r 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

with 

TK 12 443 

(Test for mutagenic properties in bacteria) 

T\NUV\K/ qoo 

GU 2. 3 

Februar y 5 , 19 82 

This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is 

the property of the sponsor and may be used only for that purpose intended by the sponsor and as 

authorized under applicable law. Every other or additional use, exploitation, reproduction, publication or 

submission to other parties requires the written permission of the sponsor, except as otherwise required by 

applicable law (e.g., TSCA). The use of these studies/documents for regulatory, registration or other 

commercial purposes [including but not limited to Registration, Evaluation, Authorization and Restriction 

of Chemical Substances (REACH)], without the express written permission of the sponsor is unauthorized 

and strictly prohibited.
- 2 -

Salmonella/MammalianMicrosome Mutagenicity Test 

No. of experiment: 811194 

Batch: EN 24375 

SUMMARY AND CONCLUSIONS 

TK 12 443 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were 

performed with the following concentrations of the trial substance 

without and with microsomal activation: 25, 75, 22 5, 67 5 and 

2025 jig/O.l ml. Since fluctuations in the number of back-mutants 

occurred in the experiments on strains TA 1535 and TA 1537, the 

experiments on these strains were repeated. 

These tests permit the detection of point mutations in bacteria 

induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria 

in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects 

of metabolites of the test substances formed in mammals would 

also be detected, experiments were performed in which the cultures 

are additionally treated with an activation mixture (rat liver 

microsomes and co-factors) (lit.1,2,3). 

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations 

of TK 12 443 revealed no marked deviations. 
- 3 -

No evidence of the induction of point mutations by TK 12 443 or 

by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium 

used in these experiments. 

Study director: 

Report reviewed and 

approved by: 
- 4 -

PROCEDURE 

The tests were carried out in accordance with the method described by AMES et al. (lit.1,2,3). 

The bacteria on which the tests were performed were the following histidine-auxotrophic strains of Salmonella typhimurium: 

TA 98, TA 100, TA 1535, TA 1537. 

The tests were performed with the following concentrations of 

the trial substance without and with microsomal activation: 25, 

75, 225, 675 and 2025 )ig/0.1 ml. The substance was dissolved in 

acetone. Acetone alone was used for the negative controls (the 

substances and vehicles used for the positive controls are indicated below). Each Petri dish contained: 1) approx. 20 ml of 

minimum agar (Agar, Difco Laboratories, Detroit, Michigan, 

U.S.A., plus salts (Vogel-Bonner Medium E) and glucose), 

2) 0.1 ml of the solution of the test substance or the vehicle 

and 0.1 ml of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 

2.0 ml of soft agar. The soft agar was composed of: 100 ml of 

0.6% agar solution with 0.6% NaCl and 10 ml of a solution of 

1-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and +biotin 

0.5 mM (Fluka, Buchs, Switzerland). In the experiments in which 

the substance was metabolically activated, 0.5 ml of an activation mixture was added also (lit.1,2,3). 1 ml activation mixture 

contained: 0.3 ml S9 fraction of liver from rats induced with 

Aroclor 12 54 (Analabs., Inc., North Haven, Connecticut, U.S.A.) 

and 0.7 ml of a solution of co-factors. 

Positive control experiments were carried out simultaneously 

with the following substances: 1) for strain TA 98: daunorubicin-HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica 

GmbH, Freiburg i.Br., Germany), 5 and 10 ]iq/0. 1 ml phosphate 

buffer; 2) for strain TA 100: 4-nitroquinoline-N-oxide (Fluka, 
- 5 -

Buchs, Switzerland), 0.125 and 0.25 \ig/0. 1 ml phosphate buffer; 

3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 jig/O.l ml phosphate buffer; 

4) for strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 50 and 100 >ig/0.1 ml DMSO. 

The activation mixture was tested with strain TA 1535 and cyclo-

® phosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 

250 vig/0.1 ml phosphate buffer. 

In the experiments without and with the addition of microsomal 

activation mixture three Petri dishes were prepared per strain 

and per group (i.e. per concentration or per control group). 

The plates were incubated for about 48 hours at 37 C in darkness . 

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration (lit.3). 
- 6 -

RESULTS 

(see Tables 1 to 8) 

In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants 

in the controls and after treatment with TK 12 443 revealed no 

marked differences. 

The slight increase in the number of back-mutant colonies observed 

in the first experiment without activation on strain TA 1537 and 

with activation on strain TA 1535 is attributed to fluctuations 

in the rate of spontaneously occuring back-mutants. 

At the concentrations of 22 5 yg/0.1 ml and above the substance 

precipitated in soft agar. 

AMES, B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. 

Proc. Natl. Acad. Sci. USA ^0, 782-786. 

^AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), 

Carcinogens are Mutagens: A Simple Test System Combining Liver 

Homogenates for Activation and Bacteria for Detection. 

Proc. Natl. Acad. Sci. USA 70, 2281-2285. 

•^AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for 

Detecting Carcinogens and Mutagens with the Salmonella/MammalianMicrosome Mutagenicity Test. 

Mutation Res. 31, 347-364. 
TABLE 1 

SALMONELLA/MAMMALIANMICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN) 

N o . of experiment : 811194 

Date : Januar y 04 , 1982 

STRAIN 1 

Contro l 

25 pg/O. l ml 

75 pg/C l ml 

225 pg/C l ml 

675 Mg/0. 1 ml 

202 5 Mg/0. 1 rol 

Positiv e control s 

dauno r ub icin-HC 1 

Contro l 

5 \iq/0. 1 ml 

10 pg/O. l ml 

4-nitroquinoline -

N-oxid e 

Control 

0.125 Mg/0.1 ml 

0.25 Mg/0.1 ml 

N^nethyl-N'-nitro -

N-nitrosoguanidin e 

Contro l 

3 Mg/0. 1 ml 

5 Mg/0. 1 ml 

9(5)aminoacridine -

hydrochlo r id e 

Contro l 

50 Mg/0. 1 ml 

100 Mg/0. 1 ml 

2-nitrofluoren e 

Contro l 

5 Mg/0. 1 ml 

10 Mg/0. 1 ml 

35 

314 

353 

Tes t substance : TK 12 44 3 

98 

29 

23 

30 

33 

32 

33 

TA 100 

104 

101 

117 

104 

107 

111 

TA 1535 

17 

16 

13 

18 

21 

17 

TA 1537 

5 

6 

8 

12 

9 

4 

TA 1538 

102 

298 

689 

8 

1157 

1590 

4 

68 

976 
TABLE 2 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN) 

N o . of experiment : 811194 Tes t substance : TK 12 44 3 

Date : Januar y 04 , 1982 

STRAIN 

Contro l 

25 Mg/0. 1 ml 

75 Mg/0. 1 ml 

225 Mg/0. 1 ml 

675 Mg/0. 1 ml 

202 5 Mg/0. 1 ml 

TA 98 

49 

50 

50 

55 

50 

64 

TA 100 

93 

100 

1 0 3 

98 

96 

1 0 3 

TA 1535 

11 

12 

19 

21 

22 

20 

TA 1537 

8 

6 

5 

6 

6 

5 

TA 1538 

Positiv e contro l of th e 

microsoma l activatio n 

cyclophosphamid e 

Contro l 18 

250 Mg/0. 1 ml 280 

2-aminoanthracen e 

Contro l 

5 Mg/0. 1 ml 
TABLE 3 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 Tes t substance : TK 12 44 3 

Date : Januar y 04 , 1982 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l 

25 Mg/0. 1 ml 

75 Mg/0. 1 ml 

225 Mg/0. 1 ml 

675 Mg/0. 1 ml 

202 5 Mg/0. 1 ml 

Positiv e control s 

Contro l 

Concentratio n A 

B 

29 

23 

30 

33 

32 

33 

35 

314 

353 

104 

101 

117 

104 

107 

111 

102 

298 

689 

17 

16 

13 

18 

21 

17 

8 

1157 

1590 

5 

6 

8 

12 

9 

4 

4 

68 

976 
TABLE 4 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 

Date : Januar y 04 , 1982 

Tes t substance : TK 12 443 

STRAIN 

Contro l 

25 Mg/0. 1 ml 

75 Mg/0. 1 ml 

225 Mg/0. 1 ml 

675 Mg/0. 1 ml 

2025 Mg/0. 1 ml 

Positiv e contro l of th e 

microsoma l activatio n 

98 

49 

50 

50 

55 

50 

64 

TA 100 

93 

100 

103 

98 

96 

103 

TA 1535 TA 1537 TA 1538 

11 

12 

19 

2 1 

22 

20 

Contro l 

Concentratio n A 

18 

280 
11 

13 

12 

8 

14 

14 

8 

6 

5 

5 

3 

6 

TABLE 5 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN) 

No. of experiment : 811194 Tes t substance : TK 12 44 3 

Date : Januar y 14 , 198 2 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l 

25 Mg/0. 1 ml 

75 pg/O. l ml 

2 2 5 Mg/0. 1 ml 

6 7 5 Mg/0. 1 ml 

202 5 Mg/0. 1 ml 

Positiv e control s 

dauno r ubicin-HC l 

Contro l 

5 Mg/0. 1 ml 

10 Mg/0. 1 ml 

4-nitroquinoline -

N-oxid e 

Contro l 

0.12 5 Mg/0. 1 ml 

0 . 25 Mg/0. 1 ml 

N-methyl-N'-nitro -

N-nitrosoguanidin e 

Contro l 7 

3 Mg/0. 1 ml 266 

5 Mg/0. 1 ml 119 5 

9(5)aminoacridine -

hydrochlorid e 

Contro l 6 

50 Mg/0. 1 ml 25 

100 Mg/0. 1 ml 389 

2-nitrofluoren e 

Contro l 

5 Mg/0. 1 ml 

10 Mg/0. 1 ml 
' TABLE 6 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE (ARITHMETIC MEAN) 

No. of experiment : 811194 Tes t substance : TK 12 44 3 

Date : Januar y 14 , 1982 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l 

25 Mg/0. 1 ml 

75 Mg/0. 1 ml 

225 Mg/0. 1 ml 

675 Mg/0. 1 ml 

202 5 Mg/0. 1 ml 

Positiv e contro l o f th e 

microsoma l activatio n 

cyclophosphamid e 

Contro l 10 

250 Mg/0. 1 m l 412 

2-aminoanthracen e 

Contro l 

5 Mg/0. 1 m l 

13 

10 

12 

12 

11 

9 

5 

4 

6 

6 

6 

4 
TABLE 7 

SALMONELL-A/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. o f experiment : 811194 Test substance : TK 12 44 3 

Date : Januar y 14 , 1982 

STRAIN TA 98 TA 100 

Contro l 

2 5 Mg/0. 1 ml 

75 Mg/0. 1 ml 

2 2 5 Mg/0. 1 ml 

6 7 5 Mg/0. 1 ml 

202 5 Mg/0. 1 ml 

Positiv e control s 

(i>ntro l 

Concentratio n A 

B 

1535 TA 

11 

13 

12 

8 

14 

14 

7 

266 

1195 

1537 

2 

38 

TA 1538 

8 

6 

5 

5 

3 

6 

6 

• 

!5 

19 
TABLE 8 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 Tes t substance : TK 12 44 3 

Date : Januar y 14 , 198 2 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l

13 5 

25 Mg/0. 1 ml

10 4 

75 Mg/0. 1 m l

12 6 

2 2 5 Mg/0. 1 m l

12 6 

675 Mg/0. 1 m l

11 6 

2025 Mg/0.1 ml

9 4 

Positiv e contro l of th e 

microsomal activatio n 

Contro l

10 

Concentratio n A

412 
Verteiler : 

HH.

( 

 Test No.: 811194 

Protection of Health 

and Environment 

GU 2.3 

Experimental Pathology 

SUPPLEMEN T TOTH E REPOR T 

(date d Februar y 5 , 1982 ) 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

Test material: TK 12 443 

Date: June 17, 1987 
-2-

RAW DATA 

Toxicitv test (table 1); 

A preliminary toxicity test was carried out on strain TA 100 

without microsomal activation with the concentrations ranging from 

0.0001 to 2000 ug/0.1 ml. The protocol used was the same as in the 

mutagenicity test, however, only two plates per concentration were 

used. The individual plate counts and the mean values are given in 

columns 4, 5 and 6 of table 1. There was no growth-inhibiting effect of TK 12 443 observed. At the concentrations of 50 ug/0.1 ml 

and above the test material precipitated in soft agar (*). 

Mutagenicity test (tables 2 to 5): 

Copies of the original computer outprints showing individual 

values per plate and mean values per group are given. 

Study director: 

Date: May 22. , 19 87 

Reviewed and approved by; 

Date: June 17, 1987 
Table 1 

MIKROBIELLE MUTAGENITAETSPRUEFUNG 

Bestironunq der Toxizit&t 

Vers.Nr.t.^Jj^?.H.... . Testsubstanz : . X )?-. V^?i. Charge^. ^.^^. ^>?tr . 

Teststamm: TA lOO OK: .|$^ XX^ V . . Vehikel:. fi<Vi>ytK\ . . 

VersuchsdurchfQhrung (Datxun/Visum) : . ^.-.X^-.^V . . /. ^ ... . 

Versuchs auswertung (Daturo/Arisum) :..%%.: }Xi.%K ../ . .ilH... 

Substanzverdiinnunqen; 

LSsung 

pg/0. 1 ml 

-

9xx>o 

\oc»o 

Sbo 

}oo 

teO 

\o 

I 

« . ; 

aoi 

eiooA 

1Kontroll e 

Verdiinnung (ml) 

L6sg.+Vehike l 

i A) 

* * ; 

>H-M 

> * « 

;*s 

Qf.-tw.: 

^ ^' tv \r 

o,f-t ^.r 

'^a * ^. ^ 

<\i-t k^ ' 

Dos i s 

pg/0. 1 ml 

JLooo 

;oo o 

£Do 

LOO 

5 0 

\o 

) 

^ } 

o.oi 

2*»QL -

0,oo*J 

0 . 1 ml 

le 

X-

«• 

}t 

*k 

Kolonie n 

1 

 

 

/Platt e 

2 

X 

jiJ.0 

St.o 

<3o,o 

«t,0 

;ot.r 

8itO 

jola 

j^<..r 

)0>iT> 

<n.,r 

^J^if 

S&io 

Positive Kontrolle 

4-Nitrocruinolin-N-oxi d 

VehiJcel: P.P . 

_ , Konz. 

Platt e 

1 

2 

X 

K 

jos: 

)Ar 

no.o 

OJ.25 pg 

UU 

S«l 

^ | 0 

0 . 25 pg 

=Klt 

t^r 

X««..o 

Kontrolle (Datum/Visum) ...k-.d:f:^./.L^ 
Tabl e Z 

TABLE A (RAW DATA) 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHCXJT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 

Date : Jan . 04 , 1982 

STRAIN 

Contro l 

25 pg/0. 1 ml 

75 jjg/0. 1 ml 

225 |jg/0. 1 ml 

675 jjg/O.l ml 

2025 pg/O. 1 ml 

•• 

Positiv e controls 

Contro l 

Concentratio n A 

B 

811194 

TA 98 

29 

23 

30 

33 

32 

33 

32 

35 

314 

353 

Testsubstan c 

Apparatu s cc 

TA 100 TA 

104 

101 

117 

104 

107 

111 

84 

102 

^^6^i 

: e : TK 12 

intro l (10 

1535 Tl 

17 

16 

13 

18 

21 

17 

9 

8 

1157 

1590 

44 3 

10): 

i 1537 

5 

6 

8 

12 

, 9 

4 

7 

4 

68 

976 

Visa :

99 

TA 1538 

^cCtk ILMJ^ . 

UU.^' ^ ^ 
Tabl e 3 

TABLE B (RAW DATA) 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 

Date : Jan . 04 , 1981 

Testsubstance : TK 12 443 Visa :

Apparatus contro l (100) : 101 

STRAIN 1 

Contro l 

25 pg/0. 1 ml 

75 pg/0. 1 ml 

225 pg/0. 1 ml 

675 jjg/O-l nil 

2025 jjg/O-l ml 

' 

Positiv e contro l of 

t h e microsoma l activ a 

Contro l 

Concentratio n A 

PA 98 

49 

50 

50 

55 

50 

64 

tio n 

TA 100 TA 

93 

100 

103 

98 

96 

103 

• 

—"_-• 

1535 TA 1537 

.6-/A 

^iiS j 

19 

21 <^/L 

22 X 

20 

18 

280 
Table 4 

TABLE A (RAW DATA) 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITHOUT MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 Testsubstance : TK 12 443 Visa :

Date : Jan . 14 , 1982 Apparatu s contro l (100): 100 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l 

25 jjg/O.l ml 

75 pg/0. 1 ml 

225 pg/O. l ml 

675 pg/O. l ml 

2025 Mg/0.1 ml 

Positiv e controls 

Contro l 

Concentratio n A 

B 

11 

13 

12 

8 

14 

14 

7 

266 

1195 

8 

6 

5 

5 

3 

6 

6 

25 

389 
Table 5 

TABLE B (RAW DATA) 

SALMONELLA/MAMMALIAN-MICROSOME MUTAGENICITY TEST 

EXPERIMENTS WITH MICROSOMAL ACTIVATION 

NUMBER OF BACK-MUTANT COLONIES PER PLATE AND ARITHMETIC MEAN 

No. of experiment : 811194 

Date : Jan . 14 , 198 2 

Testsubstance : TK 12 44 3 

Apparatu s contro l (100): 

Visa:

100 

STRAIN TA 98 TA 100 TA 1535 TA 1537 TA 1538 

Contro l 

25 pg/O.l rol 

75 jjg/O.l rol 

225 pg/O. l rol 

675 pg/O. l rol 

2025 Mg/0.1 rol 

Positiv e contro l of 

t h e roicrosoroal activatio n 

Contro l 

Concentration A 

13 

10 

12 

12 

11 * 

10 

412