies  have U-d  to  the development  of  a model
 system  for the  rational design, synthesis  .ind evaluation of  Insect Icl.Ul
 compounds wlch reduced genetic  hazard.

      Various  ha logem ced liyd roca rbons  and  their analogs were designed and
 synthesized for this  study.   The  test bysteras  employed for  ev-i 1 ,n tlon" of the
 iaan-nallan  jnd  insect  effects were:   .1)  nor-ail  (CI «ind  153)  ,..id  SV-40
 cr.iiisfornn.-d  (VA-4)  hundn flhroh lists;  b)  D»\  repair by UTS  and  BUdR
 Photolysis;  c) .ne-.sj ru-ncnt  of  association  of  »*C-l.nbaled syntheClc mvilops
 with  genetic  naterlil; d )  cytoto ;yptl).
            sta-iies  MMo.Ced  chat .nod I f ica t I on of  the  6,7-double bond of
.il.lrln aaJ  the  6,7-epocide  of dlellrin could  lead  to potent  ! nsec t ic Ida I
agones  with  reduced cytotox 1 c I ty .  [>::.\ repair  in-l  |)\A association.  These
studies  Included  the sv.ithesls and ..-vaUnclon of  three distinct series of
pesticide .inalo(-q  Incl.Miag two series  of ha loKcn.i ted cyclodienes wlch
nodif-e- i-olectron  ch,ract.,r and a  sorle-, of  .-.ronwtlc  py re t lir i n-re la Ced
agents .

     This  report was,  submitted  in fulfillment  of  Grant  No.  R-805008 by  the
..3..KC Orouii unJer tlie sponsorship of r!ic U.S. Pnvl ro.imuntj 1 Protection AP.-...CV
cover lns the period  July 13,  1977 to J.,lv I/  1980.                           '

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                                    CONTENTS



FOREWORD	

P REPACK	     lv

ABSTRACT 	      v
                                             )
FIGURES	'.  .  .    vll

TMJLCS ....'..	vlll

ABBREVIATIONS   	     ix
                                                )
I.    Introduction  	      1

      Objectives  	      1

      Sum.ua ry of  Results	      ^

IT.   Conclusions   	     ^

111.  Narrative	--•- .-.-.—.-.-.— .-.  .- .—.-.	12_

IV.   Overview	     37
REFERENCES
                                                                              38
                                       V]

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                                     FIGURES


 1.   Structures of llalogenatcd Cyclo-lienes  for  SAR  studies - Aldrin and
     Dleldrln Analogs	

 2.   Chemical structures of Iwlo8cn«tcd cyclodlenes  -  electron  rich
     3H31O£S   ••••••••
                                                  t
 3.   Extended  aromatic  system - pyrcLlirln related analogs	     6

«.   Synthetic  and  Precursor Components for Extended Tr-Systen, Analogs  . .     8

b.   Structures of  the  co.mnercl.illy important pyrctl.rlns evaluated   .  .  .     9

6.   pesticide  Induced  cytoxlclty In VA-4 cells 	    17

7.  PestlciHe  Induced  cytotoxlclty In 153 cells  	         17

            dy:  'net;lbolisia of -'Ihydroaldrln by S-10 fraction of rat
                                                                              36
                                    vii

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                                      TABLES


  1.   Cell surviv.il  of  liu.vin flbrobl.iscs treated  with pesticides   ....     18

  2.   L'DS in VA-4 c. Us after treat incut wUh  <:  series of chlorinated
      hydrocarbons   .........................         jg

  3.   Relationship of  1/Mw of VA-4 cells treated  with 10 un pesticide  -
      12 hours  .............................     20

  «.   Repair of pesticide  induced  DMA damage  In 'the  C.I hurian cell
      line  ...............................     22

  5.   Effect of dieldrin . nH  tildrin in scheduled  SNA synthesis and
      daughter DSA   .................  '.  .........     23

  6-   -  C-Chlorinated hydrocarbon  assocl.iLion wit'.i hurwn cell  OKA  ...       25

  7.   Effect  of dialysis on  1/4C-pcst Ic Ide association  with  calf
      lliymus  I)N\  .........................      ^     26
 8.  Toxfclty  of standards and  precursors LO adult  nixed  - sex
     •niisci  doraesL ica and a odes  .lop.ypti  larvae  .............    29

 9.  IisiicLlcid.il activity of cyclodiene  .i.lducts  in house flies   ....    30

10.  Toxlcity  of oruanochlorlnus  to  house flit's   ............    30

II.  OrganochlorliL-s:  In-ierslon  Lo-:lclty Lo ^lu-ies  lo^ypti  Jarvje  ...    3L
12.  Quantitative  .nijly&it. - InnS.iLlon  of  .1 Ihyd ron l.l r in with  S-10
     fraction oC  rat llv-r .......................    32

13.  Tm-nerslon  toxiclty of py rcthrolils with /iLli nstir  nomjuito  l.irvde,
                    ...........................    33
     Toplcil jppltcitlon uf syialiesized  pyrol.liroi.ls with house  flies:
     mortalities  it  discrete doses
     Topical applications  of  IIL phony le.io pyri.-Lhroi.1s Lo hou«u> files:
     active com;io:.in.!s  and  -tLandirds  ..................     35
                                i

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                                 LIST OF ABBREVIATIONS




                            :<1 Envtronnental  Rese.ircli
  "-ISO -  01r»lhyl,ulf0»We
  MA - Oe
              -I apeceroacopy
 J -( Joules
 LC50 -  Lethal conceacra t Ion .  50*
 Li>50 -  Lethal -lose,  50X
 MfO  - Mlxe,l  function

                 buffer  s.illne
               o,!ecyl  Sl,lf=,
TCA -  rrlchluro.,<.etlc ^c Id
                                      ix

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                                 SECTION I


                                INTRODUCTION
 OBJKCFIVES

                                                        °r
                                                                      ot



     3)  Evalua-.e the ^n vitro rummallan cellular effects.
                                                              '-
SUMMARY OF ..KSULTS



Synthesis and Selccttoo of 1U logcn>i tec! PestlciHo
                       ^^
                        ^^^^
                                 ]

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  molecules.  The  synthetic approaches were extended to  the  pyrethrin classes
  (Figure  3) of lnsectlcid.il agents  based on results (4) obtained early  in  the
  grant neiiod.  The chemical approaches resulted la the synthesis of a variety
  of  novel chemical agents with  observed Insectlcldal  activities.   Several  of
  the agents synthesized were  radiolabeled (5)  for further macromolecular
  Interaction studies which led  to the  eventual synthesis  of  chemical  agents
  which retained  their insec11cIda 1  action while demonstrating  a markedly
  reduced  deleterious  effect  on mammalian systems.   The structures  of  the
  chemical agents  selected  and/or  synthesized  for these studies are summarized
  in  Figures 1-4.

  Insect Tcxicity  Studies

      All  chemical agents and  EPA  standards were evaluated for  their
  insectlcldal action in house fly  (Musca .domestlca) and  mosquito  (Acdcs
  aegypti)  test systems (6,7)  utilizing both  topical  and oral administration
  roates.   Additionally,  experiments were  carried  out to determine the  effects"
  of  synerglstlc Insectlcldal chemicals (8)  and  the effects  of  metabolism  onthe
  observed  toxl-cltles  of  pesticides from  the several  classes  involved  in these
.  studies.

      The studies  undertaken indicated little direct  relationship between high
  halogen  content, aronatic ring content  and  Insecticidal  action.  The
  halogenated  cyclodiene  analogs  (Fig. 2)  (1)  demonstrated only very weak
  Insecticidal  action  by either  o.-al or topical  routes  of administration.  The
  compounds  with  extended -i-electron rich  syste.as  (utilizing pyrethrln-related
  agents) (Figure  3) failed to demonstrate decreased mammalian toxic!ty  while
  retaining  effective  pesticldal action.  Analogs  of  aldrln and dieldrin (Fig.
  1;  provided  the most  effective  lnsecticld.il  agents and demonstrated the
 greatest activities  when used In  conjunction with synergistlc agents  such.as
  PRO.  The  r-jsults  of these studies  clearly  demonstrated that structure-action
 correlations could be established  base.-, upon probable routes  of  metabolism In
 the  Insect  species.

      Our  studies  further demons traced a  stereochenical selectively  of cis
 isomerlc  pyrethrine-related analogs as  the  most effective  insectlcldal  agenTsT
 Naturally  occurring  and other synthetic analogs usually  favor  the trans
 Isomanc structures (Figare 3).  Additionally, the most effective  Insectlcldal
 analog of  aldrln, 6,7-dlhyd--o=i Idrln  (Figure 1 ,_4 ) was shown to be converted by
 mammalian  liver hcmogsnates to aldrin  (Figure 1,1), dieldrin  (Figure 1,2) and
 6-hydn :ydihydroa Idrln (Figure  1,14).                                  ~~

 In Vltru Marvnallan Cellular Effects

      The  cytotoxic  effects of the chlorinated hydrocarbons  synthesized were
 determined  in normal fibroblasts (C-153)  anJ  transformed human  flbrobl.ist
 (VA-4) cell line-.  Aldrln,  dihydroxyaldrln (Figure 1 ,_6) and dieldrin at a 100
 uM concentration  reduced  the  colony  fornlng  ability to 0% in both C-153 and
 V\-4 ftbroblasts.  Other structar.il modifications of  the active moieties of
 the  chlorinated hydrocarbons greyly  reduced  cy totoxlc i ty,  but not  to the
 level ooserved with the above three  compounds.   Allethrln and  pyrethrin

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(Figure 5), of the  pyrethrold class,  exhibited a high level of cytotoxlclty
resulting in 0% survival  at  100 u£l concentration in the C-153 and  VA-4  cell
ll.ies.  All compounds tested exhibited typical  cytotoxlc curves dependent  upon
dose.

Induction of DMA Repair

     Dieldrin  Induced  UDS  in  the VA-4 cell  line (9),  while It showed
comparatively  li?ss  'JDS  In the C-153  and  Cl  (10,11)  (normal human  skin
fibroblasts) cell  Lines.   Aldrln did  not  show any UDS  in the cell lines
tested.  Of the  structural analogs tested for UDS, those containing a rcHCtlve
moiety at the  6,7 position Induced UDS.  The  other analogs did not  appear  to
Induce significant  levels of UDS.  Repair  was  also measured  using  the  BUdR
photolysis technique.  Aldrln and dleldrln both showed pbotolyzable sites  In
the CI cell line,  while dlhydroaldrin did not (11).  Aldrin and dleldrln both
inhibited normal DN'A  replication.  Pyrethrin  showed repair while allethrln and
permethrln did  not.

Radtolabeled Agents  for Assessment of DNA-Associatton

     In  order  to assess  the  relationship  of ^pestlcldal association with
macrornoleculn r cell  components, ^ C-aldrin,   C-dieldrin, * C-dihydro.ildrln
and  l-*C-d ihydroxydihydroaIdr In  (5),  were  required at different  stages  of
research in this project.  The  l^C-aldrln and l*C-dieldrln were obtainable
through comraerci.il  sources at 80 mCi/tnM.    These agents  were  used  for
biological studies and  served further  .is  the starting  materials for
radlosynthetIc  procedures   that  resulted  in  novel  synthesis  of  the
14C-dihvdroaldrln and  l4C-dihydroxydihydroaldrln (12).  Coincident with UDS
data,  l*C-dieldrlnf .-as found  to be associated  with VA-4 and  153 cells to the
greatest extent.  liC-aldrin and ^C-dihydroaldrln did not  associate with the
DNA of the VA-4 and  153 cells while ^C-dihydroxydihydroaldrln did  associate
to a limited extent.   Activation studies utilizing  S-9 rat liver mlc rosu-.nes
Increased the hlnJing of ^o-aldrln and  i:"C-d ihydroa Idr In in both VA-4  an:l 153
cell  lines.  It was  also  demons t r-: ted  that  l^C-dieldrln  associated  with
single- and double-stranded Isolated calf thymus DNA.

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                                     FIGURE 1
           Structures of II.iloqcr.ated CycloJienes for SAR Scudies
                          'Vidrin and Uieldrin Analogs
                       Cl,        '          X
                                                7
                                           '    -6
  1)  X = d!2; A-B is CII = di (aldrin)

  2)  X = di,; A-B is d!2 (dihydroaldrin)

  3)  X = CII,; A-B is di-CII (cUeldrin)

  4)  X = Q12; ^-B is CIKOIO-CIKOH) (dieldrindiol)

  5)  X = 0; A-B j.s QI=ai (oxyaldrin)

  G)  X = 0; A-B is dlQKdlOU  (dihydrodihydro>Yaldrin)
                     s°\
  7)  X = 0; A-B is CII-^1  (oxydicldrin)

  8)  X = 0; A-B is CII{Otl)-CH(Oll)  (oxydieldrindiol")

  9)  X = dI2; A-B  is dirai2

 10)  X = dI9; A-B is dlFdlT
                                 0
 11)  X = d!2; A-B is  (dIOFI) aIOda^3  (naioacetox^-monohydraxN';

 12)   X = di,; A-B is  (dIOCCH3)9  (diacetoxydihydro)

 13)   X =  O!2; A-B is i-'-ai, (6-o:-:o)

 14)   X =  dI2; A-3 is ai2GI(01l)  (rxDnolv drox\-cihydro)

 15)   x = CIL,;  --y is di2cr0

 15)   X = CH,;  A-i3 is auclKxtu, (monascetox" dihvdroaldrin)
                     . o-o     J             "    '
 17)   X = C!I9;  A-3 js'-C-C1-

^3)   x = en,,-  VB j<3 cr,cr
                        z

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                                  FIGURE 2

             Chemical Structures of Halogenatec! Cyclodienes
                          Electron Rich ,\naloqs
19)

20)

21)

22)

23)

24)

25)

2G)
R1=R2=C6H5    R3=R,(=OCH3

               R3=Rj,=H

               R3=Rj.=Cl
R1=R2=Ci.'3

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                                FIGURE 3

                       Extended Aromatic Systems -
                       Pyrethrin-Related Analogs
               CH,
                  ~
                  H H
                         C = C
                 /CH3

                   CH3
                                                                         01
                   \/
29)   CH.-CHOC f*C_C""C =

                 H  H
                               CH
                               *-**
                                          CH,  CK,
                                         O  3  / 3
                           30)           n  >.
                                  CH.-CHOC  cfjC   C
                                    3 -     a  H   ft
 3


!I3
CH3  ,CH3
                             CH_
                                                      CH   CH
                                                   II    / v       _x
                                                  OC n" C  C '" C » IT
                                                       CH.,
           33)     CH  CH

                                                            CH_
                                          II    / \         /
                                          C»-C_C"" C = C
                                               H   It '  H

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Figure 3 (cent.'d)
                         CHOC
         A          CH3
        cOc^.c-cx
                                 CE3  CH

                             o    ^c'
                          CH0OC mi c£Jj "
                                  H  H
                     CH,
               C
               ft
/


 \
                „..
                36)
CH, O

 I   "
 CHOC
                                CH.  CH.
                                  V X  3
                                             CH
                               «~C-^,,,,C =C
                                           N
                                             CH3
                 17)
                                   "3  CH3
 nu  o    ^c
  I   "    / *
  CHOC'" C—C
                                               CH_
                                         C =
                                         4
                                         H
               38)
              on
   CH-   CI
     ^- /
  O
  II   /  '•
  oc*»-c-cr
                                             C1L
                              cm   c;>
                                           CH_
                    ®
     H  !i    H    CIL

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                              FIGURE  4
  Synthetic and Precursor Components  for  Extended ';-Systei" Analogs
     CH,  CH
  o    V
                   CIL
HOC*
      H  H
«1 )   CH    CH
  0   V
 HOC,,l(cljC-"C
       H  H   H
                                                                    CH.,
                                               43)
          CH OH
             CHOH
                                                          CHOH

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                                      ync  5
            Structures of Commercially  Irsoortant  Pyrothrins Studied
  Pyrethnns  (naturally occuring)
           R
                = C  u
                               , co2a.3
 Pemgthrin

                       ^3  .aI,
        Cl          H   /^X
                              II
IiUPlLTJ.'!  (synthetic)

          sco pyre^hrjns  (above)     R =

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                                     SECTION H

                                     CONCLUSIONS
       1.   Aldrin  and  dleldrln  as  the  parent  compounds  of  a series of
  ndlOReiw.tfil pesticides  .ire cytotoxlc  Lo  .in.nm.'llan colls  in vitro.

  •LA  i2"  i"11"'  net'lhollc  'ctUatlun of  .ildrln produces a product  with greater
  •J>\  damping potentl.il  thin the p-ireiit compound.

  n-  ,?',   'lodtfiic h,7-dl»iydro.il.lrin Imlioitul tint  re.lucixl  in... 	„..
c.. .otoxi :ici..-s  .T:) l)\',\  J.niRo could he obtilnod  while ret-ilninp, ,1  hi?n levl
 V  insi;-t  tfKlci'.y.                                               '"     a   • -


 ,«  .I1'','  r,"!11'rs  >f.V,11''  ""ly Su''P°rt! chl>  fo.isl'»nicy of che wclonal .,,-,,.,„
 ^pc-s.i.-.. ..-, t.nj. Jill ,,,t  Induce genetic  :M.niKe  rcMiltin.-*  l,i nnf.ig jnea I s,
«• ir.: i ITU.- '..-; is  i v!  ./Loto-:i.:i ty.   The selection  of i limited nunh-^r of model'
...|-.«K,,I..S  .irh  nolifle-l rh,.,lr,l  fun-tlon.,1  ,;rOUl.b  :.or;eJ  .is  jn"erfeccl«
l..>ls  o-  ilp.,i:-yln):  c-u- -pr.jl,.,!,lo  sites  of  re.u-1 I vl cy  responsible  for
ii^ctln.-!  .,,»! -.-.miM,, .ollul.r erects  1.1  t'te  i 1 d r , .-,/d ie Id r i n  cl.iss  of
n i. O;;»T».->[>_•-•  pi-st Ic idr-s.

                                        10

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                                   SECTION  III

                                    NARRATIVE
 MATERIAL A.\T> METHODS

 Cell Culture

      The  VA-4  cell  line, a  SV-40 transformed human  skin flbroblast was
 obtained  from Dr.  Janes  Blakeslce  In the  Department  of Veterinary
 Patnoblolosy.  at  The Ohio State University.   This  call  line was originally
 established by Dr.  A.J. Clradl  (13).   The  CI and  C-153 cell lines  were
 c'stibllshed In our  laboratory  from  neonatal  fpresklns.  The cell lines were
 maintained In nt'lEI supplemented with 5? KCS and  100 ug/ml  streptomycin, 100
 u£5/-nl  penicillin,  and 100 ug/ml  fungazone.  Cells were lncub..tcd at 37°C In a
 huml'ltfted 52 C02:   95Z air  atmosphere.
      Cells  were seeded  onto 1 I  x  22 m.n coversllps contained  In a  100  mm
 dlaneter glass petrl dish  at  , density  of  1 x  10* cell, per <.*2.  Twelve ,„„„
 later   after cells  had  attached,  the media  w.is  removed .1 iid  fresh  mediun
 containing 5. CS ami 2  mM  HU  was added  to  Inhibit scheduled IJVA synthesis.  At
 the  time of chenlcal addition,  the ned 1.,  was  removed- the  fresh-medlum-
 conLaning  sS CS,  2 uCl/-al [ 3,,, c:,ymldlne  (specific activity  5 Cl/aM)  and  2  mM
 HU was  addcrl to the pl.iles alonu with  the  compound to be tcslcM.  F^r  cells  to
 be Irradiated with  UV radiation,  the  cells were  washed  twice  with  PBS and
 irradiated  -Itl, 254-nai  radiation  at  ,   flucnce  of  1 J/B2/sec.   Tiamed Utely
 following irradiation, medluTi cont.i lnlnS,CS, [ 3(| J tnymid lnu .,nJ  UU  were added
 as described above  with the chemical.   Cover slips  wer.»  Pull..-d ac the  time
 points  In-Jilted  in the  flSur,s,  legends and  tables,  and  wished in  Hank's
 ouffered  salt solution, flved in 45% ethanol:  57. acetic Held  (9).    Slides
 were washed and  rehydrjted  by dipping in 95* ethanol,  70X ethanol  and
 distilled  water.  .\fter air 'Jr/inS, the coversllps were  mounted onto  slides
 and dipped  in 2-fold ,ltl.jt.>.l  Flford  ''A  emulsion (i-ast-nan Kodak) an.1 kept  In
 the dark four days at   i°C.   Slides  wore  then developed  In  D-19 Kodak
 Developer, fixed .,,vl  ..talnel  with Harris he-wtoxyltn and eosin.  ApproxImteU
 100 cells were randomly  sel-.-cted and the number of  grains over nucleus were
 counted and  corrected for -jac-k.-.round grains.   All grair  ,-ount'ng wis
 us inK an Ariex ,m..lol 380  co.inf.-r  with an  Httichl  video screen connected
 Lelt/. -\us Jena  Doc-jval microscope.   Pesticides wre dissolved in DMSO
 hlShest concentration of D'lSO  used was  I.OX.  Rt-3l,lt3  obtained with
agents Jero conjp-ired tc controls  containing »MSO only  and no IHSO.
i. one
 to a
 The
 test
                                      I 1

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Cytotoxictty

     The  cytotoxiclty of  the chlorinated  hydrocarbons was  determined  by
assaying  for the number  of surviving  cells following  treatment with  the
compounds.   The  assay as described previously (14)  Is based upon the capacity
of  a  cell  to replicate  and  form a  visible colony after  staining.
Approximately 600 cells  were seeded  In the 100 mm diameter tissue culture
dishes (Corning).  Following attachment various concentrations  of chlorinated
hydrocarbons were added  to the cultures and incub.ited  for 12 hours at 37«C.
The raedla was removed and  the  plates were washed twice with  10 ml of  PBS  and
fresh  mMF.M containing 5X  FCS.  After 7 days of Incubation at  37°C the plates
were washed twice with  Hank's  basic solution and stained  by  Incubating  for 10
minutes  with .1  saturated  fileiasa  solution.  After  rlnslr.g, the  number of
colonies  per plate were  counted  using an Artex  model 880 counter  with a
Hitachi  video screen.   Three counts were made, on  each  plate  and at least 10
pl.ites were used  In determination  of each point on the survival curves shown
In  the figures.   Two controls  wore  used  In each experiment,  one with media and
the other with DMSO as  a  solvent.

BUd'K Photolysis

     The  BUdK photolysis  -aethod was described previously (15,16).  Cells were
labeled  wit'i cither  13HJ  or  [IAC] thyraidlne.  After labeling, the cells were
Incubated  with  pestlcl-'e as reported above.  The  ->H-labeied  cells were
Incubated with  0.1  uM BUdR,  while the  14C-labolod cells were Incubated with
0.1 mM thyraidlne  for  15 hours.   The 3H-  i> nd  '"C-labelod  cells were mixed
together and  Irradiated for 0, 6,  and  9  minutes  with 313-iui radiation.  The
DNA was  sedlmented  on alkaline  sucrose gradients and the  number of
slngle-!>rrand breaks (Photolyzable sites) calculated (16).

ON A Association Determination

     Cells  were Incubated  with ^C-labcled  pesticide.   At the  time  Indicated,
cells  were  scrapeJ from the plates and the  DMA harvested using  SDS-lsoarayl
alcohol  (17).   The purified  DNA was precipitated with absolute alcohol  and
counted  for  rad loact UlLy .  Calf  Thynus  UNA  which was reacted with radioactive
pesticide  was  precipitited  with TCA ,  washed  with alcohol  and  acetone  and
collected on Whatman U3 paper discs (18). The  discs after drying were counted
 for radioactivity.  Single-stranded  m'A was  prepared by heating at  100 C  for
 15  minutes  followed by rapid cooling  at  -10°C.   The  amount  of pesticide
associated  with  D'.'A was calculated fio.n  the  specific activity after correcting
 for dilution  of  Isotope a.iJ quenching.
 Insect Tov.ictty  As->iys

      Housefly  -  Topic*! Application

      Contact  roxlclty  was  determined  as  Ln50 by  toplcil application of
 chemical  dilutions  to mlxe.l-sc< house f I les ,  Mu^r.a domcsttca (6,7)  using
 re-.,jont  gra-U> acetone ..s  solvent.   fne-.nicroH tcr droplets of each solution
 were' applied with  .in ISCO mlcroappl leal >r to the thoracic  region of  20  adult


                                      12

-------
  files,  3 + 1 days of age.  Dead and norlbud  flies  were  recorded  at  24 and 48
  hours.  Reconstituted powdered at Ik  was  offered  as  lood .luring the observation
  period.  Acetone only was administered to  the  control  files.
                                                   %
       Housefly - Oral Exposure

       Oral  toxicUy was  deternined by  feeding  expert cents on alxod-sox house
  rues.   An appropriate volume of  acetone dilutions of  each chemical  was
  untfornly  mixed with gr inul.i ted sugar, and  the  solvent  was evaporated to
  provide a w/wZ concentration In the  food.  The 48 hour LC50 '-'as determined  for
  each exposure group containing 2'J files  in a ventil.iteJ container.   Dead  and
  •moribund  files were counted .it 24 and 43 hours.

       Mosquito  Larvae - I-merslon ToxlclEy

       Immersion  toxlclty wJS determined  hy exposing 20 fourth  Inst.ir mosquito
  larvae (Ag^os aeRyptl)  in  water containing the  chemicals  to be  assayed.
  ,-hemlcaH  were Jissolved  In  acetone anJ 0.5 -.1 of hot solution was added  to
  500  ml  of water.   Dead  and  moribund lirvac.- were  recorded after 24  and  48
  nours.  Control  groups were exposed  to acetone and water.

  Determination  of Tottctty Values,  All Insects               "   "

      LD50 ,,nd  I.C 5|) values were  interpolated  from  regression  lines of problt
 Tiort-illty  vs.  l^r.io  "1°-ie  "l'«n  graded  responses  occurred.   Other.-lbe   the
 :aiKlr,u.o response «  tho hl^hesc .?ose w.is  recorded.   Insecticides  of  known
 potency (aldrln.  dleldnn,  al letl.r tn)  were  l.icl.Mod  In  each experiment as
 standards of toxicity.
 Tn Vitro "li-MboH^ of Chen1c.-tl<; Sy  S-10  Fr-ictlon  of  ilit Mvi.-r

      Incubation a.H K
      Chemicals  wore  Incubated  with  S-IO liver  fraction  and  the  ncresviry
 co-.actors (NADP.  lsoci:rn«.  Uocltrat... dohyd roB«.i.i -•« , Mg+2   :m+2)  for
 virlous  periods  of tine.  The mKcaro  was  .-xtr.-.ctoJ  three  times  with  petroleum
 etner,  dried wit.i  Nn2SOit reduced In volune an]  anal /zed.   In  SODC  cases  the
 ext.nct  -i:. ..urlfled by colu.n:! clironMto^riphy using  silicic  acid and  florosil.

      CLC Amlysl-i  of .'loc^Tol i ti.-s
      Petroleum  ec-ier extracts  w«re .in.il/zcd  by yas-Hquld  ch romoto^raohy
•iEtll7.1iiK  olnctrm capture detection.  Qu-i II t it I w .,n.lv,ls  was  icccmp 1 1 shed
by co-nparln,;  ret»..itlon Lines of M-V,nowns ...,J sta.viar-ls.  Q.ISi,iL It -.t Ive an-.l.sls
 -   obtalm-d  by  pt-ak  holghr  neasuronents.

      a 1 Synthesis  of  Staa'irds!  .- I  An., logs
    /|TloU» qy'1r!;il0sos 1>f  l'u-  Pl'vcl  :'no.;ca-ited  cy.:l,v!i..nes !,,vc been reported by
us  xl,2).  fho s/-,LSi,.sls  -,f  the  rUiol-ihelel  .Icrlvitlvo ' 'C-6, 7-J ihyd roal-i ri n
bis ilso  ,.:«,! p.i-ji:..liH  (3).   r(n-  synthouc  n-t.n.is .iLill^ol f->r rlie pyretli-tn

-------
related  f.inlly of  agents have been described CO  and  literature preparations
were  utilized  for  the  synthesis  of  oxoaldrJn (3),  oxodleldrln  and  the
derivatives (Figure  I)  of  6,7-dihydronldrIn and  6,7-dihydroxydlhydroaldrln.
All chemical/samples were analyzed  by GLC chromato^raphy  for  purity, and  were
purified  as required by preparitlve tliln layer chromatoynpliy.  Sanp1(as were
evaluated for chemical composition  by  standard spectral  analysis ( IR,  NMR,
TOa as  spec} and  microanalys I s  for  carbon  hydrogen  and chlorine  where
applicable.  MJSS  speciroscopy was  e.aploycrj ID needed  for  metabolism  studies
and conclusive Identification of  reaction mixture conponents.
                                     U'.

-------
                                    RESULTS

 gytotoxlclty
      The  effects of chlorinated  hydrocarbons and pyretheld pesticides  on  the
 colony forming ability (survival)  of VA-4 (Figure  6) and C-153 (Figure  7}
 human  flbroblast  ls  shown In Table 1.  The cytoto
-------
                    C-153  SURVIVAL
                 VA 4 SURVIVAL
§

I
   10
                                         • ALDRIN
                                         o DIELDRIN
                                         « CXHYLiflOALDRIN
                                         0 WhrDROXYOIHYDRO-
                                            ALORIN
                                         I 6-OXYWHYDRO-
                                            ALORIN
                                         A ALLETHRIN
                                         A PYRETHRIN
                                                                 10
                                  • ALDRIN
                                  • DIELDRIN
                                  « DIKYOROALDR1N
                                  0 WHYOROXYDIHYORO-
                                     ALORIN
                                  • 6-OXYOIHYOROALORIN
           20    40    60    80    100
                 DOSE  (>uM)
         20    40    60    HO    100
               DOSE (>iM)
  Figure 6.   Pesticide  induced cytotoxicity
              in  VA-4 cells
Figure  7.   Pesticide induced cytotoxicity
            in C-153 cells

-------
                     TABLE 1






Cell Survival of Human Fibroblasts to Pesticide.
Compound
Aldrin



Dielnrin


Dihydroaldrin


Dihydro:"'-
dihydroaldrin

6-Oxydilv'dro-
aldrin

Allethrin


Pyrethrin


Concentration % Survival
UM VA-4 C-153
30
60
100

30
60
100
30
60
100
30
60
100
30
80
100
_
50
100
_
50
100
' I
;'' 73.8 ± 6.6
60.0 ± 9.8
0
1
73.3 ± 5.1
33.5 :. 2.0
0
72.3 = 6.5
33.1 i 3.3
0
91.1 ± 5.5
79.4 ± 6.4
76.7 ± 6.1
68.0 i 5.4
56.1 ± 5.6
40.2 ± 3.2
—
-
-
_
-
-
61.4 ± 4.3
53.7 ± 3.2
0

68.3 ± 7.5
23.9 ± 2.4
0
67.7 ± 6.1
24.5 ± 2.7
0
91.8 : 9.2
--81. 2 ± 5.7
80.9 = 7.3
± 3.1
53.7 ± 2.1
15.2 = 0.8
^
77.0 - 9.2
0
^
71.5 ± 7.9
0
                          17

-------
                                          TAIUE 2
Unscheduled DNA Synthesis3 in VA-4 Cells after Treatment with a Scries of Chlorinated Hydrocarbons
            Yrojtnent
                                      Dose
Exp. SI
                                                                       Exp. 82
Average
Co.'iLio! (17. IViTO)
IV- -
Ojcldrin
A] drin
DiM^lraildrm
Di h\ -d ro: :\ d i h , d roa Idr in
Mjnoaco to: :• -a ] < i r i n
I'Jiicvtiv:1 aldran
Giyaldrin
Oxydicklian
-
1 OJ/M2
100 ', M
100 -_n
100 ',jM
100 i-M
ion I-M
100 ;M
100 ..M
100 ;iM
0
15
11
0
0
0
11
10
9
1
.8 •
.4 i
.0 i
.1 i
.4 i
.9 J.
.2 i
.7 i
.3 ±
.0 i
0.
1.
0.
0.
0.
0.
0.
0.
0.
0.
4»
0
8
4
6
4
8
8
o
8
].
20.
13.
1.
0.
1.
10.
12.
6.
6.
D
4
6
7
2
4
7
4
3
3
• 0.
: 0.
.' 1.
i 0.
i 0.
-l 0.
i 1.
i 1.
i -1.
i 0.
4
7
6
5
4
3
1
0
0
9
1.
17.
12.
1.
0.
1.
10.
11.
7.
6.
j
9
3
2
3
2
9
6
8
7
''riqurcs rc.-;.;rcso-,t the mean number of grains/nucleus  of 50 cells from which background was
               Treatment time = 5h.

-------
                                    TABLE 3
Relationship of 1/Mw of VA4 cells treated with 10 uM pesticide for 12 hours.
      Pesticide
     Dihydroaldrin2
     Aldrin
     Dieldrin
     AUethrin
     Pyrethrin
     Permethrin
Photolysis
time
nq.n
0
6
9
0
6
9
0
6
9
6
q
0
6
9
0
6
9
•"•/tow


0
0
.44
P
.018
.104
0
.025
' .135
0
0
Q
.015
.028
0
0
0

-------
Allethrin and peruethrln did  not*

     Repair  uas measured  In the CI cell  Line by  UDS  and BUdR  photolysis.
Aldrln and dleldrln both Induce.]  repair  (Table  '•) as rae.isured by UDS and  BUdR
photolysis.  The nunber of  rep.ilr  sites  Induced  In the Cl cell Lines by aldrln
and dleldrln was greater than  In  the VA-4 cell line.  Other  studies using
Inter passage of CI cells,  passage 17, showed that aldrin and dleldrln Induced
a lower Level of repair in  the  presence  or absence  of S-9  liver  mlcrosoroes.
Dlacetoxydlhydroaldrln, 6-acetoxy-Jlhydroaldrln, oxyaldrln and oxydieldrln all
Induced significant levels  of  UDS.   The dlhydroaldrln and  6-liyJ roxya Id rl n
compounds Induced  UDS only  in  the presence  of  the S-9  liver  :nlc rosoma I
fraction.  The  taonofluoro  (Figure I ,j)) and  6-oxo analog of  dldrln did not
induce UDS while they retain  pestlcldal  activity.

Effect Upon PSA Replication

     Aldrln  and dleldrln  were tested  for their ability to Inhibit scheduled
DNA synthesis and their ability to  replicate daughter DNA.  For this study  we
used the  Syrian hamster cell  system  described previously (19) for poly:aroma tic
hydrocarbons.  .As shown In  Table 5,  aldrln and  dleldrln Inhibited scheduled
DNA  synthesis  In  a  dose  related  nanncr.  Also,  both aldrln and dleldrln
Inhibited  the ability of cells  to  synthesize daughter UNA  (Table  5).  These
data indicate  that  .ildrln  and  dleldrln  act  as blocks  for  normal DNA
replication.
                      /
\ssoclatlon to Cellular DNA
     The  extent of  l ^C-pcs t Ic Ide  association  with the DN'A of VV-4 and 153
cells was determined 12 hours after  addition of the conpound.  Even though the
cells and oxtricted DNA were extensively  washed,  we prefer not to use- the term
"blndlns" In the Interpretation of  thes.>  results.  Association  to the  DNA  Is
used  here  as  a  broader  term to  Include  interaction  with DN'A as  well
Interealit Ion with D\A and covalent binding  to DNA.  As  shown in Table d
radioactive dleldrin associated with the  DNA of both  VA-4 and C-133 cells.  On
the other hand little of the radioactive  aldrln,  wihtout metabolic activation,
associated with DfJA.  These data  correspond with  the  above UDS studies showl-ig
that dleldrln Induced UDS while aldrln  d^id  not.   Two analogs,  d Ihyd roa Id r i n
and  d i Tyd r oxyd Ihyd roa Idr in,  wore ^C-synthes 1 zed and  tested for  their
.issoclntlon with cellular DNA.   Little  association oC  these  compounds  to DN\
was observed.

     When cells were incubated  with  compounds  In  the  presence of the  S-9 liver
•ilcrosoTMl fraction, the level  of aldrin  and d I liydroa Idrln associated  wlrh thu
DNA  increased.  The s.n.ill amount  of  dlhydroxyd ihydroaldr In associating with
DNA without actlvitlon, decreased to background with  the S-9 fractions.

     It  is  Interesting to note that  the  compounds  testel for association lo
cellul.ir  !)N^  a) diel.lrln, aldrln,  d 1 hydroa M r in are cytotoxlc  to the  cells:
and b)  only dieldrln associated  to ;i  great extent  with DNA.  These results
suggest that alcliouft'i UDS (in Indirect  mc-.is.ireraunt of UNA damage) parallels
the  r.i.l lo-ic t I ve association of  the  chlorinated  hydrocarbon, the cytotoxlclty


                                     20

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                                     TABLE -'.
        of  Pcsti  ido induced D>iA damage an  the C-I human cell line.
/Vjcnt1
Dielclrin
.Mar in

IJV
Cone
iCO .'•'.
100 'M
i
lOJ/n-
liDS2
•5.8
7.1

12.2
Photolysis
0.15
O.OG

0.2.
           dissolved  m I3TISO.

"•"•'.'crnqcj nir'Jxjr of r-ivurs ror  nuclei.
 Cv. 11 s v.-crc  i :-.cailj. i tcv!  w i t_h fo'; 11 ^ i L'.O 6 hj:.

-"- :\-i  tor 103 dciltoivj.   t.cllij  \.ur incubatocl  for 1-1 lir.
 •.-.•idi  [XJGticidc anti  BUiK.

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                                TABLE 5
Effect of dielclrin and aldrin in scheduled DNA synthesis and daughter DNA.
   Corcound
Cone
 ..M
% Inhibition
of scheduled
DNA synthesis
                                                      Ratio  of neutral
                                                      daughter DMA co-
                                                      sedimenting with
                                                      control DNA
   Aldrin
 10

100
                                       37.5

                                       70.0
                         0.91

                         0.77
   nieldrin
 10

100
                                       34.0

                                       50.0
                         0.84

                         0.77
   krcnpounctewere dissolved in DMSO.

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does  not .ippear to be directly related to either  UDS or  to Mndlng.

^ssoelatlon to Cell-free DNA

      To  determine whether tlie association  observed  with cellular DNA was due
to  covaljnt  b 1 nd1 ng or  to  soae other  factor,  we  perfor-aed a  series  of
experiments on purified c.ilf thymus DNA.   In these  uxperl.nents  single-stranded
and double-stranded DNA wore Incubated with  rndto labeled dieldrln and  aldrln.
The  nraount of ridlolabcled compound wis  assayed  following ,t 12 hour period or
allowed  to dl.ilyzc against buffer for an  extended period of tl-ne.  As shoc!i

-------
l~C-Ch]orir.3ted hydrocarbon assocuici.cn with hurvin cell DNA.
Conjound •
Dic-lcirin
Alclrin
Dilv/droalclri n
Di-. i':rox/c:." dro-
aluriiv'1
VA-42
+S9J -S9,
96.2
23.0 3.1
3-1.0 1.5
3.9 12. C
C-1532
+S93 -S9
32.5
27.1 8.5
24.0 1.3
1.2 8.3
 Ccn.ound \ro:-::ri:itol'.  90i pure.

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                                        TABLE 7
                                  11,
            Effect of Dialysis on   C-Pcsticide Association to Calf Thymus DMA
Single Strand
.Treatment
no
14C-Dicldrin
14C-Aldrin
l4C-Dihydroaldrin
^•4C-Diir. clroxydihydroaldrin


dialysis
19.013
10.68
12.65 "
10.79


dialysis
16.72
7.15
J.41
3.45
i
Double Strand

% lost'* no dialysis
12 , 50.01
33 9.46
73 11.08
68 6.41 ~
— • —

dialysis
26.00
3.32
1.88
1.34


% lost3
48
65
S3
79

$ lost = (no dialysis - dialysis)/no dialysis



-.•moles bound per mole nucleotide

-------
Toxirity of Standards and  Precursors

     The use  of aldrln,  dleldrln And certain  other Insect Icicle 1 ly active
co-apounds  confirmed the utility  of  che study and  established  them as
sufficiently sensitive  assays  of loxlclty (6,7).  None of the precursors  were
very  toxic to  houseflles .is  extern.il doses.  llexachlorocyc lopentad lene,
1 ,2 , 3,4,7,7-nexachloroblcyclo (2,2,1)  2,5 heptadlene and  cxo-2,3-cpoxy
norborrune  were highly to
-------
ft,7-dihydroildrln Co 6-h/J roxyd i hyd ro<* Id rl n  (Table  12;  Flgur.-  8).  Or
Inlrtg.iiny phenomenon,  L!ie appearance of  .ilJrin and dicldrtn  tn  incuhjcIons  <
dlhydrod1 Irln  with r.i t  liver  .ui crosoraes ,  was  confirmed  in two  separat
ovnf*r * n^nre _
cxperInencs

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                                                                        I.Mil I   H

                                  l.ixlilt\  .it .Si.inJ.inl-. .iml I'ri. ur .in -  to AJiilt Mlxul-Sux Xn .. .1  J...i,
                                                (limn.  IU)  :in.l  Ai ili .  .m,y|i| I  (ranvpilin  Inry.ii.).
1 .. , 	 ll' 1. I..I
i: Ii i .
.li. l.li MI
inn

1. (,-.!li\. I.-PIMI i.li.iif
(J. n I.j. J.'.-ULI.I, l.l. >i,.l. nt i I.
1.1. .i,. f: .• n .', i-iu '.i.ijun,
in \ ii t i.ii iii i.it . nt i.i u in
i, :.!.•../ M . • i.iii.-i,. M, \. i.. ( • M) _•.»•, ii. pi 1.1 1, -in
A'1'"1 '.'
fur. in
Linlrfn
L\I'-J. 1-1 |'llv Mllll Ili'l II. Ill |
1. _'-• p.i%t- i. i. l-i i i. liliii.i|ii..|.ni, i

li. ' ' 1. III. II* . '.'\ Illc
i
i. I'H/i i.pri- H5 m:/il«
H.ii'.


ill . R/f U
''.') i-s/fly
•"'.0 UR/flv
I.' ny.it \)
'".D IIR/I ly
:o nr./fi>
-li) ur./tly


> .0 us/fly
•W n(;/fl%




nr.il ll.
n- '
O.S ppra
II. S ppm
II. H |\lclt\
"'.„• '•'«•
O.fllfi
D.lllli
l>. HI J

•10 p,in
>m pi -;
••U) ppra
•0 '> ppn
- 1 ppm
>l(l ppm
•5 ppm


0.4 ppn
• 111 ppn




i.   [till  l«|-it  .,|mii.f«  of l-'Uflll  in .til  r-»t:,»julln  liiv.ic.

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                           TABLE 9


      In-i-cticidal Accivlty of Cyclodiene  Ad.lurt.s in
               House-flies (Musca domestica).


Compound
J9
20
21
22
23
24
25
26
B-c!:loid.-mc-C
aJdrinc
lieptachlorc
'j'opical Appli-
cation:
LD50' ng/fly
>:>Ua
>20J
>2Ja
16
>20a
>20a
>20a
12
0.05
0.02
0.04

Oral:
LC50> PPm
;• 1 2 000
/!, b
'12 000b
10 000
10 000
10 000
JO 000
12 000
3.0
0 5

a. Or: .,,. rL...'i:v  r,i  2" ui;/fly.  h.  0% mortality at 12 000  ppm.
c. Source:  USLPA  standards.
                   TARLE JO

Toxic I ty of r.r';anoc!)Jorincs  to
                                            I'Jics,
a-™.!*!!

Aid r in
1) i <_• 1 ,7 Dili,. dro::ya l
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                            TABLE 11





Inversion toxicity of organochlorines  to Aedes aegypti larvae.
Chen i cal

Aldrin
Dieldrin
Oxyaldn'n
D i hyc ro> > d i hyd roa 1 d r i n
Oxydieldrin
LC50'
alone
0.016
0.035
0.048
J.-T.ig/L - Of:
0.18
mo/L
+5 Vnq/L PBO
0.015
0.0*8
mortal ity
0.13
.Notes
/
Mo syne rg ism
No synergisn
No syneroism

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                                                TARI.E 12
                           Quantitative Analysis:   Incubation  of Dihydroaldrin
                                    With S-10 Fraction  of Rat  Livers.
jation Time
lours
0.25
0.5
1.0
2.0

Dihydroaldrin
10.8
14.3
26.4
9.2
'/' of Initial Dihydroaldrin
6-hydroxy
dihydroaldrin
5.0
7.7
16.3
7.1
a,b
Aldrin
0.2
0.1
-0.1
0.1

Dieldrin
0.04
0.1
0.1
0.2

Total *.
Recovery
46
22
37
17
 Corrected for recovery efficiencies after florisil  column clean up.'
 Recovery efficiencies were dihydroaldrin (94?,), aldrin (51"'),  dieldrin
 •44;) and 6-hydroxydihydroaldrin (24.).


 Uncorrected for extraction efficiencies  from incubation mixture.


C0ualiutive analysis Kisod on GLC retention times.

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                          TABLE  13

Iprr.ersion Toxicity of  Pyrethroids witn  1th Instar ilosquito
                   Larv.-.e,  Aedes  aeoyptj.
cJI£!]li£!\L Mortality at Concentration, mq/L
Alone + PBO (5 mo/L)
10,;:n/L = 0
36 lOmg/L = 0
3-3 IQmn/L = 0 ';
33 lOmg/L = 0
27 lOmci/L = 0
LC50 Va1ilt;
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                          TViLt
     Topical  Apolication of Synthesized Pyethroids with
         House Flies:   Mortalities at Discrete Doses.
Chemicals

40 (trans)
,'42.
41 (cis)
36 (trans)
34 (trans)
33 (trans)
27 (trans)
32 (cis)
31 (trans)
39 (cis)
30 (c-.s)
29 (trans)
% Mortal ity
Alone, 40 i:q

80" '
o-':
20' !
0,
30.
0' 	
10-
20 gg = 0 M
20 ug = 0 H
20 ug = 0 H
20 ug = 25 M
10 ug = 0 M
at Given Dose
+ PRO (5 uo)

40 ng = 100"
10 ug =' 40"
40 uq = 70
10 i.q = 0
40 uq = 90-
10 ug = .10 ..
40 uq = 70"
10 uq = 30'
40 ug = 100 '
10 uq = 40
	 40 ug =— 40---
10 ug = 0
LD50=1.5 g
(Ratio >25)a
2 ug = 35' M
2 ng = 40'.M
2 ug = 30" H
2 ug = 90" H
2 vj = 50"-1
           I.D-Q alone
a. Ratio = ,-o—•—-n*
                              33

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                        TABLE 15


Topical Application of Biphenylene Pyrethroids to House
         Flies:  Active Compounds and Standards.
Che;;;ic.il
28
35
27
34
Pyrethrins
(Standard)
* Svnprai<;M'r
Alone
20
20 ug = 20 M
40 na = 40' ?!
40 ur, = 30'.M

0.22
PAtm. L.D50a1°ne
+ PBO (5 i.q)
0.2
0.8
1.5
10 ug = 40'

0.006

Ratio*
100
>25
>25


37

                  LD
                     50
                           V,

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                                           rirnrr n
                                           i i »ILM.L '/
~




r_
      10
                    -£-
                   .25
                                              Til.;-.1 'j I inly  "o til bo! i CM of cl ihyilro.ililr in



                                              by S-10 fiMrtion  of  >Mt livcc.
                                                                     Q-  6-OH  dihydroaldrin
                                      oieldrin =  0
                             = o
.50
                                                         1.0
                                                                                               2.0
                                            Tine  of  incubation, hours

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                                 SECTION IV

                                  OVEKVIKW

     The  results obtained  from this study  support the hypothesis  that .1
 Limited number of rationally designed and selected analogs can be  of  value In
 determining specific  chemical loxlcltles based  on funct lonal groups.  The
 choices of a Id r In and dlol<1rl:i provided  us with  A  chemical model  which  was
 readily  manipulated for  appropriate cellular  biological .ind ,lnsectIcldal
 studies.   It  Is  Important  to emphasize  that  Che project  was  funded  at
 approximately 50% of  year  01 requests  and that the budget remained cons tine
 despite large Increases  (over  300% In some cases)  for supplies, chemicals,
 :nediura and  personnel.  There si.ll!  remains additional work for:  a) detailed
(cellular  studies  oC  the  model  compounds of greatest Interest and  b)  model
 ecosystem evaluations  for  this particular 'class  of agents.   It  Is  clear
 however tint this Interdisciplinary approach  Is  readily adaptable  to  other
 chemical  classes  of  environmental agents.

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                                   RCFERKNCKS
1.  N.J. Lewis,  D.H.  Knight and W.J. Collins,  "Dlels-Alder adduces of fulvenos
    and halogenited  dienos:  Synthesis  and  Insect Lcldal activity" J. Mcd
    Chcm, 22,  1505-1509 (1979).

2.  N.J. Lewis and  D.R. Knight, "Dlels-Alder 'adduces of fulvones and halogen-
    atcd cyclopentrid lencs :  Application of  proton  .ind ^C-N'MK to structure
    decermlint Ion"  Abstracts of the  l«th Midwest meeting of the A-aer. Cliero.
    Society,  Fayettevllle, Arkansas  (1973).
                                               I
3.  M. Kleln-in,  U.S.  Patent no. 2655513 (1953).

4.  N.J. Lewis,  K.  Ekwurlbe, R.W. Hart  *r.d 'J.J.  Collins, "Synthesis biological
    activity  of  novel  pyrethroid-type jn.ilogs" Abstracts at the A-ner. Client.
    Society Congress,  Honolulu, Hawaii  (1979).

5.  N.I. Lewis .ind  P.O. Ca^er, "Facile  mlcro-nol ir  roil action ot" (1,2,3,4,10-)
    (14CJ Aldrln to  6,7-dihydro (1,2,3,4,10-)  [14CJ  AMrln" J. Lihol.
    Conpoun.ls  Rjd lopha c n-jceut let Is ,  18,  471-473  (I9o0).

6.  J.R. Butvlne, "A  critical review of  cechnl>|iu-s for lesllng Insectlcltlcs"
    Comiaonwc.t Ith Agrlcultur.il Bureau, 2 IK! edition, Slouch, T.ng. (1971).

7.  11.11. Shepar>i, "Methoi'.s of testing chenloals  on Insects" Vol. I, Burgess
    Pub. Co., Minn.  (1953).
8.  J.E. Caslilei,  "^ixecl  function oxldase  Involvc/menc In the biochemistry of
    Insecl.le synerglsts" J ART Food Chcm jj,  753-772 (1970).
9.  F.K. A'lmeil ,  R.W.  Mar. and N.J. Lewis,  "Pesticide InJucud DMA damage .11"!
    Its rep.ilr  In  cult ired lian.ui cells" Mutit.  Kcs.  42, 161-174 (1977).

10. K.Y. Hill,  F.B.  Daniel, N'.J. Lewis, S.M.  !)'/\mhroslo ^nJ R.W. Hart,
    "H.i logon-iced hyjrocirhons Induce I)N\ di.n-u;e,  rep.ilr and nutagenesls"
    Abstracts of the  145th Aner . Assoi . Afiv.  Scl .  meeting p. 120
    (197«J).

11.  S.M. D'fVnbroslo, K.Y. Hall, R.W. MirL nml  N.J.  Lewis, "Pesticide  In.luced
     OVA d.innjje" Fed. Proc . 38. 539 (1979).

12.  N.J. Lewis, F.D. C/izer jnJ N'. Kkwurl.le,  "Kacllrt -alcromoLir synt'nesls of
       C-pe';l Iclile ilerl/itlvcs ml iaer Tool 1 tps" Ahstricts of Lhe 7th
     rcglon.il .nee ting of  the A-ner. Ch'jn. Soc . ,  Col., Ohio (1979).

-------
                  M  J>iM°n "? "'  Koi'rowsk1'  "SV-« i-*J«c«l  transform : Ion  of
      05   fi9-«           '          '"1J rec°Vwry"Cell Con..
      ~~
      F.:..  Ah-nod. N.J.  Lewis .md R.M.  Mart, "Pesticide- Induced  ounb.iln.re-
                           1'111"08'0 l"nsllir V7y colls"  Chen. Biol.  Interact..
 15.   S.M. I) ', V-ihrosio.  F.B.  C-inlcl .i.id  R.'J. 'KirL,  "CelluLir repair  of DNA
      d.sa.j'.e  Induco-J hy  7 . 1 2-11 noLhy H,enZ(., ).n,i.ir.,COno --m. Its  fluoro analoCs
      {•"' R':!:.i"'  "'^ S'-'clow  -ln'J «'"•  '-=y.  "-•'•M-n.'l and  defective repair of
      .i,:n,«ed ,INA  In  „„,„ c,lls:  A Se,sUlve .,Sr,,y  utlll.l^ the  photolysis
      of  '.rono.io.TciirMin..  jj^^ VKj^  Acad^ Scj^ ^SA. ^8,  708-712  ( 1971 ).
17.   F.B.  nmiel .  L. ,  Wo-,,.. C.  Or.vcc. F.b.  Cir«r, A. WinRl  S.«. D'Anhroslo.
       ;,'  ',"" airjf!)-r-  •'-^"k.  "Bl,»che,lc.,l  studies on  tho  metaboUs.  and
         --  of  3XH\ -ml sor,,-  of It.  mo,,»f|,,oro dorlvatlves of va
1*.  W.I..  C.,rrl«r -,nd  K.«.  Setlow,  "I'.,per strip netl.o.1  for ajaaymK gradient
                  "11"1"1  riJl°      '  '-"                T O.
     r'nilr1   ''   ">">   -W"ll3rl.  K'D-  Cj^r-nd D.T.  WltUk,  "DNA
     rop.lr  I.,  ,yr  ,:, I,., ,st,r  o.n'.rvo  c«lls ir.Mtod  w. t h- 7, 1 2-d ( meihy lh,.nly(a )
     h  "s'^M  (1-  -v5" W'Jt«onl»: *-n..or,, .....loa" Cancer Lett..

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