# r SALMONELLA/MAMMALIAN-MICROSOM E MUTAGENICIT Y TES T wit h T K 1 2 44 3 (Tes t fo r mutageni c propertie s i n bacteria ) 

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Salmonella/Mammalian Microsom e Mutagenicit yTes t No . o f experiment : 81119 4 Batch : E N2437 5

SUMMAR Y AN D CONCLUSION S T K 1 2 44 3 wa s teste d fo r mutageni c effect s o n histidine-auxo trophi c mutant s o f Salmonell a typhimurium . Th e investigation s wer e performe d wit hth e followin g concentration s o f th e tria l substanc e withou t an d wit h microsoma l activation : 25 , 75 , 2 2 5 , 6 7 5 and 202 5 jig/O. l ml . Sinc e fluctuation s i n th e numbe r o f back-mutant s occurre d i n th e experiment s o n strain s T A153 5 an d TA1537 , th e experiment s o n thes e strain s wer e repeated . Thes e test s permi t th e detectio n o f poin t mutation s i n bacteri a induce d b

ychemica l substances . Anymutageni c effect s o f th e sub stance s ar e demonstrabl e o n compariso n o f th e numbe r o f bacteri a i n th e treate d an d contro l culture s tha t hav e undergon e back-muta tio n t o histidine-prototrophism . T o ensur e tha t mutageni c effect s o f metabolite s o f th e tes t substance s forme d i n mammal s woul d als o b e detected , experiment s wer e performe d i n whic hth e culture s ar e additionall ytreate d wit h a n activatio n mixtur e (ra t live r microsome s an d co-factors ) (lit.1,2,3) . I n th e experiment s performe d withou t an d wit hmicrosoma l activa tion , compariso n o f th e numbe r o f back-mutan t colonie s i n th e con trol s an d th e culture s treate d wit hth e variou s concentration s o f T K 1 2 44 3 reveale d n o marke d deviations .- 3 -

N o evidenc e o f th e inductio n o f poin t mutation s byT K 1 2 44 3 o r byth e metabolite s o f th e substanc e forme d a s a resul t o f micro soma l activatio n wa s detectabl e i n th e strain s o f S . typhimuriu m use d i n thes e experiments .

Studydirector :

Repor t reviewe d an d approve d by :- 4 - PROCEDUR E Th e test s wer e carrie d ou t i n accordanc e wit hth e metho d de scribe d b

yAME S e t al . (lit.1,2,3) . Th e bacteri a o n whic h th e test s wer e performe d wer e th e follow in g histidine-auxotrophi c strain s o f Salmonell a typhimurium : TA98 , TA100 , T A1535 , TA1537 . Th e test s wer e performe d wit h th e followin g concentration s o f th e tria l substanc e withou t an d wit hmicrosoma l activation : 25 , 75 , 225 , 67 5 an d 202 5 )ig/0. 1 ml . Th e substanc e wa s dissolve d i n acetone . Aceton e alon e wa s use d fo r th e negativ e control s (th e substance s and vehicle s use d fo r th e positiv e control s ar e indi cate d below) . Eac hPetr i dis hcontained : 1 ) approx . 2 0 m l o f minimu maga r (Agar , Difc o Laboratories , Detroit , Michigan , U.S.A. , plu s salt s (Vogel-Bonne r Mediu mE ) an d glucose) , 2 ) 0. 1 m l o f th e solutio n o f th e tes t substanc e o r th e vehicl e an d 0. 1 m l o f a bacteria l cultur e (i n nutrien t broth , Difc o Labo ratories , Detroit , Michigan , U.S.A. , 0.8 % plu s 0.5 % NaCl ) i n 2. 0 m l o f sof t agar . Th e sof t aga r wa s compose d of : 10 0 m l o f 0.6 % aga r solutio n wit h 0.6 % NaC l an d 1 0 m l o f a solutio n o f 1-histidine , 0. 5 m M (Fluka , Buchs , Switzerland ) an d +bioti n 0. 5 m M (Fluka , Buchs , Switzerland) . I n th e experiment s i n whic h th e substanc e wa s metabolicall yactivated , 0. 5 m l o f a n activa tio n mixtur e wa s adde d als o (lit.1,2,3) . 1 m l activatio n mixtur e contained : 0. 3 m l S 9 fractio n o f live r fro mrat s induce d wit h Aroclo r 1 2 5 4 (Analabs. , Inc. , Nort h Haven , Connecticut , U.S.A. ) an d 0. 7 m l o f a solutio n o f co-factors . Positiv e contro l experiment s wer e carrie d ou t simultaneousl y wit hth e followin g substances : 1 ) fo r strai n T A98 : daunorubi cin-HC l (DAUNOBLASTI N, Farmitalia , Montediso n Farmaceutic a GmbH , Freibur g i.Br. , Germany) , 5 and 1 0 ]iq/0. 1 m l phosphat e buffer ; 2 ) fo r strai n TA100 : 4-nitroquinoline-N-oxid e (Fluka ,- 5 -

Buchs , Switzerland) , 0.12 5 and 0.2 5 \ig/0. 1 m l phosphat e buffer ; 3 ) fo r strai n TA1535 : N-methyl-N'-nitro-N-nitrosoguanidin e (Ser va , Heidelberg , Germany) , 3 an d 5 jig/O. l m l phosphat e buffer ; 4 ) fo r strai n TA1537 : 9(5)aminoacridin e hydrochlorid e mono hydrat e (Fluka , Buchs , Switzerland) , 5 0 an d 10 0 >ig/0. 1 m l DMSO . Th e activatio n mixtur e wa s teste d wit hstrai n TA153 5 an d cyclo - ® phosphamid e (ENDOXAN-AST A, Asta-Werke , Bielefeld , Germany) , 25 0 vig/0. 1 m l phosphat e buffer . I n th e experiment s withou t an d wit hth e additio n o f microsoma l activatio n mixtur e thre e Petr i dishe s wer e prepare d pe r strai n an d pe r grou p (i.e . pe r concentratio n o r pe r contro l group) . Th e plate s wer e incubate d fo r abou t 4 8 hour s a t 3 7 C i n dark nes s . Whe n th e colonie s ha d bee n counted , th e arithmeti c mea n wa s cal culated . Th e tes t substanc e i s generall yconsidere d t o b e non mutageni c i f th e colon ycoun t i n relatio n t o th e negativ e con tro l i s no t double d a t an yconcentratio n (lit.3) .- 6 - RESULT S (se e Table s 1 t o 8 ) I n th e experiment s performe d withou t an d wit hmicrosoma l activa tion , compariso n o f th e numbe r o f histidine-prototrophi c mutant s i n th e control s an d afte r treatmen t wit hT K 1 2 44 3 reveale d n o marke d differences . Th e sligh t increas e i n th e numbe r o f back-mutan t colonie s observe d i n th e firs t experimen t withou t activatio n o n strai n TA153 7 an d wit hactivatio n o n strai n TA153 5 i s attribute d t o fluctuation s i n th e rat e o f spontaneousl yoccurin g back-mutants . A t th e concentration s o f 2 2 5 yg/0. 1 m l an d abov e th e substanc e precipitate d i n sof t agar .

AMES , B.N. , F.D . LEE , an d W.E . DURSTO N(1973) , A n Improve d Bac teria l Tes t Syste mfo r th e Detectio n an d Classificatio n o f Muta gen s an d Carcinogens . Proc . Natl . Acad . Sci . US A^0 , 782-786 . ^AMES , B.N. , W.E . DURSTON , E . YAMASAKI , an d F.D . LE E (1973) , Carcinogen s ar e Mutagens : ASimpl e Tes t Syste mCombinin g Live r Homogenate s fo r Activatio n an d Bacteri a fo r Detection . Proc . Natl . Acad . Sci . US A70 , 2281-2285 . •^AMES , B.N. , J . McCANN , an d E . YAMASAK I (1975) , Method s fo r Detectin g Carcinogen s an d Mutagen s wit hth e Salmonella/Mammalian Microsom e Mutagenicit yTest . Mutatio n Res . 31 , 347-364 .TABL E1

SALMONELLA/MAMMALIANMICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WITHOUTMICROSOMA LACTIVATIO N NUMBE ROFBACK-MUTANTCOLONIE S PE RPLAT E(ARITHMETI C MEAN ) N o . o f experiment : 81119 4 Date : Januar y 04 , 198 2 STRAI N1

Contro l 2 5 pg/O. l m l 7 5 pg/C l m l 22 5 pg/C l m l 67 5 Mg/0. 1 m l 202 5 Mg/0. 1 rol Positiv e control s dauno r u b icin-HC 1 Contro l 5 \iq/0. 1 m l 1 0 pg/O. l m l 4-nitroquinoline - N-oxid e 

### Contro l 0.12 5 Mg/0. 1 m l 0.2 5 Mg/0. 1 m l N^nethyl-N'-nitro - N-nitrosoguanidin e

Contro l 3 Mg/0. 1 m l 5 Mg/0. 1 m l 9(5)aminoacridine - hydrochlo r

id e Contro l 5 0 Mg/0. 1 m l 10 0 Mg/0. 1 m l 2-nitrofluoren e

Contro l 5 Mg/0. 1 m l 1 0 Mg/0. 1 m l 3 5 31 4 35 3 Tes t

substance : T

K 1 2 4 4 3 9 8 2 9 2 3 3 0 3 3 3 2 3 3 TA10 0

10 4 10 1 11 7 10 4 10 7 11 1 T A 153 5

1 7 1 6 1 3 1 8 2 1 1 7 T A 153 7

5 6 8 1 2 9 4 T A153 8

10 2 29 8 68 9 8

115 7 159 0 4

6 8 97 6TABL E2

SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WITH MICROSOMALACTIVATIO N NUMBE ROFBACK-MUTANTCOLONIE S PE RPLAT E(ARITHMETI C MEAN) N o . o f experiment : 81119 4 Tes t substance : T

K 1 2 4 4 3 Date : Januar y 04 , 198 2 STRAI N Contro l 2 5 Mg/0. 1 m l 7 5 Mg/0. 1 m l 22 5 Mg/0. 1 m l 67 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l TA 9 8 4 9 5 0 5 0 5 5 5 0 6 4 T

A 10 0 9 3 10 0 1 0 3 9 8 9 6 1 0 3 TA 153 5

1 1 1 2 1 9 2 1 2 2 2 0 TA 153 7

8 6 5 6 6 5 TA153 8

Positiv e contro l o f th e microsoma l activatio n cyclophosphamid e

Contro l 1 8 25 0 Mg/0. 1 m l 28 0 2-aminoanthracen e

Contro l 5 Mg/0. 1 m lTABL E3

SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WITHOU TMICROSOMA LACTIVATIO N NUMBE ROFBACK-MUTANTCOLONIE S PE RPLAT EAN D ARITHMETI C MEA N No . o f experiment : 81119 4 Tes t substance : T

K 1 2 44 3 Date : Januar y 04 , 198 2 STRAI NTA9 8 T A10 0 TA153 5 T A153 7 TA153 8 Contro l 2 5 Mg/0. 1 m l 7 5 Mg/0. 1 m l 22 5 Mg/0. 1 m l 67 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l Positiv e control s

Contro l Concentratio nA

B 2 9

2 3 3 0 3 3 3 2 3 3

3 5 31 4 35 3 10 4

10 1 11 7 10 4 10 7 11 1

10 2 29 8 68 9 1 7

1 6 1 3 1 8 2 1 1 7

8 115 7 159 0 5

6 8 1 2 9 4

4 6 8 97 6TABL E4

SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WIT H MICROSOMALACTIVATIO N NUMBE RO FBACK-MUTANTCOLONIE S PE RPLAT EAN D ARITHMETI C MEA N No . o f experiment : 81119 4 Date : Januar y 04 , 198 2 Tes t substance : T

K 1 2 44 3

STRAI N Contro l 2 5 Mg/0. 1 m l 7 5 Mg/0. 1 m l 22 5 Mg/0. 1 m l 67 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l Positiv e contro l o f th e microsoma l activatio n 9 8

4 9 5 0 5 0 5 5 5 0 6 4 TA10 0

9 3

10 0 10 3 9 8 9 6 10 3 TA153 5 TA153 7 TA153 8

1 1 1 2 1 9 2 1 2 2 2 0 Contro l Concentratio nA 1 8 28 01 1 1 3 1 2 8 1 4 1 4 8

6 5 5 3 6 TABL E5 SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WITHOU TMICROSOMALACTIVATIO N NUMBE ROFBACK-MUTANTCOLONIE S PE RPLAT E(ARITHMETI C MEAN ) No . o f experiment : 81119 4 Tes t substance : T

K 1 2 44 3 Date : Januar y 14 , 198 2 STRAI NTA9 8 T A10 0 T

A153 5 T

A153 7 TA153 8 Contro l 2 5 Mg/0. 1 m l 7 5 pg/O. l m l 2 2 5 Mg/0. 1 m l 6 7 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l Positiv e control s dauno r ubicin-HC l Contro l 5 Mg/0. 1 m l 1 0 Mg/0. 1 m l 4-nitroquinoline - N-oxid e Contro l 0.12 5 Mg/0. 1 m l 0 . 2 5 Mg/0. 1 m l N-methyl-N'-nitro - N-nitrosoguanidin e

Contro l 7 3 Mg/0. 1 m l 26 6 5 Mg/0. 1 m l 119 5 9(5)aminoacridine - hydrochlorid e

Contro l 6 5 0 Mg/0. 1 m l 2 5 10 0 Mg/0. 1 m l 38 9 2-nitrofluoren e

Contro l 5 Mg/0. 1 m l 1 0 Mg/0 . 1 m l' TABL E6

SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WIT H MICROSOMALACTIVATIO N NUMBE RO FBACK-MUTANTCOLONIE S PE RPLAT E(ARITHMETI C MEAN ) No . o f experiment : 81119 4 Tes t substance : T

K 1 2 44 3 Date : Januar y 14 , 198 2 STRAI NT A9 8 TA10 0 TA153 5 T A153 7 TA153 8 Contro l 2 5 Mg/0. 1 m l 7 5 Mg/0. 1 m l 22 5 Mg/0. 1 m l 67 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l Positiv e contro l o f th e microsoma l activatio n cyclophosphamid e

Contro l 1 0 25 0 Mg/0. 1 m l 41 2 2-aminoanthracen e

Contro l 5 Mg/0. 1 m l 1 3 1 0 1 2 1 2 1 1 9 5

4 6 6 6 4TABL E7

SALMONELL-A/MAMMALIAN-MICROSOMEMUTAGENICIT Y TES T EXPERIMENT S WITHOU TMICROSOMALACTIVATIO N NUMBE ROFBACK-MUTANTCOLONIE S PE RPLAT EAN D ARITHMETI C MEA N No . o f experiment : 81119 4 Tes t substance : T

K 1 2 44 3 Date : Januar y 14 , 198 2 STRAI NTA9 8 T A10 0 Contro l 2 5 Mg/0. 1 m l 7 5 Mg/0. 1 m l 2 2 5 Mg/0. 1 m l 6 7 5 Mg/0. 1 m l 202 5 Mg/0. 1 m l Positiv e control s

(i>ntro l Concentratio nA

B 153 5 T A

1 1 1 3 1 2 8 1 4 1 4

7 26 6 119 5 153 7

2

3 8 T A153 8

8 6 5 5 3 6

6 • ! 5 1 9TABL E8

SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WIT H MICROSOMALACTIVATIO N NUMBE RO FBACK-MUTANTCOLONIE S PE RPLAT EAN D ARITHMETI C MEA N No . o f experiment : 81119 4 Tes t substance : T

K 1 2 44 3 Date : Januar y 14 , 198 2 STRAI NT A9 8 T

A10 0 TA153 5 T A153 7 TA153 8 Contro l 1 3 5 2 5 Mg/0. 1 m l 1 0 4 7 5 Mg/0. 1 m l 1 2 6 2 2 5 Mg/0. 1 m l 1 2 6 67 5 Mg/0. 1 m l 1 1 6 202 5 Mg/0. 1 m l 9 4 Positiv e contro l o f th e microsoma l activatio n

Contro l 1 0 Concentratio nA 41 2Verteiler :

HH .( Tes t No. : 81119 4

Protectio n o f Healt h an d Environmen t

GU2. 3 Experimenta l Patholog y

SUPPLEMEN TTOTH EREPOR T (date dFebruar y 5 , 1982 )

SALMONELLA/MAMMALIAN-MICROSOM E MUTAGENICIT Y TES T

Tes t material : T K 1 2 44 3

Date : Jun e 17 , 198 7-2 - RA W DAT A Toxicit vtes t (tabl e 1) ; Apreliminar ytoxicit ytes t wa s carrie d ou t o n strai n T A10 0 withou t microsoma l activatio n wit hth e concentration s rangin g fro m 0.000 1 t o 200 0 ug/0. 1 ml . Th e protoco l use d wa s th e sam e a s i n th e mutagenicit ytest , however , onl ytw o plate s pe r concentratio n wer e used . Th e individua l plat e count s an d th e mea n value s ar e give n i n column s 4 , 5 an d 6 o f tabl e 1 . Ther e wa s n o growth-inhibitin g ef fec t o f T K 1 2 44 3 observed . A t th e concentration s o f 5 0 ug/0. 1 m l an d abov e th e tes t materia l precipitate d i n sof t aga r (*) . Mutagenicit ytes t (table s 2 t o 5) : Copie s o f th e origina l compute r outprint s showin g individua l value s pe r plat e an d mea n value s pe r grou p ar e given .

Stud ydirector : Date : Ma y22 . , 1 9 8 7

Reviewe d an d approve d by ;

Date : Jun e 17 , 198 7Tabl e 1

MIKROBIELL EMUTAGENITAETSPRUEFUN G 

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TABLEA(RA

WDATA ) SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT Y TES T EXPERIMENT S WITHCXJ TMICROSOMA

LACTIVATIO N NUMBE RO FBACK-MUTAN TCOLONIE S PE RPLAT EAN D ARITHMETI C MEA N No . o f experiment : Date : Jan . 04 , 198 2 STRAIN Contro l 2 5 pg/0. 1 m l 7 5 jjg/0. 1 m l 22 5 |jg/0. 1 m l 67 5 jjg/O. l m l 202 5 pg/O. 1 m l

• • Positiv e control s

Contro l Concentratio nA

B 81119 4

T A9 8

2 9 2 3 3 0 3 3 3 2 3 3 3 2 3 5 31 4 35 3 Testsubstan c Apparatu s c c T A10 0 T

A

10 4 10 1 11 7 10 4 10 7 11 1 8 4 10 2



# ^^6^ i : e : T

K 1 2 intro l (1 0 153 5 T l

1 7 1 6 1 3 1 8 2 1 1 7 9 8 115 7 159 0 4 4 3 10) : i 153 7

5 6 8 1 2

, 9 4 7 4 6 8 97 6 Visa : 9 9 T A153 8



### ^cCt k ILMJ^ . 

### UU.^ ' ^ ^Tabl e 3 TABL EB (RA WDATA ) SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICIT

Y TES T EXPERIMENT S WIT

H MICROSOMA

LACTIVATION NUMBE ROFBACK-MUTAN

TCOLONIE S PERPLAT EAN D ARITHMETI C MEA

N No . o f experiment : 81119 4 Date : Jan . 04 , 198 1 Testsubstance : T K 1 2 44 3 Visa : Apparatu s contro l (100 ) : 10 1 STRAIN1

Contro l 2 5 pg/0. 1 m l 7 5 pg/0. 1 m l 22 5 pg/0. 1 m l 67 5 jjg/O- l ni l 202 5 jjg/O- l m l ' Positiv e contro l o f t h e microsoma l activ a

Contro l Concentratio nA P A9 8

4 9 5 0 5 0 5 5 5 0 6 4 tio n T

A10 0 T

A

9 3 10 0 10 3 9 8 9 6 10 3

• —"_- • 153 5 T

A153 7

.6-/ A ^iiS j 1 9 2 1 <^/ L 2 2 X 2 0

1 8 28 0Tabl e 4 TABL EA(RA

WDATA ) SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICITY TES T EXPERIMENT S WITHOU TMICROSOMA

LACTIVATIO N NUMBE ROFBACK-MUTAN

TCOLONIE S PE RPLATEAN D ARITHMETI C MEA

N No . o f experiment : 81119 4 Testsubstance : T K 1 2 44 3 Visa : Date : Jan . 14 , 198 2 Apparatu s contro l (100) : 10 0 STRAINT A9 8 T

A10 0 T

A153 5 T

A153 7 T

A153 8 Contro l 2 5 jjg/O. l m l 7 5 pg/0. 1 m l 22 5 pg/O. l m l 67 5 pg/O. l m l 202 5 Mg/0. 1 m l Positiv e control s

Contro l Concentratio nA

B 

### 1 1 1 3 1 2 

### 8 1 4 1 4



### 7 26 6 119 5 

### 8

6 5 5 3 6

6 2 5 

### 38 9Tabl e 5

TABL EB (RA WDATA ) SALMONELLA/MAMMALIAN-MICROSOM EMUTAGENICITY TES T EXPERIMENTS WIT

H MICROSOMA

LACTIVATIO N NUMBE RO FBACK-MUTAN

TCOLONIE S PERPLAT EAN D ARITHMETI C MEA

N No . o f experiment : 81119 4 Date : Jan . 14 , 19 8 2 Testsubstance : T K 1 2 4 4 3 Apparatu s contro l (100) : 

### Visa : 10 0 STRAI N T A9 8 T

A10 0 T

A153 5 T

A153 7 T

A153 8 Contro l 2 5 pg/O. l rol 7 5 jjg/O. l rol 22 5 pg/O. l rol 67 5 pg/O. l rol 202 5 Mg/0. 1 rol Positiv e contro l o f t h e roicrosoroal activatio n

Contro l Concentratio n A 

### 1 3 1 0 1 2 1 2 1 1 *



### 1 0 

### 41 2