)

j

·1 . \". \

\ .... ~--- :, .

. ·· .. -.:. .. -~ - ·.' _:-·.: .

f\ )I') I 1 (.f,(I. I:.·) 1 (, .I' ()i : 'i' , 1 j \J

Rotenone Reregistration

EPA Administrative Number 6704-Q

Toxicology Oata

(Guideline No. 84-2)

April 24, 1987

VOLUME 3 SALMONELLA/MAMMALIAN-MICROSOME PLATE ' INCORPORATION MUTAGENESIS ASSAY {ROTENONE)

(Guideline No. 84-2)

26 pages

. \ '. - ~· ... ,. ;~}}":'.->!.:.~ -

)

Study Title

Salmonella/Mammalian-Microsome Plate Incorporation Mutagenesis Assay (Rotenone)

Data Requirement

Guideline No. 84-2

Authors

Study Director: Steve R. Haworth, Ph.D.

Study Completed On

November 3, 1978

Performing Laboratory

EG&G Mason Research Institute 1530 East Jefferson Street Rockville, Maryland 20852

Laboratory Project .!Q

Study #019-563-165-1

Page 1 of 25

)

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA §lO(d)(l)(A), (B), or (C).

Agency ---------=U-=-·=-s::..•..:.F..:.i.::.s:.:.h....:a:.:.n:.::d_W::..1::..·1.:.d:..:.l..:.i.:...fe=-=S.:.e.:...rv.:...i.:.c:.::e _______ _

Agency Agent: Fred P. Meyer

Di rector National Fisheries R.esearch Center La Crosse, Wisconsin

~age 2 of 26

Date:

Si gnat e(J

)

;

,. '•

GOOD LABORATORY PRACTICE STATEMENT

This study meets the requirements for 40 CFR Part 160

Submitter U.S. Fish and Wildlife Service

Sponsor U.S. Fish and Wildlife Service

Study Director Steve R. Haworth

Pay~ 2l of 20

?~ EGc.G MASON RESEARCH INSTITUTE tSJO EAST JEFFERSON STREET, ROCKVILLE, MARYLAND 20•S2

Tel. {]OJ) 770-4400

SALMONELLA/MAMMALIAN-MICROSOME PLATE

INCORPORATION MUTAGENESIS ASSAY

STUDY 1019-563-165-l

Notebook #MRI-WL-21 Page 236 240, 242, 248 Archived at EG&G Mason Research Institute, 1530 East Jefferson Street, Rockville, Maryland 20852

Sponsor: U. s. Fish and Wildlife Service Box 818 La Crosse, Wisconsin 54601

Test Article I.D.: Retenone Crystalline Code #864200

Test Article Lot No.: 735-RAP-1502

Compound Description: White Crystalline Solid

Storage Conditions: Room Temperature in Carcinogen Room under Desiccation

Date Received: 11/3/78

Study Coordinator: John L. Allen U. s. Fish arid Wildlife Service

Study Director: Steve R. Haworth, Ph.D. EG&G Mason Research Institute

Report Date: 6-29-79

... . . .

!::: . . " (.; c

t •• .. ,

• • •

... Steve R. Haworth, Ph.D. Study Director

. Lawlor Dat'ei'. ...

Clam,,,, K sl::Vi Jeaaene Smi Technician

Date

I'• •

ff '?'.:Pt .(/2..:-:.C:,,~:::. :?,,U ?/7 7 Nan . W1_ iams ' Da'te

terry'Reichard Data Technician

Technician

z-;;T-i.l,;: 1 '. I 5,£(,. LA< (,-,-<_?· )'I Patrick Bur);:'.e Date Date Technician

(.} . /, - (/' . \,,...,,' Ir~ ~,,..· '- C. / 1::r'.1.&:::::z::::vv: ✓John Cameron Laboratory Assistant

page 4 of 26 ·,,• ,, .

( ·._,;.-)

) ,.,

Introduction

u. s. Fish and Wildlife Service Retenone Crystalline

Code 1864200, Lot 1735-RAP-1502 (MRI 1165) was received

on November 3, 1978, for testing in the Salmonella/

mammalian-microsome mutagenicity assay at six dose levels

using five tester strains, TA98, TAl00, TA1535, TA1537

and TA1538, both with and without metabolic activation by

Aroclor induced rat liver microsomes. The test article was

assayed twice with a minimum of two weeks elapsing between

assays.

Materials and Methods

The experimental protocol (see Appendix) is a modifi­

cation of that described by Ames, B. N., et al. Methods for

detecting carcinogens and mutagens with the Salmonella/

mammalian-microsome mutagenicity test. Mutation Research

31:347-364, 1975.

Pa9e 5 of 26

-1-

)

RESULTS

Pa9e 6 of 26 -- 2 -

--- ( , . .,

~~-J

)

d,':,Gaca MASON R£5£~f'.?S:/-{ INSTITUTE

Preliminary Toxicity Determination

of Test Arti,:;le

Table 1 ·

Lof ii, 7.35"·1?/IP-/SO~

Study Number fuie :ti-£<. 'to"IOO 6'e tcmoe- c'r--:lsta, Test Article Identific tion

Test Compound TAlOO TAlOO T/\100 Concentration Viable count/ Spontaneous Background

µg/Plate Plate Revertants/Plate Bacterial Law,

. .(/) ,,1 ,[)mS,,., 35 3 /0-3 ,,,Ar-a/

IOOLJ' .amso I},'(. //) If- - ·,/17{1 ·-, • 11 35.;; IO{, A -/

,s/l ,-:?7/ q.3 MA--/i/

IS.O • :? ':13 /0-7 - A -,nr. I

.ll 9. 0 .317 9. ':( 11 orffltl I

15 "'.,,.. .J 4. " <7S , _____ , /_ ---;.,,., ,, .

4?9.a o:?9 / /CJ-I 1,-,-----/···/,.,, ----:,'.,,,, ~ -1-

L , - -- ~ I '"" I - - ; : ..... / 56.3/1 3.::?q .?/ 'i:" .I"

-,

w/hcavt(. Cooo.o J'IY '17 ,. ··r __ ,..., _.... -•c_,.,,

-t ;/' .,

't) O'°"; 0 /; (2/J y_lf · w//,et? vy ,;ll/.t, JJ.s- 11orrnt1I "'--,(':a , , , -

Date Plated S'/1 /7 9 j '

/11Ctt!/,;a-e. Counted By:

-f Ac,,,d c.oo,.,,.. d,.1e ro preeir7i 1 . f..e

g . .J.=·d, sht1'/

Eight serial half-log dilutions of the test compound ar~ plated with TAlOO on rninim;il agar plus lOXSA and on minimal agar plus lXSA. Equal numbers of cells are seeded 011 each plate in the presence of the test compound. The percent survival of an appropriately diluted TAlOO culture on the LOY.SA supplemented plates is determined by comparing numbers 9f colonies on the solvent control with those on the plates containing test co~­pound. To:d.c ity on the lXSA supplemcn tcd plates is detectable by a decrcni:c in the nu,~J.,er of. revertant colonies occurring per plate and by a thinning or disnppearancc of the background bac tcr ial lm•m. The highest concentration of test compound used in tlie broad range mutagenesis assay is that which_gives a dctectublc reduct.: nn of !;pon taneous rcvcrtun t!; on the !XS/\ supplemented plates.

. -- ... Porm No. \·/L-107 5/79

Page 7 of 26 -J-

.- ~ . ,,, · .. . . •·::-., .. . . ._ .. ·.. . .

, -:y);[~l! {1 ·, ... __ .' ,-- .

j

-0 I 0,

A <O I ro

Q:)

0 -h

N

°'

,.._,,,, . ; .I

l,, taa=a MASON RESE:ARCH :Nsrtrure· TABLE 2

SALMON!::~;;:,;,. X!JT/\GEllESIS ASSAY

H e:/-c11a11 c <:..r-yst-<1.l//11 e cJ/1- s-c 3- /G_S--1 Lai- II 73 6"-RAP-/,:i-oi1_ t;;.,J,.}1!:-l~ J/ B,oo

~ tuCy r:u.~c r Test Article Identification

Solvent uM .. <"O ...s_g_ ..LE.2. 3.Jo /()() .,,J_

,::<in, T,i 'If Revert:ants c,zO .JO .:).S- --<.. (., Oote ?lated,..£.{f":-77-_ per .;is- c<.S- 38 l<j ::o. o! Cells Seec!cd: /. 3 x108

Plate :-:et:A!:lolic Ac~ivatlon; i?d f J.;.-er .:z (., ~.? .:,_7 ~,

.s--;,_,.7 r .l .\ver~gcd di_,,d c~~n:cd ~y, ?. :r. 3,,,., R~vc:.·ttnt::r .,,l 'I- .) 'x 7/J ,;, 4

-:olc~;«ions bp,,ij /:';if'!/{.,?,,,.f}~/,.:i/7q Sta:i.da:d ,./. I Ocviat!.on -3 3 7

Strdn, T/i 'f :i Rever:.ants /7 17 /G /7 O;itc Plate<!, S"-/::,":. 79 per /8 I? 17 /~

l:tc,. of C'lll3 5ec~~d: / .. 3' xl08

Pl,1te ,x~ /.:,- 16 17 I :•:ie:t.lbolic Activation: J '"J1 e s=-.v-1'1 d Ave:D.ged

:? I I I{,, IS° //,,,, d co~:1te~ oy, P. ..r, :, • ~Pvr.rto=its 17 c,lcuiations by, h' ,{-':,.(e,l!t.,at-' ,,p.:,/79 Standa:-~

O~vi4t'!.on 'f I ? I ~

•

ro1:::i. No. 1-1!.-ln"

,~_)

- ..

So - /Ol doO ~ .Oos~ Ra.r.9e

Conccnt=<!t:ion

/()00 -,=- ~ I';', CtJO --<?<.1 .:2.S- .:2 C. I ;)._ 9 o< -1/ ~o /(. J.3 ~'f

I I

;

-< l, J 4 o22

b I 3·

/ '-/ /7 g

17 I '1 /0 I 16 /( &

Jf,., /&, 9 . .J..... ~ _.,

Ir.·

I V> I

.}

\~· '<,·

i. '.{,

,.

i-·;,.;.: ·; is/>; r f;,~..-r•(

}!Ff

j\_ I

"" "' -o (1)

'° 0

""' N

°'

'--

'" I=G,G MASON ReSeARCH INSTITIJTe TABLE 3

SAU:ON:::LLA MUTACENZSIS ASSl\'i

011..- 5""6 3- t,t,, 5- L ,f ~ ,,_.,✓,,,,,,,, C'rysfq11 :,1c.-

t:.•"" efz~·,.-11:.t!.t:.- 1£{!,:i. <!,,,1,!_ r;x_~,d0i2. .St.ui!y ~u.~cr Test Article Identification

Solvent D/1')50 ,,,..~,.JJ _,3Q_ ..Lro

:;:=ab: "T/J !00 .Rcv!!:rta:it.s I .:J /a I'/-/ J,.;J,,Y ~.:. ?latec: ,2·12-79 per ~o. o! Cells Sec~ed: !.S x10 8

Plate 101 /,~ .3 I I l/--

:-:e:a!>olic llctiv•tion: J?/!r 1,. r vEP /,;i. 1- I :Zn /.::i :1, 4'-J.J-7'1

J-1 ""c( Ccun :•:<! ':J'l: r! :r. [?, ,,, J " A.vc=aqcd //9 I:/ I Revc:-t.~:i.t.s /-;:?.()

-::•!cd•~ioas by' if /Z,•1e.tu~l! .:,-/2~ /., 1 Stl?.nCa:d q Cevlatio:, 10 ')

S":.:-~in: --;-.4 ro.? Rcvertant:s I :,:j i57 /,;l-0

O•t•· ?la:cc:_£:Lj°"..:.1.f_ per 1</- I 135 1~7 8

~o. o! Ccll.s s~eded: t•,'l xlO Pl.J.te ;a.. 'I

~:etabolic Activai:.io:1: ,',/(},<.JI" .. 171 /(,;:;).

~ -,J,r?r i, Ave:aqed ;--/ ,r-nt,{ co~ntcd by, /J..7, l?11~ >h J",'() ,,, ,, 15 3 Revert.a:, ts

Calcula:ion• by, j1/ JPu;, J:t_,,.fJ_,r-/:,,~/7<? Sta:,dar~ ;f{ /.:}-, Devia.tio:, /o{

,F~~ ~o. h'!..-105 r .,-.-

!f (_. --

30- 10. QQQ e12 .::>osc P . .cngc

1

Cor.cent:atio:'l

~ !COO ~ 1aooo 1

/_r.;"t} /~/ I tt..1- L '.:33

I It'! _97 /::'{,.:} /,:J 7

I;;,. I I.~ ;r' ;.;i.9 /() 7

!35 II Q I~."> I ,3,. cl

ol.3 I 9 10 ,~-/33 /{-CJ I 19 I/(.. . ;S1- 170 1-~~ J/5"

,sY J_tf_ 7 !3'6 I I f./-

-;,,,_y /5.;). I =3 (') //5"

1.3 ;/2 /() I

-

.

jJ

I C, l -0

'" <O (0

..... 0

0 ..... N

"'

011 - 5"(, J- IC.S-- I

S:t1.:c!y Nu.~er

st"in, p11rrr.. O<tc ?lo~e~:S-•l.f;-79'·

ro. of Cc~ ls Socdec, /, / ><108

:-:e:o:,ouc ,.c~ivation, R~t J..,v.:r .r-;u-7 'l

1/q ad Cour.~oC:. ~:/1 ?. .:r, .,., ·· • J • i:,:c~ia:!oos by: £,' 11,, >//, ,,Jl ,/4;,/2,;

'

s:rain:01£3~

O~tc ?l;,.:ed: S'°-($-- ?7 ::-:,. o! C•!l!:. 5ec2.cc!: /,/ Y.108

~te.t.a.~olic l'-ctiva:.io-n: ,110 .. ,..

1,'I g lj J .;-~r?Y ff,,

co·,mte:c! byr >!J., ;iul'>.

Calcul>tlons by,:d._ /-f,:,;,i.',,~,,1' -/,.~/-,<;

ror:'I\ ~o. tn .. -105 S/79

'

-------·

Jlc,_EGcG MASON FU!:ScARCH INSTITUTIZ" TABLE 4

SALMO~~LLA M~TAGE~ESIS ASSAY

Ji_

If(. f c: no, (. C r-ys 1'4. ///11 <: .30- ro, oao 1.. ct l!' 7 3 s-- &.-1. P -1 Fo:d, i:: ad,.. "'S' r; -'I.). • o .,..t,-,v.:::

7 Test .:\rticlc Idcnti!1<:atlo:i . Dose Ri.r.i;-e

Co:iec:icration

Solvent J;N.f Ci 3<t /00 .330 · ~ <::Clo .5 300 1"0,000 /o~•~ :::-<..::...:.. --

Rcvert.Ant:r II /I /0 /-1/ /-1/ '7 s-p•- IJ.. I)._ 7 -- /t, '7 /0 7

Plate /0 16 1.r 8 7 /0 ;t,

"vc:.1gcd R~·,c.:-tcnf;.5 I.:? q /;)__, If /f'l Cf 9

s:zinCa:-d ;;: ·3 1-/-Deviation .3 .:2. :2... (o .

Re.vertants If ,2;? .)"/ 31 .::! 3 33 /3' per 3S- J._ 1 3J. <-< f' ;)_ LI I ~} 13

Plate 3,2. .)_ / /6 ..]{, «-1 o{ 0 I 17 .F.Ve:'Aged ,q ,.,,.. 24- ?r5 ,21.l -">.<;°' I LJ. . Re.vertt\n.ts S~andar~ q :? g (~ I 7 _,

n ... vi .. ,t:ion l

j_l

,.,, --.J 0, I <.O

(0

~ 0 ..,, N

°'

tJI"(- S-G 3-/ t:.s-/

s tucy l<=er

:Strain, T(! t:,:3?

~H• naeec: ;:[-1:r:- 7'1 1:0. of Cells SecCcd: o. 7 xlo9

::ct.l!:!olic Activation: 1?,cf- J..i,,er

. ll<rnt{ ,r-)./-l'fi,

Coun tcd l>y: />. J, fJ . .u,z.,.

,:1alc~i•t1ons l:>y: ,4 I'.,; fin ,fl, .f(/).:;/29 \ I .

!i~r,in: Td IS° 37 :,heo na«c, s--1s--=? r

I ? a :•r• of Cells Seccd: . 0. xlO

:-:i;:taOolit;: t\CtiV.ltion: i./rJ>l e I r-.:i..1-'lr L /fgn c( Cocnted cy, ?. .r. 8 --~

C4lcu1Hions l>y: ,Y A'f!i/,,el ~-,/2_;/7(i . .

~

l ~i~ :;o.

W!.-105

f,,1'GcG MASON RESEARCH INSTITUTE:'

TABLE 5 SALMON~LLA MUTAGENESIS ASSAY

r<ercno>i <:!. Cryft,,,///,1e

J..o-t/F'7..li= ;?di'- IS'a~ C"'k""'if'.6 </.) oa Te3t Article Identific~tion

Solvent ~MSb ;,•o~ 30 L£Q. 330

.

Reve.rtllnt3 G CJ ? c; per g 7 10 /3

Plate

"I ~ g 3 Avc:-.1qcd

:t~vcrt~r.ts C. 7 c; //) St~:1c!a:-d

Devia::.io:i. .,

<><- I • --:j

Revert.ant,: (, g 7 g. per /0. 9 b /()

Pl~to 8 7 /0 /0

A.ve:aged g R 'x q Revertn~ts Stand.J.rd

,;J_ Df!viacion I ;l.., {.

,I': 'll_,

30 -/o,,ooa ..-<.L<:1-I

.Oose Ra:i.9c

Concent::-.1tion

1,000 3j.?oo /01 00:J --/o 7 ~ ~ '7 1'

i 1 s-

<l' 7 s ,2, :? I

9 7 7 7 b b

7 ? ~

<l 7 s I I I 3

I 00 I

11

"Cl ,,, "" ro ,_. N

0

"" N Cf'

·'-...--·"

!~ J:G,a MASON RESeARCH INSTITUTE,

TABLE 6 SALMON~LLA MUTAGENESIS ASSAY

Kere1101e C r7J r-,,_ //,i-t <.

~t _,•

0/'i-S-'-s- I C.S--! ,1,.t II" 7 3S"-R,,f?-IS-a;, ( c,{~¢,S,G '1/ d_oO 3d - /&, (lOO ~~ Stu~y Num!ler

itnin, T/l IS-3 8 tl&te l>lAted, ,')-/,- 2 '7 ~.'.o .. of Cells Sected:- I,,; xlOS

i:eta:iolic J\ctiv•tio~, xa -f- J.ivcr s--.V-71

a~,,,( Coun~cd l>y< P.,T. "' • J/., C>lc~l>t!ons by: .,6 #A_,;,__/1,,_ ,_/) _,,.j,~/7t:;

/ -

Strain, T,1/_;Jg o,co ?late,, $"-/,£- 7f ~:o. o! Cell.s Se,;:ded: I,'-/ xlOa

t-~etabolic P..ctivation: Noll.,: s-:u-7? ;J,

l:t:4._11.J__ Co\!ntcd by 1 'D • ..r, ? ..... •

C.ilct:1ations by: d. f.'r-;:,,/.JJ.rJ:S/2dh'7

Fon i:o. WL-lOS 5/79

,

Test ~rticlc !dcnti!icatiofi

Solve-::\t J:,NJd

.2Q_ .LQQ_ /0'1 ,,,J

RevertAnt~ 17 ~J -< 3 per ~o 19 19

Place

3(, J..( ,:Z? Avc.:-.1g~!d

,;,. 1,/. ,,)._/ -?3 R>!vcrte:it:s S t~nCa.:-d

<f-Oe-viatio~ /{) ~

Ravertants /9 13 /(, per //) ~J_ /6

Plat:c 13 -=<J. /0 Averaged

I+- tCl tlL R.evcrta.~ts Standa.rd

< I < .1 ncvi4t1on

.Oosc R.!.::.ge

Co:iee:ntrAtion

330 - ;~ 3,~dO /c1 acc 1 __

o< 3 ~7 ,< J./ I/ I -I

:2.1 ..Z? I g I 7 cl;l - dJ /I) .,zo

_:) ::i. .,)S 17 /7

I t/- 7 .-<. /C, /?, C /~ IS- /';l /0 g

/,r' /{, ,',S- 'I

,s- !, n•, II I I . ,< t.1-

I

I .

I \0

I

II .JI:

-0 0,

'"' ID

.... w 0 -s

'" en

I aIq-se,3-10s -1

S tuC.y ::'.!..-:-~~r

D~:e S::.-ain Ch-::::i.ieal

I I .t;. IS· 79 7;\~~ 2-l-..., !.:\Oa:'l t!-:!'l:C"?:'l~

Is.;_,,. 7q T:,:e l 2-rH-:.=-o!luo:enc

5 .;.:,. 7q :'A!OO 2-Ar.lir.cc~~~r~c~n~

t~~OO I !.l-?rooooo Sultor.e

[s./s · Z'Z 7,\,5;5 I !.l-?roe,ne s•,l~ene

Ls. ,5 ,7 91 T:.15]7 9->.:n!r.oacrlcir.e

5.15. 79 7;,1518

.:'O!":J :-:o. ;,;!..-105 ':,/7 £'

::-:ii': :-of'! \!O':cn~

I

L'

' 1 ~ 4•·

··---·

,:.~ GGcG .MASCN F/ESEARCH INSTITUTE

SJ\L~:C~ZLLA t,::.:tAGENZSIS rtSS.A.1

'

Po3i~ivc Co~t=ols

'!'ABLE_ 7 tfe tow,,,r:: C,y::, >¼ / l,ne.. c1c1e :r. ,rc,4.;,oc, Lor:# 73:S-~AfJ-15-Z).,:,__;

~est ~=t~clc Ic!cnti!icatio~

Conce:1tr4tion Hetol.bolic J\vc:-ac;cc! I cvc:-t.:.:'1-:.~

?er plate Aetiv.it!o:,, Rcvc:~ants/?late ~ l~ s,cr P.:..1-:-:: \ S.O.

Rat 1.0 :,g Liv-::!'

10.0 uq NO:'J.C

R>t 1.0 ug L~vc:

o. 04 µl None

0. O< u l :-lone

75 µg t-:one

10.0 '.:g Nor.~

,--:i,;,..< 1 LU -, • I II- .1-'.? I l/- '.? 0 I 12fl 1033

I 10,,t.q 1117 / ✓-l(,,, ,,_ -st,,- I

c2 .3.;i~ I ..2 v&,l, .:; ., ..,,,_ ,c / 7 j:, 7 51 qc;<;I 11¥-0 I/(,, .'s 1100 ~ IOYi:o I <iS3 977 Jrlrl-'\ 71,

S11 7 :;-3 /'., -;:i I 7~.:;, ' Cj(,,

't.-'.3 q < / I., Oq 1039 1-'f 7

I

-

t!lr''HIN( Co-.:ntcc! b;r: _// ;/(/.c;.:..V ~/-,,j,, ca_ .il~.tions br: ,,./l@Ua.ufl C/:;,._'J/7 q

_JI

I -,, ,...,,, QCl ,ro ., ,~

C, -~

"" en

QI~- 51--2· ll.S-l Stu(!y ~ur.-.!>er

3t=.:i:.i~ :rn '1 s ::ia:c ?l.::~<!: Gl:i!J :J ::~. o! C~lls SccC:cd: (.::,.. x108

:.!c:~olic Ac:.iv,1:ior.: R,+-1 · ""'

1-\-,md ·:J Cou:,tc-:1 by: at-.. ,. • ,_.., ~•!cuiations by, J../. fir1cJa~c;{J 1.,/;.;:,./7'1

, .

s::.:!n: Jil:::l s o.i::c Pla:.eC: GlsjJj

:--:o; o[ Cells Secaded: , • :i.. xlO 8

:-:c:.z.!:iol!.c Act:.i.·.:t:.o~:· Nt.ot

H-~,r"'i~ Coun ~•~ by, C \-1- :..;.J ,. I .. I~-.

calculations by: i/ l(e/c~tudJ (;.//;J./79

ro=. No. ;,::.,-!OS s;;9

·,...._,.

/::,.E!GcG MASON R!2SeARCH INSTITUTE'

TABLE 8 SALMON!::LLl\ HUTAG!::NESIS ASSAY

- L.t • ·ns.· R.nP- 150.:t.

Ber=~;!. ~,,. ,~,k, Q...r),., t:1 !u:'l:2..co .., Te~t ;\rttele Ident1(1c.o.tion

Solvent

loo.ii.. :t'.>rr,~O

.. \o.c ~ ~

rlc\·e:-~ants 1 li .2.o ,"Cl :l.;:,.

per :.,.o :l:l.. ll 17

?l.: tc

~=!I . ::>. '-l ll, :i.s

A•,c=aqcd -<.3 ~.l. _, I

R'?.VC:"t.l~t~ c<o<. StanC.a=d

7 'J Oc\•fa::!on .,1.. 'I-

Rcvcrtants :J. "l 11. Ii I'?

per ·:to .'x' .?."i .11 · .?la:.c

H J'I 1, :J.5

Ave::-.ag~d ,q II I q '.? I A.ever:a:'lts Standard

If. .3 -,L 1-Oevi~1tion

tt .. __ ..

~-0 ~ lo 1ooc..o -.. (I

.Cose R.o.ngc

Co:,ccntration

I oco.o ~o /~O --.:ti ::i. S' 8

"I IC/ ,,.. .,.1 -i.:; I >-/

.~5"' ;2.3 1..3

- .s-. (,, ..:,

.::it. ::i 0 J 'l

:l,i 13 '" IG ..1, I .J

,;i_ I 1? II~

.< .If- :z

11

I ,_. -,::, 0, ,_.

<O I (D

,_, "' 0 -,, N 01

QI'.'!- St; ·;- 11,5-1 St.1.:c!y N1.!.·:,,!:u:r

:;t:ain: ::mioo -,.,. ?l<tec,_u~;IJj

::o. o! Cells Sc~l~d: /.3 xl08

~!c-;.~ol !.c Acti va ti.on• p,-.i.. L' .• ~

\.J:0,nc\ Cocn:oc by: <::J.:.,,/ ,., .. ,,~ • ji_l'_.,:/- ✓ - Il/4/11/7q C.l:c\!.1.atior.3 by: . I.:?. . .

Strain: --n,. 100 ,-

Doto Platec,_.1.:._1.~)J~

~o. o! Cells s~~~ed: ,, ~ xl08

~:c:a!:lol1c Act!.vat!.o:i: Ne-•

l:l--ol Co•on<cd by,<-.:_.]_.:.;./ ,_,.,, -~ ::.lculations by,/./. / r.,";{f -,,d) t,,f'l-;,../7 q

r·on. ~o. h'L-105 li/:CI

l,,t=G,a MASON RESi:ARCH JNSTITVTe TABLE 9

Sl\r,KONELLi\ XUTAGE:lESIS ASSAY

ii •.__..•

L .. "1,, 715 ~ r-.A r. 1 sc~ R;t«:,·,p< C r,~·,\-. .. \\·,,.,s.. s:,;J, " '.\u•-n.cc 3<:::i.o ~Io. oo<>, o --..

0

Tc5: Article Idc,iti!ic.ition .Cose R4ngc

Cor.ccntr.:itio:,,

Solvent ICO-l ~ ~ ;t),l"l~O ~ ,,cc: . ..: 3 "}CO.O /~c

Rcv~:-tant5 111 95 'i'S' Jo:,. JC/ to.3 (o~

per

Plate 9 l, "r--1 103 113 lo;> ,,s '?9

11-, lc9 99 11 ~ lo, 9o "11 .A.vcr.:i.gcd

/{)(~ qq 93 Ill lrJ6 /() :? 'i'.3 R'!:vcrtant5 St~:'lc!a:-d

'l OcviatiO!"I q 3' g 2, 1..3 I <J·

Rcvertant3 lo I Ill, 11--1 I :i. \, l::i. 'j 100 I c,., per 117 1,~ Jc, !OS 1-:i.?; lJA fog

Pl.l.tc l Jio I ::i.9 J ::i.~ I Jlo 131 ,~q /07

Ave=uged I() q I I I., tit_. ;;;..9 10 7 /t')7 Rc.v~rta:1t:s I:, I su.ndzrd I

D~vi.3t:.ion \s 7 fl II o<..> ,_ I

--

. I

I

I

.!\ I I

l

I (.

!

.,, 0,

I "" ro ,_. N .... I 0)

0 -,,

N 0)

'-_..·

j_,,12GoG MASON R!!:SE:ARCH INST/TUTe· TABLE 10

SALMOll!::LLA 11UTAGEllESIS ASSAY

LT .. 735- R~r- ,sc.-2

1[ -· -~

O\:J-f)L,3-1\::5-{ Rs.i:5:nc'nt (c:.r;,.&:mt ~-,\1 .... 3b':11S:Q 3o,c-'>lo,m,c ..... e;. . '

S~\!C.:r ~Ut:l!)cr Test Article Identification .Dos-: Range

.

Conccnt::-ation

Solvent i::oJ. .:i,n,~ ~ ~ 3·1c.o (coo.a ~ le coo.o __

:;:=do, ]l'r 15-l.S l\e•,ertMts 11 I '-f "I 11 5 ~ 8 Ja:, Pla~•c:_J,IS/J2 per I

::a. o! Coli. sccced, f,7 x10 8 Plate G Ii '-I It., 11 I/ Lt

:·:e:uol:c Ac:ivation: Q,,t L·,.,u- IC. q :to lo le 5 re I ...---, •1 '..i/ Avc=oocd . \+,- n•l Co~n.cc b,: -.....,/... ,. , .. I,-, ~e,•e=t~nts / / 1 I I I I.::? 9 J? 7

:a:cciuions br: // f',;J1{,,.£J /4/,~/7C/ St•n~a=d ,,. .., 0 ~ ..., . ' Oc\·1at!o:t :") '1 l) V 3 ..:3 .. ~

s:rdn::rf\1S3.5 Revertants :l.7 .ll ::J.5 .:l..l I~ -11 I:,..

o,:, na:ec:_J,i.SIJ5 8

per ::l.3 10 11 If ;:i_ 7 ;:i:,_ 17 !:o. of Cells Sc-:t:!ed: J.J xlO Plo1tc

~:c:abolic Ac:iva:ion: j\).,, 11 , J.c l'l I '3 ::l.l 19 .:l.o "I

/'l J . ;! ;,.ve=a<;~d r/ · . 3 l:h-ocl Cocnted by:<..! .... .:./.'I ,,1,-1~-; ~cvereo~ts ..J..3 d).t1 I if .;io di I c". ! I

Calcda:io~sby,,A.'.i'-}i,/,7,,/l;:,/,.;z/7; S~o_r:d•=c ,JI / 7 , _,,.- / 1- J Dcv1~tion -, . e,.(J 0

Fon ~o. h'L-10S

I I-' w I

'lLI Ii .

.. ' ·'

-0 0.,

<Cl ro ..., _, 0 ~, N

°'

019- St,3- l!,S-1 S:uCy t❖.1.":'ll:)cr

3~=.ain: :rn: 1~,:;7 ~•« na:ec:_LJS/ 77 ::o. o! Ccl~.s SccC'1d: ,,.5 xl08

:-:~:~olic ·Ac':.i'\·atio:1: O,ct- l · -•u-

,, . ; \·bo.J coun:oc b:t: Cd,.::+t ,.luh~,

!:a:c~~•tcons !>y: ,L/ f', i/..lt> ,,r} l.fl::J.f7q '

s:r~i~: 1B 1.537 0,):~ ?1.-:':.c_C: I· !f;IJ~

xl08 ~o. o! Cells Sc~Ced: j,S

.:-:ctabol!.c Ac::lvat!.o:i: · 11c-•

Co~~:cc by,(""' ,f_ • ,-! ,. I .t~r, 't:b.::iA C•lc'-llacion• !>y: ,l(j' ;((1./.Lv.c{/ r;h,;i/-;,

,

ton N'o. h"L-105

,J11 ··--~- ~.

J'_,,l=Gca MASON R!:SZARCH INSTITUTE. TABLE 11

SALMON~LLA XUTAGENESIS ASSAY

l..;,i- - 7.'.JS • P<:l\fl. 1,0.:,_

Rm=0t C,-~._+,,\\'.oS' (1.-rh A 'i.'J-/4J..cc .3010-::> ,0,000.0 .... j·

':'est 1\rticle Id.cnt.i!ic.c:.tion .Dos~ Range

Cc-ne-:::'l:r.a:io:'\

Solvc:\t loo-q_ ~ 3::;c.c ·.uu.._o

"J::in1~0 ,~ 1,c"'r,.o ~-0 ---Rcve=tnnt3 (,, 10 7 (, ~ s y

pe_r

Pl.1tc /0 q ..., 7 7 ~ {.

'.i' t .. '-i s '-I I"- ,1

Avc:.igcd R~vc:-::ant.s f 'x (P (, & 7 4'.

S!~:'ICa!'d d.., I ~evia::ion ,;:), ~ I .;J ,_/.. -<J .

.1 '-{ lo 7 ' s -!\eve rt.ants

per 7 <l' lo Si' -~ c:: , Plate

l? .., (o c; 9 ,::: 9 Ave=aged

G, s L -

7 t:... r <,, Rcv~rtzi:1ts Sta~da::d

..3 «!., Deviation 0 ,;? ' '-:i {) Lf

I ,_. A I

ll

0/"J.- S,~3-/<.S--/

Stu".:)' th: .. -:-.ber

:J.:.: C s::-a.in Chc:nico.l

i &·!J-·7--> 7,\C)~ l '--A.~i:ioar.t~=a~~nc

" ":'i\~8 2-~ i :=ofluor~nc

"' '° ro

~/4-~-7<)

. .I< .... 7 <)

I~~-~- 7., 7,\10() 2-~~i~o~~:hr~cc~~

,_. 0:,

0 -t,

N er,

':'.t..100 \ 1. J-r':-coane S-.:1 tone

lt:.-s--,, T,\15~5 ! 1 J-?:-o~~ne S•.ll::one

.. ~,. ,-. 7 ., ':"AISJ7

e,.,;-.7-, 7111 S )8

Fo:~ Xe. XL-lOG SIi~

9-1\.~ino~c=ldinc

~-N i::rofluo:-enc I

·-

,:,°-,, IEG:G 0

MASCN RE:S£ARCH INST/TUT£

SllLHO!:E:LLl\ 1-\UTAGE:NZSIS ASSAY

Pozitivc Co~t~ols

,. ~~

1 .. ·:,II! lJ}

lot' iZ,S• Rfh-,,_ l!ioo..'.2. c...:..clc.. -c- S-e;.'f:t....:i 0

,,P ,£. < ~ ,£.t' ,</ £ /' 0::1'.~ 2/.!:6 "-~c£.E

7cs~ A=~iclc I~cn~i!i~a~ion

Avc=a9ed. } Conccn::r4::ion Mcto.bolic Rcvcrt.:i.n:,

pc: plate Ac:iv.,,::ion Rcvcr::an:s/?l~tc per Pla:c ( s.o.

Ra.!. 1<t1 Q I 10.-<: LC ~g Live:- /:5"""7-r' /JS7 /S<-S-

10.0 Ug ~one //(. - /J7S" /.S-/.S- I~,< I 17 7 Rat

l.O Ug Liver /.307 ///S ,,..,,,. 79 1300 I% ,,)J

0.04 Ul ?-:one //.,,.,,. /,3 ~---, f/&-J I , ;;i l.f-7 1:l.O

o.o.; l.11 - None /J7o /,Ls/ /-1 J ,, !3'-1-q IOI

75 Ug None /~ .,,-e, /7S" /C, ..7- ,<"Li. :? ,.)65"

10:0 ug Mon~

l

- I

I I I

I

'{"&(/,</-c" Ccunt~c! by: p/.lcti~ w( ";l'//?'7 Cal cul a tb:is ~y: _j!, ,(,,l,ie-/ut.<.Jl /_ // ., /-,o

1ij

"'Cl 0,

<O I 11>

..... ,_. V\ '° 'o .....

N

°'

·~----

f_,, E2G=G MASCfV R!::SlaARCH INSTITUTE TABLE 13

SALXON~LLA Y.UTAGENZSIS ASSAY

Lci't ~ t3C- IUtP- JS',)"l..

olj- $L,3- /GS-I l3t.tl1''i",C( c ''1:t,.\\·."' c~,.\7 .IS ':fb'-f~c.,o

. S:·a~y l<o:'"'c= Test ~r:.icle Identificntlon

Solvent 1c0 .J,..

-~-0 1=i:l_ -:i>f'I'\,; 0

3t:a!.~~ :::te:15:1} Rcvert.o.nts .2 I .'l:J. .:> I

~a.:e ?la:.cC: !·P:1.b2 per ~o. o! Cells Sec~~~: I. I xl0 8

Plate ::i..s f-; )1

:-:ctUolic >.c-:.!...-at!.on: ~",. I ... ~ :.t I ;l..,, ,:, ....l:J:,:.,:iL Coun:cd ~y, ,,J.,;::J ,. l:,c,ho

Avc:.:?.ge:d :'.2 J. . ;)... j '.2 7 R,~VCt"tl!'nt!'I

-:a!cciations br:/J. C.~?-?"'P---- t: /2c/)..- St~:tC.1::-d )..

I 3 I, ,,, /

, !:)t?viation I

s-:.r<!in: TI 1SJS Rcvc::-tants l'i' I lo ,, OJ.':c:: ?la:.ed: ~ 11~/J'.] per 1.3 IS '{ ~o. of Cells SccCcd: I. I x10

8 Pl.itc

:-:cta~ollc >.ctivatio:i: !:,la o !.c I c; IS I'<

co~nted by, <, f - / Gf:,cl= >.v-:=~ged -\·bed !l~V'!!rtA:"lt.3 /j !5' I 1

0 (/ ,/, Standard -calculations by: ( G"?-,~,,__" ·~/- ~ I ., , , . Dcvi,3tfon ~

fo::r. ~~o. h'L-105

Jt

.':o.c--=> fo,oco.c, "l .Dose Ra:i.gc

Conccn:ratio:'l

.7.7c. c-- 1,oc:C 0 t.! ~ ,c,cc.o.o ---·J. '.l IC • ;ll, ?.O

::w ::r.S .'J.9 \li' I )l, :i.c ::i. \., 9 I

1 ,,- 1.0 :J. 7 /6 3 6 ;i_ /.,

19 :t.:, 17 I '"1

I c; 1-1 ]'-{ 17

\\, I ", '.),_ 13 . 17 If, I 7 I~-

1 3 .3 '].. I

.-,:, 0,

'° ro

N ,.... 0 C\

0 I ..., N

"'

iU, ) I I

i: I

• i I .

I !

r I

i I

:>z.:.e

1-11~/,~

_ _r,1,q);q

Ql:l- SL·l-it.S-1 St\.!C:y ::1.:.-:-::icr

s::.:-a!.n Che::,ical

:,•-.91? 2-;...~!:-ioun:.h:-ac~:-ie

7A"?U 2-:: !. ::-o!luo:-cne

7AlCO 2->..~i:"lo~:"lth:-~cen~

';'.';..loo · I 1. J-?:--::oa:,"! Sul:.6:,.-:

7,\!.S) S l 1 !-P:-oo!.r.e Sl:l :.on~

I I 7).!.5J7 1:-,\::-:~:-io.:c:-!Cinc

.-.. -.!.sJe l 2-:Ht::ofluorcn'!

I I

I t·o=ra ~o- ~~-le~ ~/7~

,_ ' l'ij' iJJi -

,:.'::, lEGcG .MASCN RE:SE:ARCH /NST/TUT!r

s11 .. no1::a:LL~. M:JTAGENZSIS ASSAY

.

Positive Co~t=ols

TABLE 14

Co:i.ccn:.rA':.ion x~:.~olic ?er ;:>lace J\ctivatio:,

Ra':. l.O ~a :..ivct"

10 .o uq :-:c-nc Ra::.

1.0 UQ Liver

0.04 Ul Kone

0 .0( ',Jl ~one

75 U<; }:o:,e

10.0 Ug N'On'!

L..-t.,. 13,5- ~fip .. ISc.':l.

R~t"sx:is:JJs: ~ ~" ·::t-:,.\l ·, o• CcA1 " y.:.::f::::ioo •

Test A=:iclc I~cn:ific~:.io:,

A.vc=.:;c-:! I P,cvcr:..in:.s ,

Rcvcrt~:its/?late pc·c Pl>to j s .~.

I I I

_",t., 7 S' 3'.ioo .'.><\CO ?701 I ', , !

I I 995 I .,s '1:1.:3 '1 ? I I; o

-

' LJ rn-, .. ):i,~l a 1-...:....L.. ,_, ,, ,.,~ dcda tio::-.s by: y . (?-z:,:;'--t.-T"'-- ~/,,. ( /7 '7

(, )

-·- ( ~

)

Conclusions

The rcsul ts of the Sa lmone l ln/mrunmu lia n-microsome plu tc

·incorporation mutagcnicity assays indicate that FWS test

article netenonc Crystc1llinc Code aB64200 (MRI llGS) did not

cause a significant increase in the number of revertants per

plate of any of the tester strains neither with nor without

metabolic activation by J\roclor 1254 induced rat liver

microsomcs.

In the exp er i"mcnt of G/5/7 9, the TJ\15 3 8 cul tur-c was

grossly contaminated . The str-ain was retested on 6/19/79.

--- -~. - ... --·- .. ... ~•---'-·•· .. -·- ,---~~-..,,,·-· - .. _ -:-.__.:---.:·~·•---'-

Page 2 i of 26

- 17 -

)

APPENDIX

Page 22 of 26 -18-

)

Media

EGE, G M/\SON RE::;E/\HCII HJ!;TJ. TUTE Protocol for S,1lmo11·cl lu/t1u111111<1liun Microsome

Plate Incorporation Mutugcncsis /\ssay

Top agar is initially prepared with 8 g/liter Difeo nacto

Agar and S g/liter NaCl. After autoclaving, the molten agar

is distributed in 100 ml aliquots into sterile bottles where

it is stored at room temperuture. Immediately before its use

in the mutugenesis assay, the top agar is melted and suppl~­

mented with 10 ml/100 ml agar of a sterile solution containing

0. Sm11 L-histidine and 0. Smf.l biotin (lXSA). Twenty-'five ml of

sterile deionized water is added per 100 ml top aga'r when it

is used in assays without metabolic activation. This insures

that final top agar and amino acid supplement concentrations

are the same on plates with or without metabolic activation.

-l0XSA supplement contains S.0mM L-histidine and 0.SmM

biotin.

Bottom agur is the Vogel-Bonner minimal medium E described

by /\mes.

Nutrient broth used for growing ov~rnight cultures of the

tester strains contains 25g per liter of Nutrient Droth No. 2

(Oxoid) .

Storage and Preparation of Tester Strains

All tester strains are stored in liquid nitrogen, and

fresh cultures are inoculated directly from these frozen

stocks. Broth cultures are grown overnight at 37°c with

shaking. If necessary, the cultures are then centrifuged and

resuspended in their culture me<li11m to give an appropriate

final cell concentration. Each culture is routinely checked

for crystal violet sensitivity and nmpicilli!l resistance·

before use. in the mutagenesis assay.

Page 23 of 26

-1~-

)

Toxicity Determination of Test Articles

Each test article is checked for toxicity to the tester

strains up to a concentration of 10 mg/plate for solids and

up to 10 µ1/plate in the case of liquids. Eight serial half­

log dilutions of the test article are plated with TAlOO on

minimal agar plus lOXSA and on minimal ag~r plus lXSA. Equal

numbers of cells are seeded on each plate in the presence of

th_e test article. The percent survival of an appropriatel;zr

diluted TAlOO culture on tl1c lOXSA suppleme11tcd plates is

determined by comparing numbers of colonies on the solvent

control with those on the plates containing test article.

Toxicity on the lXSA supplemented plates is detectable by

a decrease in the number of revertant colonies occurcing per

plate and by a thinning or disappearance of the background

bacterial lawn. The highest concentration of test article

~sed in the broad range mutagenesis assay is that which gives

a detectable reduction of spontaneous revertants on the lXSA

supplemented plates. The results of the preliminary toxicity

study are reccrded on Form No. l'IL·-107.

Plating Procedures for Mutagenesis Plate Incorporation Assay

Routinely, the test article is prepared immediately before

its use in the mutagcnesis assay. Six doses of the test article

are first plated with all five tester strains (TA98, TAlQO,

TAlSJS, TA1537, TA1538) with metabolic activation, after.which

they are immediately plated on all tester strains without

metabolic activation. All positive controls, solvent controls,

and test article dilutions are plated in triplicate. With-

out metabolic activation, SO µl of tester strain and SO µl

of solvent or test article are added to 2.5 ml of-molten top 0 agar at 45 C. With metabolic activation, 50 µl of tester

strain, 50 111 of solvent or test article, and o. S ml of S-9

mix arc added to 2.0 of molten top agar at ~s 0 c. To achieve

the desired maximum concentration per pl.ate of the test article

it is sometimes necessary to plate 200 ul or 100 ul aliquots

Page 24 of 26

-20-

. ( 7·

)

·-~

instcu<l of the usuu.l 50 . µl aliquots. J\ppl."opriate r;olvcnt

controlr; ure included in these instances. /\[ter vortexing,

the mixture is poured onto the surface of 25 ml of bottom

agar contained in a 15>:100 mm plastic petri dish.

Positive Controls

Positive controls arc run \·1ith cu.ch tester strain. ·

All combinations of positive controls und tester strains

arc listed on Form No. WL-lOG .

Tester Strain Titers ·

Tester strain titers are determined by viable count

assays on lOXS/\ supplemented minimal. agar plates., The number

of cells plated per plate is reported on Form No. WL-105.

Preparation and Storage of Liver Microsomal Enzymes

Liver microsomal enzymes are routinely prepared from

male Sprague-Dawley rats that have been injected with Aroclor

125~ at 500 mg/kg. The Aroclor is diluted in corn oil to a

concentration of 200 m~/ml. Five d~ys after their i,?, injc~tion

with the /\roclor, the rats arc sacrificed by decapitation,

and their livers are excised. The rats are denied access to

food for 12 hours immediately preceding sacrifice.

The preparation of the microsomal enzyme fraction is

carried out with sterile glassware and solutions at 0-~0 c.

The liver from each rat is exciied and placed in 20 ml of

O.lSM KCl contained in a pre- weighed beaker. After weighing

the liver, it .is transferred to another beaker containing

3 volumes of O.lSM KCl (3ml/g of wet liver) where it is

minced w:ith sterile scissors. The minced liver is homogenized

in a Potter-Elvehjcn apparatus with a teflon pestle. The

homogenate is centrifug12d at 9000 x g [or 10 minutes in the

SS-J'1 rotor of a Sorvull SS - 3 centrifuge. The supernc\tant

(re(c.:rred to by /\mes as the S-9 fraction) is decunted, and

small aliquots arc distributed into freezing arnpul~s whic.:h are

sto~c<l in liquid nitrogen.

Page 25 of 26

- 21-

, ___ _:_ :-f ·,

One ml of the 1nicrosomal enzyme reaction mixture (S-9

mix) which is added to the soft agar overlay contains the

following components:

S-9 o.os ml

0. 4M l-tgC l 2 0.02 ml

1. 65M KCl 0.02 ml

0,04M N/\DP 0. 10 ml

o·. o s11 Glucose-6-phos~iate 0.10 ml

1. OOM llaH2PO,, pl! 7. 4 0.10 ml

II 20 0.61 ml

LOO ml

Evaluation of Mutagenesis /\ssay Data

For a test ~rticle to be considered positive, it must

cause at least a doubling in the observed revertants per plate

of at least one tester strain. This increase in revertants

per plate must be accompanied by a dose response to increasing

concentrations of the test article.

Pace 26 of 25 -22-