# 1110 

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# • 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. Departmentof Commerce 

### NationalTechnical Information Service

# One Source. One Search. One Solution.
Providing Permanent, EasyAccess to U.S. Government Information



## The National Technical Information Service is the Nation's largest repository and disseminatorof government-initiated scientific, technical, engineering, and related business information. The NTIS collection includes almost 3million information products in a variety of formats: electronic download, online access, DVD, CD-ROM, magnetictape, diskette, multimedia, microfiche and paper. Search the NTIS Database from 1990 forward 

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..S

.. DEPARTMENTOFCOMMERCE TechnologyAdministration NationalTechnical Information Service Springfield, VA 22161

# L COOING FORM FOR 

### SRCINDEXING 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 MStreet.; SW Washington, DC 20460 Attn: 8(d). Health & Safety Reporting (Notification/Reporting) Dear Sir: -J '-"' 0

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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, 

### »-!#•l-tV 

### £ 

## J./o/"

**~** Dennis L. McFadden, 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 

### studyin. 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 solutionTetrabromophthalic 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

,. 

### il1L2-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/Sb10)) Acute inr.alation toxicity in the alb1no rat after pyrolysis (ABS• resinjSb2 03)

**-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 

## JThe bioaccumulat:i.onof 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


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## 5 11UTAGENICITY EVI\Llii\THlN
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## 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 

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### LBI PROJECT NO. 2547·;_;,, APRIL 30, 1975

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## ,, TI\BLE OF CONTEIITS OBJECTIVE NATEP.IJILS ., ol\,

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•• E. Test Compound ·Indicator Hicroorganisms 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 

### .,

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

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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 1-1ithout 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:** Uhite**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 £_ercvisiil_i:!_, straio 04 nacteria Strnins:**SnlE~o_n_e_l_!_a_lYJ'!!ii•!uriUia,** strains Tr.-1535 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 

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,, ' 6 ,,moles 35 •"uolcs 213 ,.moles

2 pmoles

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### -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 llethylnit.rosoguanidine (r-:rmG) 2-IHtrofluorene (NF) SOLVE11T Water or Sa1inc DimethylsulfoY.ide•** Quinacrine mustard (Q:·i)

**l·l~tcr·** or saline Activation 2-Anthramine 

### ('F.;mr) 2-Acetylaminofluorcne (AAF) 8-AminoquJn'bline (A:{Q) Dimethylnitrosamine (Di'.ilA) " Dimethy'l..

**~u**1 foxida*r.* Dimethylsulfoxide***' Dimethylsulfoxide*** Sa1ine 

## * 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 

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**~.** 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 p1 Jced in i

**ce~co**1d 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 uppropriate 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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. , 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/plate**

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, D4*

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 102 lGO 97 91 .• )00 lOG· 48 54 t.2 43 51 54 I -'-' '-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 1iver microson:ill enzYme pt*epnY"ations from r.-**Aroclm~-induccd** rats" The following results Ht!rc obtained: 

### A.• 

### B. Taxicity 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 plote. Nonactivution Tc5t Results The resu1ts of the tests conducted on the compound in the**ab~cnce** of a motobolic 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·· 

# \\.-c**~ ~-** **~'---** Davld Orus1cf:, Pn.O. Diraetor 

# rn BIONET!CS Litton Department of Genetics ReviCl·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 sma11 numb!'"··of cc11 divisioris permits potentia·l

**muta~e~s** to ac) on **r·ep!ica~ing** DtiA."<Jhich is often more sens1t 1ve than'lnonr·cpltcat1ng 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 normdlly 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·itcria.

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



# .. 

# rn BIONETICS littonr 

### -7- 

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



# rn UUon 

## c.

• Contro1 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 metubalic 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 towhich 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 intcrr.;ediate 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 possibililj' 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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