1110 

llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll • 

OTS0523257 

One Source. One Search. One Solution. 

MUTAGENICITY EVALUATION OF 421-328 (FINAL 

REPORT) WITH TEST DATA AND COVER LETTER 

08 MAR 1990 

U.S. Department of Commerce 

National Technical Information Service 
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U ..S .. DEPARTMENT OF COMMERCE 

Technology Administration 

National Technical Information Service 

Springfield, VA 22161 
L 

COOING FORM FOR SRC INDEXING REVISED 10115186 

MUTAGENICITY EVALIJI'\TIQH OF 421-328 <FINAL REPORT> WITH TEST 

DATA AND COVER LEITER 

" 

HEXABROMOCYCLODODECAIE 13194-55-6> 
c:onlains NG CPi 

·;' 

Cbemic:ll Corporation 

:·-. - ' 0 :J 

~0. DCIX22QO ~ HIGHWJ'-'15:H'w • ~SJLAFAY£i1'E,.IN·.Ut01i ~ PHONE.::Itl-1117-6100 • f'Aa.:~_ll"""lll~ ~ Tt1LX:2NJ«2l • eAbt.E:c;.t.MCH[Ut.N'A\'£TTE 

March 8, 1990 

Document Processing Center (TS-790) Room L-100, Office of Toxic substances 

Environmental Protection Agency' 401 M Street.; SW 

Washington, DC 20460 

Attn: 8 (d). Health & Safety Reporting (Notification/Reporting) 

Dear Sir: 

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In response to the TSCA Section 8 (d) rule published in the December 

12, 1989 Federal Register, the enclosed information_is b~~ng sub-

-mitted on the following substances: .. .--- -~ 

2, 4 , 6-Trib;·omophenol Tetrabromophthalic anhydride Hexabromocyclododecane 

1,2-Bis(tribromophenoxy)ethane 

Decabromodiphenyl ether 

Pentabromodiphenyl ether 

Octabromodiphenyl ether 

CAS Number 

118-79-6 

632-79"!1 

3194-_55-6 

37853-59-1 

1163-J.S-5 • ' 

32534-81-9 

32536-52-0 

The studies on the first four compounds are submitted on behalf of 

·:.> the followifl9 manufacturing facility: 

Great Lakes Chemical Corporation Route 7, Box 529 

El Dorado, AR 7l.730 

The studies on the last three compounds are submitted on behaif of 

the following manufacturing facility: 

Great Lakes Chemical Corporation Highway 15 south, p;o.Box 1878 

El Dorado, AR 71731-1878 .,, 

;::' 

' 

-.. ; 
There are no known additives to these compoundo:;. Pentabromodiphenyl et;her consists of hexa-, penta·-, tetra-bromodiphenyl ether. 

Octabromodiphenyl ether consists of nona-; o.,ta-,, hepta-, and hexabromodiphenyl ether. We are not aware of any ongoing studies with 

•any of the listed compounds. If you have any questions concerning 

this si!bmission, please call me at (317) 497-6391. 

Sincerely, 

Dennis 

»-!#•l-tV 

L. McFadden, 

£ J./o/" ~ 

Ph,.D. 

Product Safety Coordinator 

DLM:sb:030890 

Enclosures· 

,, 

l• 

" 
TEST DATA 

2,4.6-Tribromophenol 

Acut~ toxicity studies in rats and rabbits 

Primary skin irritation study in albino rabbits 

Eye irritation study in albino rabbits 

Acute inhalation toxicity .in th~;albino rat 

Acute dermal toxicity0in male and female albino rabbits 

Acute oral toxi~ity (LD50) study in albino rats 

Dermal sensitiz'ation study in 'the albino gui'nea pig 

Bioaccumulation study in fat tissue with 2,4,6~tribromophenol 

48-Hour dynamic aquatic toxicity study with 2,4,6-tribromophenol in Daphnia · , Four-day static aquatic toxicity studies with 2,4,6-tribromophenol in rainbow trout and bluegills 

Screening test for microbiological inhibition with 2,4,6-

tribromophenol Acute dust inhalation toxicity study with 2, 4, 6-tribromophenol, lot number 3106 in rats 

28-Day subacute dermal toxicity study with 2,4,6-tribromophenol in albino rabbits (10/27/77)" · 26-Day subacute dermal toxicity study wth 2,4,6-tribromophenol in albino rabbits (12/12/77) 

Pilot teratology study in rats 

Mutagenicity evaluation of 2,4,6-tribromophenol, l'6t. number 

3287, in the Ames Salmonella/Microsome Plate Test 

Acute oral toxicity (LDsal study in. rats . The bioaccumulation of 2,4,6-tribromophenol in bluegill 

sunfish 

2,4,6-Tribromophenol biodegradation (9/30/75) 

(TBP) biodegradation (6/1/76) 

Pharmacokinetics study of 2,4,6-tribromophenol in rats 

Pbotolysis of 2,4,6-tribromophenol Partition coefficient of several flame· retardants and 

industrial chemicals = 

Water solubility of several flame retardants and rndustrial 

chemicals 

Photolysis of 2,4,6-tribromophenol in aqueous solution 
Tetrabromophthal ic Anhydride u 

Acute oral study Acute dermal study Acute toxicfty studies of tetrabromophthalic anhydride Primary skin irritation study in albino rabbits 0 Eye irritation study in albino,rabbits Acute inhalation toxicity in the albino rat ·" Dermal sensitizati,on study in the ~lbino guinea p~,g 

Acute inhalation toxicity in the dlbino rat after pyrolysis (3/12/75) 

Acute inhalation toxici;ty in the albino rat after pyrolysis (.3/26/75) 

28-Day dermal toxicity study in rabbits 

2l··Day inhalation toxicity in rats 

Human repeated insult patch test with Firemaster PHT4 

Mutagenicity evaluation of 859-74-4 ~· 

Pilot teratology study in rats 

The acute toxicity of Firemaster. PHT4 to the water flea, Daphnia maana Straus-, .. 

The acute toxicity of Firemaster'PHT4, lnt number 6332-B 

to the rainbow trout Salmo gairdneri Richardson 

The acute toxicity of Firemaster PHT4, lot number 6332~B 

to the bluegill sunfish Lepomis macrohirus Rafinesque Acute oral toxicity study in.mice V 

Comparative inhalation toxicity tests 

Evaluation of the inhalation toxicity of combustion products of selected polymers II 0 -

The bioaccumulation of tetrabromophthalic anhydride in the bluegill sunUsh 

Persistence of PHT-4 in soil at l and 1.0 Jl.g/g Photolysis of PHT-4 

Effects of PHT-4 on soil microflora at 1 and 10 Jl.g/g Pharmacokinetics of PHT-4 in rats 

Partition coefficient of several flame retardants and 

industrial chenicals ' 

Water solubility of several flame retardants and industrial chemicals 

., 
" 

• 

Hexabromocyciododecane 

" .1.Hexabromocyclodci'decane: 28-day feeding trials with rats and , 90-day feeding trials with rats 

Vln vitro microbiologicial·mutgenicity studies of,J'our CibaGeigy Corporation compounds Pilot cataractcigenic study in chicks 

CataJ:actogenic study in··· chicks 

Mutagenicity evaluation of 421~32B ~ 

AC:ute·toxicity studies in rabbits ad rats 

Mutagenicity of two lots of FM-100, lot 53 and residue of 

lot 3322 in the absence and presence of metabolic 

activation . . ··" . The acute toxicity of HBCD lot number 990-17 to the bluegill sunfish Lepomis macrohirus Rafinesque _ ~ ~ .. 

Partition coefficient of dicamba, endrin,'· VEL 3510 and ·· several industrial chemicals and flame retardants 

Water solubility of several industria.hchemicals, flame 

retardants and a herbicide VEL 3Sl0 '" 

Hydrolysis of Firemaster 100 

HBCD - biodegradability study 

,. 

il 
1L2-Bis<tribromophenoxylethane 

-Water solUbility of several flame retardants and industrial chemicals 0 

-Photolysis of Firemaster 680 

-Pharmcokinetic study of FM-680 

-Rate of hydrolysis studies 

-Partition coefficient of MC-·680, chlorendic anhydride , and chlorendic acid 

-28-Day rat feeding study -Acute oral toxicity study in the albino rat -Acute dermal LD50 (1 level) . -Acute inhalation toxicity in the albino rat -Acute oral toxicity study in beagle dogs · -i\cute'•toxicity (TLsol study in the bluegill -Acute toxicity (LD50 ) study in the rainbow trout ,, -Acute heated vapor inhalation, toxicity study in rats 

-Modified Draize multiple insult test in male human~ 

-28-Day dermal toxicity study in rabbits ~21-oay' inhalation toxicity in rats -Acute inhalation toxicity in the albino rat after pyrolysis (ABS resin;FM-680/Sb1

0)) 

Acute inr.alation toxicity in the alb1no rat after pyrolysis (ABS• resinjSb203) -14-Day~ range fnding study in rats '28-Day·toxicity study in ~ats __ .. "'-Biodegradation study with "

14c-tagged,-MC-680 . . -;:eiodegrad_ability study with 14c-tagged MC-680 ''Primary skin irritation test with,mother liquor 680 . process, code ,no. 7-29-76-B in albino rabbits 

-Skin s·ensiti:iation test with mother Mquor 680'process in albino guinea pigs c 

-~o-Day subacute oral toxicity study with Firemaster 680 in albino :i:ats (Jan. 6,,"1977) .;-, 

"'-·90-Day sUbacute oral to_xicity study with Firemaster 680 in albino rats (Mar. 10, 1977 )· 'MUtagenicity evaluation of 859-74-5 

-Test report (method. for testing the 

of chemical substances in fish . in fish) 

final report degree of accumulation .. body) (Bioaccumulation" 

d'\Acute inhalation toxicity study in the albino rat after pyrolysis (ABS resin/Sb2

oycontaining 680) !) Pilot teratology study in rats 

'-Teratology study in rats , , 'Tisl!lue residue accumulation/depletion study with Firemaster · 680 in albino rats 

D 

Cl 

c !! 
Decabromodiphenyl ether 

' \Acuts toxicity studies in rats and rabbits 

'\Mutagenicity ev:aluati6n ~of-·.compoUnt!""·364-5A~c=;'==~--=·-·r , 

, '-28-Day toxicity study in' rats (6/12/74) [,);. " ~28-Day toxicity study in rats ,.{6/24/76) 

'1,28-Day toxicity study in rat,; {3/2/77) 

··'Mut;:agenicity evalua~ion of compound 27\::rlD 

Pentabromodiphenyl oxide 

~utagenicity evaluation of compound 345-76A final report '' r, 

'\

'-rhe acute' oral toxicity, of pentabromodiphenyl ether to rats 

Acute toxicity studies in rats and rabbits 

\28-Day to;icity study in rats 

"The bioaceumulation of compound S-5l2 by carp 

"-30-oay dietary study in rats with pentabromodiphenyl, oxide -~., 

. including recovery periods of 6, 12, and 24 weeks 

'\.9p-Day dietary study in rats with pent.abromodiphenyl 0 

oxide (DE;:-71) 

octabromodiphenyl oxide 

J The bioaccumulat:i.on of compound S-511 by carp ~cute toxicity studies in rats and rabbits ~Subacute inhalation toxicity study in rats 

J!'lutagenicity evaluation of compound 345-79A final report 

Jin vitro sister chromatid exchange in Chinese hamster ovary 

.. cells with octabromodiphenyl ether (DE-79) ~Unscheduled DNA synthesis assay, compound DE-?9 

•28-Day toxicity study in rats , · '13-Week feeding study in rats 

'-A range-finding teratology study in rats with DE-79 

" 

;i ' ·) rr. 
• 

' 

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\~ 

{f J BIONmCS 

Litton 

HAY 4 '" i9i5 

Contains, NQ CB1 

,S, EPA,;DTB 

lmfillftDBDBI/1111 000b3b8~0R 

't)'~ -"10c:oo <hi b 5 

11UTAGENICITY EVI\Llii\THlN 

') '(.:' 

.Qt:_ 1"1 1'1 bRlGlNAL RECOR,D 

421-E.!! c DO NOT REMOVE 

FHlAL REPORT 

' 421-328 = Hexabromocyc1ododecane 

' No. 76040318 Dispersion 

CI4S.l± 3/f4-S!:-b 

·SUGi'lJTTED TO 

ClBA-GEIGY CORPORATION 

l1RDSLEY, NEH YORI( 10502 . :· .• c··"·•-·n·· . VELS!CUL GHH.iu}l .LJ;{rJ.!:{n!luE 

SUBHITTE!l BY 

----.,-

LlTTOtl GIONET!CS, lt:C. 

5515 NIC!IOLSON lANE 

"'0' u, i .. "n"'r.' d'i .. J\:. ... r••n·y;ll~· ~!..t.·.• ~~ ""T"-1 .::;!.!,.;;,.::: 

B ..I. s J-l\SO~ ., .. : .. : 

ORIG! ~Ac C()?Y D~ !Ci R~MOI'£ 

" 

0 

PASE.. c PAG<S ......... OF ............ · ·-

KENSINGTON, HARYLAt!D 20795 

~ ~ 

LBI PROJECT NO. 2547 ·;_;,, 

APRIL 30, 1975 

0 

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·utton 

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TI\BLE OF CONTEIITS 

OBJECTIVE 

NATEP.IJILS 

., 

ol\, 

&~ 

c. 

D •• 

E. 

Test Compound 

·Indicator Hicroorgani sms 

Reaction Hixture • • • • • ,. Tissue Homogenates and'Supernatants 

Positive Control Chemicals 

EXP~RH:lENTJIL DE.S·IGN • • • .. • • • 

11: Preparation of Tissue llomogenates and 

9,000 x 9_ Cell' Fractions ••• 

B. \:' Plate Tests (Overlay folethod) • 

C. Recording and Presenting Data 

. SUt-ii·t!\RY OF PLATE TEST RESULTS" •. ·• • 

'" ,, 

HlTERPREjATION OF RESULTS AND CO:lCLUSIONS 

., . 

' 

. ' 

. ,. 

EXPLANATION OF E;IILUATiON PROCEDURES FOR ~Lf,\TE ASSNYS 

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3 

4 

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sro;;soR: 

K;T[R !IlL: 421-32G 

SU!!Jf.CT: Flli1\l !tEPOi!T E:UT(,G[I;JCITY I'LliTE fiSSf1Y " 

l. OilJECTIVE 

The objective of this study Has to evaluate the test compound for 

genetic ilctivity in-1uicrobial assay5 1-1ith and 1-1i thout the addition 

Of mammalian m~tui.Jplic uctivution prcpLlrations. 

2. BATERJALS 

IE 

uavn 

A. . .!:£ll_ Compound 

l. Date Received: April 2. 1976 

· ·· 2.,·. O~scription: Uhi te po~zder 

ll. '·.Indicator !·licroo.·qonisms ;;1 

·The follmving strains of indicator microor!)nnisms >Jere used in 

the evo1uation: 

:1. 

. '2. 

Yeast St1·ain: Saccharomvccs £_ercv is i il_i:!_, s tra i o 04 

nacteria Strnins: SnlE~o_n_e_l_!_a_lYJ'!!ii•!uriUia, strains 

Tr.-153 5 T11- 98 

Tfl-1537 TA-100 

TA-153il 

C •• Reaction !·lixtue£ 

.:·The. iollOI·Jing reaction mi>:turc Has employed in the activation 

tests: 

Comoanent ·--..1.----

. 

. 1. TPN (sodium salt) 

2. Isocitric acid 

3. T1·is buffer, pfl 7.4 <• 

4. HgCl~ 

5, Jlomogenatc fraction equivalent 

to 25 mg of 1·1et tissue 

BIONETICS 

., . 

,, ' 

6 ,,moles 

35 •"uolcs 

213 ,.moles 

2 pmoles 

-? -. 
2. 

.i .. 

rn 

Utton 

-2-

' 

I•:OUERiALS (C~ntinued) 

D. Tissu~ l!o:uc.cg~nate.$ nnd ~ernutt:nts " 

The tissue homogenatas and 9sDOO Y. 9.. syper·natants were pr~par::rl 

fr·om the livers of,S~:wilgue-Ou;Ylcy adult male rats. The anir.:a1s 

\'/ere pr·ctreatcd with f1roc)pr ]25~ {500 mg/l:g) 5 days l>~fon: kill. \:~ :: c 

E. Posith·e Co:i'trol Chemicals 

• 

>) 

Table l belo·.1 'lists the chemicals used for positive controls in 

the nonactivation and activation assays • 

Nonuctivation 

\'J 

llethyl nit.rosoguani dine 

(r-:rmG) 

2-IHtrofluorene (NF) 

SOLVE11T 

Water or Sa 1 inc 

DimethylsulfoY.ide•** 

Quinacrine mustard (Q:·i) l·l~tcr· or saline 

Activation 2-Anthramine ('F.;mr) 

2-Acetylaminofl uorcne 

(AAF) 

8-AminoquJn'bl ine (A:{Q) 

Dimethylnitrosamine 

(Di'.ilA) 

" 

Dimethy'l .. ~u 1 foxi da*r.* 

Dimethylsulfoxide***' 

Dimethylsulfoxide*** 

Sa 1 i ne 

* Concentr·ations given in Results Section 

** BPS= Base-pair substitution 

FS = Fr·ameshift . ,.· *** Previously shown to be nonmutagenic 

,. 

PP.OCfiBLE 

t:UTf\G[riJC 

SPEC!FICTT\' 

" 

BPS•* 

FS*k 

FS** 

FS*« 

IJPS'"* 

BIONETICS 

·• 
., 

.. ,,, w •• , •• 

-: ..... ·~-. 

"_; .. 

• 0 

~. 

3. EXPErm:ErlTJ\L DES!GII 

A. ::.!,.• 

p,·eparation of Tissu~ flomoqenat~s nnd 9,000 !'. !1. Cell rract·ions 

r·:ale animals (sufficient to provide the necessary quantities of 

tissues) 1·1ere killed by cran,jal bl01·1, decapitated, and bled. 

01·gans ,.,e,·e immediately dissected from the animal u5ing aseptic 

techniques· and 1·1ere p 1 Jced in i ce~co 1 d 0. 25 1·1 sucrose buffer;ed 

><ith Tris at a pll of 7.4. ·Upon collection of the desired quantity of orgcins, they ~;ere \'lashed twice with fresh buffered suc.r:ose 

and completely homogenized \'lith a motor-driven ·homogenizing unit 

at 4C. The organ homogen~te obtained from this step v;as centrifuged for 20 minutes at 9,000 x !l..in a refrigerated centrifuge. 

• The supernatant· from the centrifuged sample was retained and 

frozcn at -SOC. Samples from t_!li~. preparation 1·1er~ used for the 

actiyation studies. - · li 

;' " 

B. 'Plate .!.est {Overlay llethod) 

Apprpxinwtely 109 ,.cells from a log phas~ culture of each indicator strain \•/ere added to test tubes conlaining 2.0 ml of molten 

agar supplemented Nith biotin and a trace of hi:.tidine. For nonactivation tests, the four dose levels of the test compound v:ere 

added to the contents of the uppropria te tubes and poured over 

the surfaces of selective a~ar plates. 'In activation tests the 

9,000 x .9. tissue SLpel'natant' and required cofactors (cm·e rcac~ · tion mixture) 1·1ere added to_the overlay tubes. FDlll' dose le,·el~ 

of the test chemical Nere added to the appropriate tubes, 1·1hich 

were then mixed and the contents poured over the surf<:ce of 'a 

minimal agar (selective n1edium) plate and allm·;ed to solidify. 

The plates uere incubated for 4B to 72 hours at 37C, and scored 

for the number of colonjes gro11ing on each plate. The concentrations of all chemicals are given in the Results Section. 

Positive and solvent controls using both directly active po5itive 

chemical-s and those thut require metabolic activat.ion Here~J-un 

with each assay. ·· ' 

C. Reconlinq ~ Presentinq Data 

The numbers of colonies on each plate Here counted and reco1·ded 

on printed forms. These ra1~ data 1·1ere tt·ansferred directly to 

the report form sheets and presented as revertants per plate for 

each indicator strain employed in the assay. The positive and 

the solvent controls are provided as reference points. ' :; 

[8 BIONETICS 

Lit·ton " 
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4. .~ . ' . -· ' .. I C-· .I . , SUI-II·lARY Q£ PLATE TEST RESULTS 0 

A. Name or code designation of the test compound: 421-328 • 

B. Test date: April 20, 1976 

C. Concentrations of the test compound: (1) 0.5 pg (2) 2.5 pg (3) 5 ug (4) so ~g/p

TEST 

NONACTIVATION 

SPECIES 

Sol vent control 

Positive control** 

Test compound ( 1) 

ACTIVATION 

(2) 

( 3) 

(4) ---

Solvent Control .. Rat 

Positive control*** P.at 

Test compound (1) Rat 

(2) Rat 

(3) Rat 

(4) Rat 

+ * Irl_ convertants per plate 

TISSUE 

Liver· 

Liv~t· 

Liver 

Liver 

Liver 

Liver 

** T~.-1535 

TA-1£37 

TA-1538 

TA-9<1 

TA-100 

04 . 

1·\tlllG 

0•'·1 

--NF 

HF~~ 

I·L':IiG 

l·i~-~i~3 

10 ill/plate 

10 "1/platc 

100 "9/?lntc 

100 ~9/Pl'lt~ 

10 cl/pin~e 

10 ul/plate 

TA-1535 

11 

>]03 

7 

9 

10 

6 

35 

238 

' 45 

84 

51 

75 

REVERTAIITS, PER PLATE 

TA-1537 TA· i 538 

5 7 

>103 >10 3 

4 4 

6 5 

5 7 

9 6 

17 23 

114 >]03 

19 

17 

23 

21 

~" TA-1535 

TA· 1537 

TA-1538 

TA-98 

TJ\-100 

04 

" 

14 3~ 31 

31 

!\IITil 

Al-i() 

Mf" 

MF 

r.mu 

C:·\IIA 

0 

TA-98 TA-100, 

8 143 

>103 >]03 

a 144 

15 161 

9 1(,6 

g 155 

41 238 

>]03 tl92 

49 269 

52 283 

38 266 

41 2"1 ,_ 

100 ug/p1atc 100 ~g/piat~ 

100 "g/p1at~ 

100 ~9/plat~ 

100 ~9/p1ate 

100 "molc;fplate 

)
-5-

5. u:TF.Ili'P.ETf:'rio;: ()f' r,[SULTS fli:O C01lCLUSlONS 

TIHf test co::J;JOuml, 421-320, \lilS exilmined for mutagenic ilCtivity 

in a ser·ics of in v·itro microbial asr.llys employing Sulrr:on~lla ilnd. 

SuccharoH"~Vces indi~ m .. ganisms" The compound \·:us tcSt(:d dirr:ctly 

and in the rn·esencc of 1 iver microson:ill enzYme pt*epnY"ations from r.-

Aroclm~-induccd rats" The following results Ht!rc obtained: 

A. 

• 

B. 

Taxi city 

The compound \'l'iiS tested over a series of concentrations such 

that there I·Jas evidence of so:ne chemically-induced physiologicul 

effects at the high dose level. The 1011 dose in al1 cose:; was 

belm1 a concentration that demonstrated any toxic effect. The 

dose •·ange employed for the evilluation of this com;Jound HilS from 

0. 5 ~9 to 50 a•9 per pl ote. 

Nonactivution Tc5t Results 

The resu1 ts of the tests conducted on the compound in the ab~cnce 

of a motobol ic system 1·1ere all negative. 

C. Activation Test Re$ults 

The results of the tests conducted on the com;Jou:od in the presence 

of the rat liver activation system 1·1erc all negative. 

D. Conclusions 

The tast cou:pound, 42l-32n, did not dcmonstrntc mutagenic 

in any of the ussnys conducted in this evuluaticm and Has 

not mutagenic under these test conditions. 

Submitted by: 

ilctivity 

consid::red 

r·· \ 

Davld \.-c Orus1cf:, ~ ~-Pn.O. ~'--- Di rae tor 

rn BIONET!CS 

Litton 

Department of Genetics 

Revi Cl·led by: 

__/.frt ,, .ftrJ ,.,"",' __ ,_ .. Ro~~rt ~- \/r;;,., Ph.D. 

Vice President 

0 
:-;··· 

•0 

6. - f:Xr'LIIiU\TlON OF EVAUJIITION PROC[IlliRES FOR PLAT!: ASSAYS 

Plat'c test data consist of dil·ect revertant colony counts obtained from a set of. selective agar plates seeded Hith populations of mutant cells suspended in a semisolid overlay. Bcciluse the test chemical · and the cells are incubated in the overluy for 2 to 3 days, and a few cell divisions occut· d~~&ing the .incubiltion per·iod, the test is semi- quantitative in natur·e·/fAlthough these features of the assay reduce the quantitation of l"CSu!lts, they provide certain advantuges not con- tained in a quantitative,\ suspension test: · · 

r 

A. 

B. 

~ • The . sma 11 numb!'"·· of cc 11 d iv is i oris permits potenti a·l muta~e~s to ac) on r·ep!ica~ing DtiA."<Jhich is often more sens 1 t 1 ve tha n'l nonr·cp lt cat 1 ng DNA. · . ·~ The combined incubation of the compound and the cells in the overlay permits constant exposure of the indicator cells for· 2 to 3 days. 

Surviving Populations 

Plate test procedures do not penult exact quantitotion of the number of cells sut·viving chemical tr·cotment. /It 10\·1 concen- trutions of the test chemicol. the sut•viving population on the treatment plates is essentially the sam;, as that on the nega- five control plate. 1\t high concentrations, the surviving popu'lation is usually reduced by some f.ra_ction. Our protQcol normd lly employs sev~ra 1 doses r·onging over t1·:o or three log concentr·ations, the highest of these doses beinn selected Lo show slight toxic-ity as determined lly subjectiv, cr·i tcria. 

~ Resoonse Phenornenu 

The demonstt·ation of dosccrelated increases in mutont counts is an importont criter-ion in establishing mutagenicity. A factor that might modify dose-response results fo,· a mutagen 1·10uld be the selection of doses that are too low (usually mutagenicity· and tox-icity arc rclu ted). If the highest dose is far· lm;er· than a to:dc concentruti~m, nq increases may be observep over the dose range selected. Conversely, if the la\·1est dose em- ;; 

mutants 

played is highly cytotoxic, the test chemical moy kill ·any '' , .. that are induced, and the compound 1·1ill not appear· ,.to be mutagenic. · · ·.:c• 

.. 

rn BIONETICS litton 
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6. E>:'rLJ\IlAT!ON OF EVf,LUflil011 rhOCCfJURES FOR PLATE 1\SSf,YS (Continued) 

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Con tro 1 Tests 

Positive and negative control azsay!:: are con::.I~;Ct~·d \·lith each exp~riment und con!iist of direct-acting mutilgens for nonactiv.nt.ion assays and 1nutagen5 that require me tubal ic ldotrunsfor;;Ja- ·· 

tion in activ~tio11 assays. Negative controls ~onsist of the 

test compound solvent in the overlay agar together 1·;ith the . other essential components. The negative control plate for 

each strain gives a t·efcrence point to which the test dilta are compilred. The positive control assay is conducted to demonstrate that the test systems are functional Hith f:nown mutagens . 

D. Interpretation of Results· 

The demonstration of dose-related incr·eases in mutant counts is the mast reliable metlwd to demonstrate mutagenicity. l;utant increa5es at only one or two doses may be significant if they occur at the higher doses. Increases at lol'l or i ntcrr.;edia te 

concentrations followed by reduced mutant counts at higher c!os~s 

may indicate that the test chemical hns a narrow acti\•ity r~nge 

or tiF·t the higb dose levels were toxic and t!:e induced revertunt cells •~ere killed. We are al>le to detect th~ latter possibility by inspecting the boc_l:ground gr01·1th, and the former possibil ilj' c<n be investigated by lool:in'g at a nurrDI< series of dose levels bracketing the presumptive active ran~~. 

It is difficult to de'tect mutagen; ~·lith little m· no toxicity in 

this assay since such ugcnts ilrC generally \"ICHlk rnutt~gens and p!·oducc only t\·JO to threefold increases in mutant counts. Variations 

of b:o to threefold are often within normal fluctuations of the 

spontaneous counts, and the use oF even higher conccntrutions is often difficult because of. the 1Helihoo<l of ovel'1ouding the Sj'Stem \"l;th large quuntitics of the chemicaL To n?solve the r.!~1t~­

genicity of such a chemical, other assilys to ~1hich statistical evaluations can be applied m"y be necessary. 

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