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
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' 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
-------
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.
-------
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.
-------
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.
-------
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
-------
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.
-------
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
-------
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
-------
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.
-------
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;
-------
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
-------
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,
-------
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
-------
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).
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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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