The following criteria determined the acceptance of an assay: 

1 . The negative controls had to be within the expected range, as defined by published data (e.g . Maron and Ames, 1983) and/ or the laboratories' own historical data (see Chapter 8). . 

2. The positive controls had to show sufficient effects, as defined by the laboratories' experience (see Chapter 8). 

3. Titer determinations had to demonstrate sufficient bacterial density In the suspension. 

Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted 

if It showed mutagenic activity of the test compound. Furthermore, an unacceptable 

trial would have been repeated. 

The following doses re evaluated: 

Posillve confIR'I~"a-azide 

Positive control, NF 

Positive control, 4NPDA 

Positive control, 4-NPDA 

Positive control, MMC 

Positive control, Cumene 

Positive control, 2-AA 

Ng per plate 

0 

5000 

1581 

500 

158 

50 

10 (only TA 1535) 

0.2 (only TA 100) 

10 (only TA 1537) 

0.5 TA 

0.2 (oniy TA 110e) 

50 (only TA 1023) 

3 

' only in plate Incorporation trials 3 only In proincubation trials "hUBLIC COPY 
Test 

21 

Cd 

formulated in deionized water and formed black suspensions. 

49 "nw%wWvasdIssoIved in deionized water. The other positive controls were dis-~ 

solved In DMSO. 

The formutatkm was performed according to 

the following proce ure: were placed in a wide-necked 

100 ml Erfmeyer flask. 30 ml deion wow w added and the flask was dosed 

with it plug. The flask was set into an ultrasonic water bath, which contains water to 

fill the bath up to approximately the 50 ml mark. The ultrasonic treatment was started. 

After about 15 minutes ice was added into the water to avoid an Increase of water 

temperature. The ultrasonic treatment continued for further 15 minutes at about 

25" C. During this time water temperature was controlled and further Ice was added to 

keep the water temperature at about 25°C. Thereafter, the formulation was kept at 

room temperature and mixed prior to use wit a rotation mixer. 

The vehicle used was chosen out of the following solvents, in the order given: water, 

DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF 

according to Information given by the Internal sponsor. The order of these solvents is 

based on their bacteriotoxic effects In preincubatkm experiments. 

No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Manm and Ames, 1983) and from our own 

experience (see Chapter 8), Indicating that this solvent had no Influence on the spontaneous mutant counts of the used strains. . 

4.5. Assessment Criteria 

A reproducible and dose-related Increase In mutant counts of at least one strain Is 

considered to be a positive result For TA 1535, TA 100 and TA 98 this Increase 

should be about twice that of negative controls, whereas for TA 1537, at least a 

threefold Increase should be reached. For TA 102 an Increase of about 100 mutants 

should be reached. Otherwise, the result is evaluated as negative. However, these 

guidelines may be overruled by good scientific judgment. , 

In case of questionable results, Investigations should continue, possibly with modiflcations, until a final evaluation Is possible. 

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q"`monelf-alF~xi xosome Test 

22 

4.6. Study Guidelines 

The study was performed according at least to the folbwing guidelines: 

Commission Directive 200013?JEC of 19 May 2000 

, Official Journal of the European Communities of 8.6.2000, L 136/57- L136/64 

B.13114. Mutagenidty - Reverse Mutation Test Bacteria 

OECD Guidelines for Testing of Chemicals No. 471 

"Bacterial Reverse Mutation Test" 

Adopted: 21 st July 1997 

Health Effects Test Guidelines; United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA712-G98-247, August 1998 

OPPTS 870.5100 - Bacterial Reverse Mutation Test 

4.7 . Study Identilkation and Responsibilities 

4.7 .1 . Type of Test and Stu 

Salmonella/Mkxosome 

4.7.2 . Responsibilities 

Head of Toxicology 

Section Head Genetic Toxicology 

Study Director 

Senior Technlciart 

Head of Archives 

Quality Assurance 

Analyst(s) 

U-BLIC COP 
S. Results 

5.1 . Description of Results 

The colony numbers of each plate and mean values are listed for each dose 

may be seen, there was no Indication of a bacteriotoxic effect 

t doses of up to and Including 5000 pg per plate. The total bacteria 

coun cons ntly produced results comparable to the negative controls, or differed 

only Insignificantly. No inhibition of growth was noted as well. At 158 Pg per plate, the 

substance started to precipitate . 

None of the five strains concerned showed in the plate incorporation test a doserelated and biologically relevant increase In mutant counts over those of the negative 

controls. This applied both to the tests with and without S9 mix (Tables 1 to 5) and 

was confirmed by the results of the preincubadon trials, ables 6 to 10). 

Summary of the Results 

in the Salmonella/M 

S9 mbc TA 1535 TA 100 TA 1537 TA 98 TA 102 

without -ve -ve -ve -ve -ve 

with ~-- _Vs -ve -ve -ve -ve 

-ve = negative 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, 

, mitomydn C, cumene hydroperoxide and 2-aminoanthracene increased mutant 

counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix. 

PUBLIC COPY 
aTrrioneli-a/Microsome Test 

5.2. Tabulated Summary of Data 

24 

Summa of Mean Values Without S9 Mix 

Table and Strain 

Group TA 1535 TA 100 TA 1537 TA 98 TA 102 

1-5 

NgIPiate . 

0 11 121 6 18 198 

50 12 130 .5 15 204 

158 11 121 5 14 240 

500 10 124 8 10 207 

1581 7 111 4 9 224 

5000 8 94 3 11 212 

Na-azide 635 

NF 292 

4-NPDA 81 . 117 

MM 460 

6-10 

NgITube 

0 14 132 ~ 14 244 

50 15 133 7 15 258 

158 12 128 8 13 211 

500 13 130 5 12 245 

1581 12 128 3 9 242 

5000 12 123 3 9 213 

Na-azide 748 

NF ' 489 _ 

4-NPDA 109 127 . 

Cumene 498 

PUBLIC COPY' 
Summa of Mean Values With S9 Mix 

Table and Strain 

Group TA 1535 TA 100 TA 1537 TA 98 TA 102 

1-5 

NgIPlate 

0 11 138 10 . 28 292 

50 9 143 8 22 263 

158 9 138 8 20 203 

500 7 127 7 20 258 

1581 6 1-17 4 12 272 

5000 8 114 5 18 228 

2-AA 183 1223 229 783 841 

B-10 

ir9lTube 

0 12 188 11 25 ' 284 

50 14 188 10 21 268 

158 15 190 9 15 249 

500 11 182~ 10 14 292 

1581 12 175 5 13 288 

5000 11 173 3 12 207 

2-AA 181 1161 192 995 462 

PUBLIC COPY 
8. Assessment 

test, employing doses of up to 5000 Irg per plate, showed 

t to produce baderiotoxic effects . Substance precipitation oocurred at 158 -09 -Per plate and above. 

Evaluation of individual dose groups, with respect to relevant assessment parameters 

(dose effect, reproducibility) - revealed no biologically relevant variations from the respectlve negative controls. . 

In spite of the low doses used, positive controls increased the mutant counts to well 

over those of the negative controls, and thus demonstrated the system's high sensitivity. 

Despite this sensitivity, no Indications of mutagenic effects 

could be found ax assessable doses of up to 5000 Ng per plate in any e Salmonella .typhimurium strains used. 

Due to these resul ~as to be regarded as non-mutagenic. 
27 

7. References 

1 . Ames, B.N. : 

Identifylng environmental chemicals causing mutations and cancer 

Science 204, 587-5g3,1979 . 

2. Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee: 

Carcinogens are mutagens: A simple test system combining liver homogenates'for activation and bacteria for detection 

Proc. net. Aced. Sd. (USA) 70, 2281-2285, 1973a 

3. Ames, B.N., F.D. Lee and W.E. Durston: 

An improved bacterial -test system for the detection and classification of 

mutagens and cardnogens , 

Proc. nat. Aced. Sci. (USA) 70, 782-786,1973b 

4. Ames, B.N., J . McCann and E. Yamasaki: 

Methods for detecting carcinogens and mutagens with the Salmonellalmammalian-micxosome mutagenicity test 

Mutation Res. 31, 347-364,1975 

5. Andrews, A.W., L.H. Thibault and W. Lijinsky: 

The relationship between cardnogenidty and mutagenicity of some polynudear hydrocarbons 

Mutation Res. 51, 311-318, 1978 

8. Clayson, D'.B. : 

ICPEMC working paper 2/1 . Comparison between In vitro and In vivo tests 

for carcinogenicity. An overview 

Mutation Res. 75,205-213, 1980 

7. Glatt, H.R., H. Schwind, F. Zajdela, A. Crolsy, P.C. Jacquignon and F. 8sch: 

Mutagenicity of 43 structurally related heteroc.yclic compounds and its relationship to their cardnogenicity 

Mutation Res. 66, 307-328,1979 

PUBLIC CO'

, ~ 
8. Herbold, B., W. Buselmaier and G. Rbhrbom: ' 

Krltlsche Betrachtung zum Lebemnikrosomen-Test, einem System zum 

Nachweis von Punktmutaflonen 

Arzneim.-Fon3ch. 26,1705-1707,1976 

9. Herbold, B. : 

Mutagenit8tsuntersuchungen mit dem Lebermikrosomen-Test 

Biol. Zbl. 97,137-152,1978 

10. Herbold, B.A. : - 

Preliminary results of an international survey on sensitivity of S. typhimurium 

strains in the Ames test . 

Toxicology Letters 15, 89-93,1983 

11 . Levin, D.E., M. Hollstein, M.F. Chrisbnan, E.. Schwiers and B.N. Ames: 

A new Salmonella tester strain (TA 102) with A -T base pairs at the site of 

mutation detects oxidative mutagens 

Proc. Nag. Aced. Sd. (USA) 79, 7445-7449,1982 . 

12. Maron, D.M. and B.N. Ames: 

Revised methods for the Salmonella mutagenicity test 

Mutation Res. 113,173-215,1983 , 

13. McCann, J ., E. Choi, E. Yamasaki and B.N. Ames: 

Detection of carcinogens as mutagena in the Salmonellalmlcrosome Test: I . 

Assay of 300 chemicals 

Proc. naL Aced. Sd. (USA) 72, 5135-5139, 1975a 

14. McCann, J., N.E. Spingam, J. Kobori and B.N. Ames: 

Detection of carcinogens as mutagens: Bacterial tester strains with R factor 

plasmids 

Proc. nat. Aced. Scl. (USA) 72, 979-983,1975b 

PUBLIC COPY 
29 

10=0 cxosome Test 

~D 

15. McCann, J. and B.N. Ames: 

Detection of carcinogens as mutagens In the Salmonellalmicrosome test: 

Assay of 300 chemicals : Discussion . 

Pnoc. net. Acad. Sci. (USA) 73; 950-954,1976 

16. Purchase, I .F.H., E. Longstaff, J. Sahib, J.A. Styles, D. Anderson, P.A. Lefevre and F.R. Westwood: 

Evaluation of six short term tests for detecting organic chemical carcinogens 

and recommendations for their use . 

Nature (London) 264, 624-627,1978 

17. Purchase, I .F.H., E. Longstaff, J. Sahib, J.A. Styles, D. Anderson, P.A. Le- . 

fevre and F.R. Westwood: 

An evaluation of six short term tests for detecdhg organic chemical carcinogens ' 

Brit. J. Cancer 37, 873-959, 1978 

18. Rinkus, S.J . and M.S. Legator. 

Chemical characterization of 485 known,or suspected carcinogens and their 

correlation with mutagenk; activity in the Salmonella typhimurium system 

Cancer Res. 39, 3289-3318,1979 

19. Zeiger, E: : 

Cardnogenidty of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent cardnogenicity 

Cancer Res. 47,1287-1296,1987 

PUBLIC COPY 
SalmonellalMicxosome Test 

- 

8. Historical Controls 

8.1 . Plate incorporation Method 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Seml-O Range (OR) 

1996 

Compound Strain I 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 mix Z OR Z OR Z OR Z OR Z OR 

Water 9 

_ 

2 88 12 8 1 23 4 228 31 

DMSO - 9 2 70 8 7 ~ 1 '19 3 220 30 

EGDE 12 2 79 9 7 1 18 5 214 21 

Na-azid - 741 80 I 

NF - 224 22 

4-NPDA - 118 17 155 20 ' 

Cumene - 430 61 

Water + 11 1 103 10 9 2 35 ' 4 287 30 

DMSO + 10 2 89 7 8 2 27 6 288 28 

EGDE + 11 2 95 12 7 1 29 10 305 __34 

2-AA + 150 36 1161 208 222 62 , 1343 220 555 93 

PUBLIC COPY 
Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (QR) 

1997 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 8 2 92 13 8 1 23 5 201 42 

DMSO - 8 1 82 14 8 2 19 4 191 43 

EGDE 11 3 94 4 8 2 21 5 179 22 

Ethanol - 8 - 97 - 8 - 27 - 158 - 

Na-a7Jd - 780 90 

NF 229 25 

4NPDA - 98 21 152 21 

Cumene - 331 81 

Water + 11 2 110 13 10 3 27 5 295 64 

DMSO + 9 9 101 8 10 2 27 3 . 285 ~ 45 

EGDE + 10 2 112 8 11 3 30 5 297 47 

Ethanol + 11 - 97 7 - 36 - 185 

12-AA +1 

_ _ 

187 49 1 -1128 188 148 54 1 1475 248 503 125 

1998 

Compound Strain 

and TA 1 535 TA 100 TA 1537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 10 2 81 10 9 3 17 3 209 47 

DMSO - 9 2 79 10 8 1 14 2 187 41 

Na-azid - 692 59 

_ 

NF - 236 23 

4NPDA - 126 18 147 18 

Cumene - 387 82 

Water + 10 2 108 9 10 2 29 4 300 40 

DMSO + 11 2 97 11 10 3 23 4 278 83 

~2-AA + 235 39 1150 275 194 68 1281 195 524 113 

PUBLIC CO Y 
mo*ne crosome Test 

32 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (QR) 

1999 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z QR Z OR Z OR Z QR Z QR ~ 

Water 8 2 82 22 7 1 21 3 185 28 

DMSO 8 1 73 13 7 2 17 3 174 30 

Ethanol - 7 - 99 - 5 - 19 - 292 

EGDE 12 - 90 - 6 - 19 - 298 - 

DMF 7 - 70 - 5 - 22 - 198 

Na-azid ' - 642 79 . 

NF 217 28 

4NPDA - 105 15 137 20 

Cumene - 351 50 

Water T + 9 2 102 31 7 2 28 7 273 22 

DMSO + 9 2 92 17 8 1 24 2 248 31 

Ethanol + 7 - 123 - 8 - 24 - 307 

EGDE + 10 - 115 - 8 - 26 - 385 

DMF + 10 98 8 - 24 - 268 ' 

2-AA + 184 43 1 1290 

_ 

304 221 571 1295 1941 477 79 1 

PUBLIC COPY 
Summary of Historical Negative and Positive Controls 

' using mean values Presented as 

Medians (Z) and Semi-O Range (OR) 

Compound Strain 

and TA 1535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 9 1 81 11 7 2 25 6 196 . 32 

DMSO - 9 2 73 17 - 7 1 23 6 209 30 

Ethanol - 12 - 97 - 6 - 31 - 209 _ 

Na,azld - 629 70 

NF - 228 35 ' 

4-NPDA - 106 15 173 21 

Cumene - 

_ 352 43 

Water + 11 1 104 14 10 2 37 5 260 41 

DMSO + 10 . 2 94 20 8 2 32 7 270 34 

Ethanol + 12 ~ - 108 - 13 - 42 - 230 - 

2-AA + 207 38 , 1468 223 252 88 1524 160 539 68 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 9 3 100 - 22 8 1 39 4 223 42 

DMSO - 9 2 100 13 8 2 34 13 208 35 

Acetone - 8 - 113 - 8 ' 38 - . 233 - 

Na-azid - 633 78 

NF 264 28 

4NPDA - 94 13 172 19 

Cumene - . 

365 ' 24 

Water + 11 2 119 22 9 2 50 6 283 30 

DMSO + 10 2 125 19 9 2 41 9 267 32 

Acetone + 12 - 129 - 10 - 45 - 317 - 

2 -AA + 193 26 1614 . 97 270 78 , 1443 67 532 71 

2000 

2001 

PUBLIC COP 
34 

Summary of Historical Negative and Positive Controls 

. Using Mean Values Presented *as . 

Medians (Z) and Semi-Q Range (QR) 

2062 

Compound - Strain . 

and TA 1 535 TA 100 TA 1537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR . Z OR Z OR 

Water - 14 3 130 29 8 3 35 8 222 50 

DMSO - 14 5 ~136 15 7 2 31 9 216 35 ' 

EGDE - 21 7 148 8 8 2 27 5 262 38 

Na-azid - 735 80 

NF 323 30 . 

4-NPDA - 88. 11 156 24 

Cumene - 558 92 

Water + 11 3 151 25 10 2 48 14 275 38 

DMSO + 12 4 151 19 8 2 39 12 270 24 

EGDE + 9 3 144 19 7 1 33 6 306 45 

2-AA +- 158 30, 1506 128 258 60, 1283 139 554 64 

PUBLIC C OF 
35 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (OR) 

2003 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 14 5 140 20 7 . 2 28 4 252 44 

DMSO - 14 3 144 13 7 1 24 5 227 28 

EGDE 18 2 167 - 7 - 26 " - 243 - 

Ethanol - 14 - 150 - 8 - 18 - 221 - 

Acetone - 15 - 134 - 9 - 42 - 279 - 

Na-azid - 679 90 

NF 358 42 

4-NPDA - 91 12 157 18 

MMC 530 63 

Water + 13 1 177 30 10 2 41 6 281 32 

DMSO + 11 2 169 22 9 2 34 6 294 32 

EGDE + 11 1 184 - 10 - 39 - 280 

Ethanol + 25 - 177 - 9 - 38 - 227 

Acetone + 10 - 172 - 12 - 31 - 311 

2-AA + 170 281 1526 143 288 781 1247 138 721 1141 

PUBLIC COPY 
e Test 

38 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (QR) 

2004 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 18 3 150 12 8 1 24 7 227 13 

DMSO - 16 ' 4 135 15 7 1 26 9 199 14 

EGDE - 16 - 156 - . 7 - 26 - 263 - 

Ethanol - 16 - 135 - 7 - 27 - 218 

Na-azid - 851 121 

_ 

NF 339 28 

4-NPDA - 91 18 153 16 

MMC ~ 541 59 

Water + 13 2 158 20 11 2 42 11 255 20 

DMSO + 11 2 153 23 10 2 40 9 255 24 

EGDE + 12 - 191 - 8 - 48 . - 301 

Ethanol + 20 - 170 - 10 - 40 - 251 

2 AA + 150 29 1438 118 259 57 1164 112 573 79 

PUBLIC COPY 
Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as . 

Medians (Z) and Semi-O Range (4R) 

Compound Strain 

and TA 1 535 TA 100 TA 1537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water - 19 7 162 22 7 1 25 5 223 20 

DMSO - 18 3 142 17 7 1 24 8 214 22 

EGDE - 19 3 152 33 7 1 29 7 254 27 

Ethanol - 11 - 138 7 - 21 - 238 - 

DMF - 10 - 125 - 6 - 24 - 212 - 

Na-azld - 652 66 

NF 310 33 

4-NPDA - 89 12 149 17 

MMC 518 38 

Water + 13 3 .205 41 ' 8 1 41 8 280 44 

DMSO + 11 2 178 22 8 2 44 8 284 30 

EGDE + 18 2 152 17 8 1 43 7 310 31 

Ethanol + 15 - 127 - 11 - 31 -' 287 

DMF + 10 - 171 - 7 - 51 - 284 

2-AA + 168 28 1 1426 142 185 58 1 1157 162 1 718 99 1 

2005 

PUBLIC COPY 
8.2 . Preincubation Method 

38 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Seml-Q Range (OR) 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 11 3 100 19 9 2 31 9 252 32 

DMSO ' - 9 2 80 9 8 1 ~22 7 271 23 

EGDE 12 1 122 16 8 1 28 3 276 13 

Na-azld - 747 71 

NF 278 30 

4NPDA - 127 21 183 27 

Cumene - 602 97 

Water + 13 2 117 22 10 2 35 8 331 45 

DMSO + 11 2 94 9 8 2 28 8 337 33 

EGDE + 14 2 139 24 8 2 39 5 400 62 

~2-AA + 172 39 1318 154 164 69 1239 143 601 93 

1998 

PUBLIC COr `, 
Summary of Historical Negative and Posidve Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q. Range (OR) 

1997 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 10 2 137 23 9 2 24 4 252 24 

DMSO - 8 . 1 86 11 7 2 18 3 258 34 

EGDE 10 1 138 9 9 2 23 5 314 12 

Na-,azid - 705 98 

NF - 258 26 

4NPDA - 98 32 170 18 

Cumene - ' 564 79 

Water + 10 2 147 28 9 2 26 4 304 38 

DMSO + 10 2 100 12 8 2 26 3 333 46 

EGDE + 11 1 157 3 8 3 29 3 380 45 

2-AA -+, 

_ 

141 32, 1215 151 63 47 , 1232 

_ 

212, 548 100 , 

1998 

Compound Strain 

and TA 1 535 TA 100 TA 1537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 11 2 119 15 11 3 17 3 285 29 

DMSO - 9 2 82 8 8 2 16 3 241 29 

Na-azid - 729 68 

NF 272 28 

4-NPDA - 143 19 159 19 

Cumene - 580 60 

Water + 13 2 152 13 12 2 27 3 384 32 

.DMSO + 11 2 ~98 9 9 2 27 3 325 34 

2-AA + ~ 168 33 1269 241 155 42 1232 188 523 73 

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-&-~llcrosorne Test 

40 

------ 

. Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (QR) 

1999 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

ater - 10 2 117 10 9 2 22 6 247 32 

DMSO - 7 2 72 12 6 1 15 ~ 3 223 20 

Ethanol - 7 - 90 - 7 - 24 - 294 - 

EGDE 10 . - 120 - 16 13 - 279 - 

DMF 12 135 - 9 - 15 - 282 

Na-azid - 638 59 

NF - 258 29 

4NPDA - 136 17 139 12 

Cumene - 525 54 

Water + 11 2 144 30 12 2 28 7 294 15 

DMSO + 8 2 88 13 8 1 23 3 298 22 

Ethanol + 11 - 98 - 8 - 28 - 312 

EGDE + 11 - 173 - 13 - 22 - 329 

DMF + 14 - 158 - 12 29 - 312 

2-AA + 149 32 1190 181 203 42 1 1117 149 467 53 

PUBLIC COPY 
Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as . 

Medians (Z) and Semi-Q Range (OR) 

Compound Strain 

and . TA 1 535 TA 1 00 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 11 3 135 8 10 2 24 5 239 23 

DMSO 8 2 80 16 8 2 21 6 237 20 

Ethanol - 9 - 103 - 11 - 44 - 228 - 

Na-azid - 620 72 

NF 294 55 

4-NPDA - 138 14 178 22 

Cumene - 

469 43 

Water + 12 2 148 15 12 2 31 4 297 28 

DMSO + 9 1 90 14 9 2 29 6 298 20 

Ethanol + 13 - 127 - 14 - 51 - 211 - 

2-AA + 178 36 1353 . 213 239 54 , 1349 180 488 39 

Compound Strain. 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 MIX Z OR Z OR Z . QR Z OR Z OR 

Water - 10 2 121 24 9 1 40 8 269 25 

DMSO 8 2 107 18 7 1 32 6 229 24 

Acetone - 13 - 120 - 10 - 37 - 271 - 

Na-azid - 614 92 ' 

NF 403 44 

4-NPDA - 121 17 182 20 

Cumene - 429 48 

Water + .11 1 136 38 10 1 - 43 9 305 32 

DMSO + 10 2 123 20 9 2 40 6 281 27 

Acetone + 15 - 142 - 14 - 51 - 315 

2-;AA + 196 28 1627 ~93 294 48 1442 120 1 470 471 

2000 

2001 

PUBLIC COPY 

3 S7 
Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (OR) 

Compound .Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water - 17 5 153 9 8 2 32 13 272 18 

DMSO - 12 3 145 13 1 2 29 6 255 32 

EGDE - 18 4 158 12 9 2 33 7 284 42 

Na-azid - 740 95 

NF 447 31 . 

4-NPDA - 114 ~14 185 17 

Curnene - . ' 480 . 35 

Water + 15 2 187 20 11 2 , 45 11 322 19 

DMSO + 11 2 155 18 9 1 38 9 314 38 

EG E + 12 10 179 20 9 2 48 10_ 342 35 

2-AA +l . 170 22 1550 122 318 57 ,12 34 149 1 509 34 I 

2002 

PUBLIC COPY 
me Test 

43 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (OR) 

2003 

Compound Strain . 

-and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z OR Z QR Z QR Z OR Z QR 

Water - 18 2 154 19 10 3 31 10 246 26 

DMSO - 15 3 137 18 8 1 20 5 236 24 

EGDE - 16 - 161 - 8 - 26 - 262 , 

Ethanol - 14 - 142 - 7 - 23 - 249 - 

Acetoine - 20 - 134 - . 12 - 33 - 340 

Na-azid - 664 88 

NF ~ 463 37 . 

4-NPDA - 120 18 164 17 

Curflen _ e - 477 44 

Water + 13 5 173 32 9 1 39 11 303 63 

DMSO + 11 2 159 24 9 2 34 5 297 28 

EGDE . + 15 - 213 - 12 - 52 - 288 

Ethanol + 21 - 181 . - 8 - 39 - 308 

Acetone + 9 - 159 - 17 28 - 398 

2-AA + 187 281 1527 159 345 601 1255 144 1 613 94 1 

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lmonel a rosome Test 

44 

Summary of Historical Negative and Positive Controls . 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (OR) 

2004 

Compound ~ Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 TA 102 

S9 Mix Z (1R Z OR Z OR Z OR .Z OR 

Water 18 3 151 9 11 2 25 5 224 14 

DMSO 15 3 134 10 8 1 24 8 209 19 

EGDE ~ 14 - 148 - . 9 - 21 - 253 - 

Ethanol - 18 - 130 - 6 22 - 218 

Na-azid - 609 107 

NF 460 56 

4-NPDA - 111 18 167 18 

Cumene - 470 34 

Water + 14 2 182 10 10 2 35 5 288 11 

DMSO + 12 2 158 24 10 2 38 7 251 25 

EGDE- + 13 - 208 - 11 - 51 - 298 - 

Ethan6i + 24 - 182 9 34 - 221 - 

12-AA + 183 14 1 1515 134 293 25 1 1145 120 512 

_ 

56 

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3 b6, 
.'~ 

ii e Test 

45 

coi& 

Summary of Historical Negative and Positive Controls 

Using Mean Values Presented as 

Medians (Z) and Semi-Q Range (OR) 

Compound Strain 

and TA 1 535 TA 100 TA 1 537 TA 98 ' TA 102 

S9 Mix Z OR Z OR Z OR Z OR Z OR 

Water 18 4 ~ 158 15 8 3 29 6 241 38 

DMSO 17 4 144 18 7 2 27 8 228 19 

EGDE 15 - 144 - 8 - 23 - 239 

Ethanol - 13 - 139 - 8 - 30 - 260 

DMF 12 93 - 8 - 32 . - 270 

Na-azid - 637 .72 

NF - 451 44 

4-NPDA - 107 21 155 11 

Cumene - 428 39 

Water + 14 2 205 30 

_ 

11 2 44 8 289 47 

DMSO + 11 3 183 31 9 2 44 7 289 33 

EGDE + 13 - 193 - 11 - 32 - 308 

Ethanol + 21 - 191 - 10 - 29 - 278 

DMF + 10 124 - 9 51 - 284 - - 

2-AA + 161 18 1 1448 159 297 46 1 1142 97 807 99 

2005 

PUBLIC COPY 
p. 27 

9. Statement of GLP Compliance 

Alr Umwelt tlfid Nstiu~l ~_ Landwirtschaft und 

V~! t 

do Lande Not+lirheia-W en . 

101lt Dtrif A.Itnl+r W4- 31.1 IAf,eJ 

Gate LsbatpnsINYsed Lattepterqr Practise 

OLp-HeseAslalgnnglatatsatejlt of OLP Compliance 

(O4Mttilaseer/Ing to 11!Y Ah. 1 CheawlYalisnpssta) 

a ;Y~sat!4mpmm ~ar~~w~~ A~nufi 

r 

ttlte,I4~aVn~e~lltar 

malowuw aet~alKlt . 

'+~twwwer»o~oem - - 

"- 

Pt011+rqm nech K-apollgn Area of eNOWsm 

1."rae.wrv,ovata8oroo,rrry p ewqayaaoo,~rr.l 

Kalp'"»: erpetp : 

11"Mom Sur IlstueieaM ev tairisplwra bdtb smu 

a.rory i 

rW.q .n W s..~.~ .~, er ~e- r w1 " afta nNuINgeft .wr., 

1% rnaA1 en On vtYO wall In rpp . 

~y11 

sawlpa ho11a0m awwAIMdeG 

~Tsiambpac Ip~ePwar, trst~uudod laio.eta, *MOM, neaeOwrarlr; 

oftn a.r k,.p"on Dal . a we,p .mno~ . 

ao. Au" a. 03. s.q.IneK 2W4 on aoAmM ulwr a s.pwrb.r zao4 

1'UBLIC COPY 
p. 28 

arma .Mbander.u rn.wew~nrwaru.tre~ri,w~awhn ,r,r 

~erotta~.dwr.ww~rt~~ ~ ~ ~ 

Aur a~ GmWw,w lampel4sa+nMw wad Most lw.r ..rrOmprr.m .eita.l .a.r~tomb e+llm an w ~r ~dr nwr~rr~ rr " trarrqrrerrwarrrw. ~w.w~w.rrrr r 

~ ara~ar~~rrr~ :~~ ~ 

au` `~~wi"Mo.wa dap. 

PUBLIC COPY 
10. Abbreviations used in the Tables 

M mean 

SD standard deviation 

-S9 without S9 mix 

+S9 with S9 mix 

96 not tested 

p precipitate 

B background lawn reduced 

C contaminated 

' mutagenic effect 

"` bacterfotoxic effect 
p. 30 

49 

11. Tables 1-10 

Table : 1 Study Number 

S 

y re 

tud Director 

Technician 

Date ovW, 2 

Strain : S .typhimurium TA 1535 

------------------------------------------------- 

REVERTANTS PER PLATE TITER QUOTIENT Do 10 Dilution per ml se/Plate % 

------------------------------------------------------------------------------ Water 12 11 2 12 11 2 150 15 .6 1 .0 1 .0 12 . 12 ' 162 

9 9 ' ' 

50 10 12 2 9 9 3 ~ / 1 .1 0.8 12 12. . 

14 . 7 

158 11 P 11 2 6 P 9 , 2 ~ / 1 .0 0.8 

9P 10P . . 

. 12 P 10 P 

500 12 P 10 2 7 P 7 3 ~ / 0 .9 0 .7 10 P . 10 P 

8 P 5 P 

1581 10 P 7 2 5 P 6 1 0 .7 0 .6 6 P 7 P 

6 P 7 P 

5000 7 P 8 2 7 P 6 3 4 / 0.8 0.5 8 P 3 P 

10 P 8 P . 

Na-azide 654 635 33 ; 57 .7* / 10 597 

653 

2-AA 166 163 6 3 / / 14 .8+ 157 

167 

--------------------------------------------------------------------- 

(pq/Plate) -S9 M SD +S9 M SD 10"` 10`1 -S9 +S9 
--------------------------------------------- 

REVERTANTS PER PLATE TITER QUOTIENT 

Dose/Plate 10% Dilution per ml 

(pq/Plate) '-S9 M SD +S9 M SD 10-` 1048 -S9 +S9 

---------------------------------------------------------------------------------

Dtater 112 121 8 140 136 7 172 16 .6 1 .0 1 .0 

123 141 160 

128 128 

50 140 130 8 151 143 9 ~ / 1 .1 l .l 

126 145 

125 134 

Table : 2 Study Number 

Study Director 

Technician 

Date : oW0.0 S TES W ~ Strain : S .typhimurium TA 100 

158 111 P 121 21 110 P 136 24 ~ / 1 .0 1 .0 

107 P 142 P 

145 P 157 P 

500 118 P 124 7 142 P 127 13 $ / 1 .0~ 0.9 

122 P 116 P 

132 P 124 P ' 

1581 114 P 111 4 126 P 117 10 $ / 0 .9 0 .9 113 P 120 P 

106 P 106 P 

5000 105 P 94 11 112 P 114 2 ~ / 0 .8 0 .8 

92 P 116 P 

84 P 115 P 

NF 272 292 17 2.4* / 0 .2 301 

302 

2-AA 1269 1223 191 9,0* 

3 1013 

1387 

--------------------------------------------------------------------------------- 

U 

e,aj 

36-6 

LIC CO' 

LIC COPY 
p. 32 

51 

Table : 3 Study Number 

Study Director 

.Technician 

Date ov . , 2006 

Strain : S .typhimurium TA 1537 

-----_--------------------------------------=----------------- 

REVERTANTS PER PLATE TITER QUOTIENT Dose/Plate 10% Dilution per ml 

(pq/Plate) -S9 M SD +S9 M SD 10-6 10+8 -S9 +S9 

--------------------------------------------------------------------------------- 

Water 5 6 1~ 11 10 2 79 7 .7 1.0 1 .0 

7 , 8 74 

5 10 

, 50 4 5 1 9 8 1 0 .8 0 .8 

5 8 

5 7 , 

158 5 P 5 1' 5 P 6 1 $ / 0.9 0.7 

4 P 7 P 

6 P 7 P 

500 7 P 6 2 6 P 7 2 16 / 1 .0 0 .7 

4 P 5 P 

6 P 9 P 

1581 3 P 4 1 3 P 4 1 1 / 0 .6 0 .4 

4 P 5 P 

4 P 3 P 

5000 4 P 3 1 4 P . 5 1 i / 0.6 0.5 

2 P 5 P 

4 P 5 P 

4-NPDA 85 81 6 3 14~.2* / 10 74 ' 

83 

2-AA 268 229 42 23 .7* 

3 235 

184 

--------------------------------------------------------------------------------- 

PUBLIC COPY 
p. 33 

52 

Dose/Plate ~ 10% Dilution per ml 

(pq/Plate) -S9 M SD +S9 M SD 10-s 10'8 -S9 +S9 

--------------------------------------------------------------------------------- 

Water 15 .16 1 29 28 4 148 14 .9 1 .0 1 .0 

15 24 149 

17 31 . 

50 17 15 3 20 22 2 1 / 1.0 0 .8 

12 23 

16 22 

158 14 P 14 3 19 P, 20 1 $ / 0 .9 . 0 .7 

16 P 21 P 

11 P 20 P ' 

500 10 P 10 1 20 P 20 2 $ / 0.6 0 .7 

11P 22P . 

9 P 18 P ' 

1581 7 P 9 2 14 P ~12 2 ~ / 0 .6 0 .4 

11 P 12 P 

8 P 10 P 

5000 16 P 11 5 18 P 16 ,3 $/ 0.7 0 .6 

7 P ' 17 P 

10 P 12 P 

4-NPDA 124 117 13 7,5* / 

0 .5 126 

102 

2-AA 762 783 ~ 61 'k / / 28 .0* 

3 735 

851 

--------------------------------------------------------------------------------- 

Table : 4 Study Number 

Study Director 

Technician 

Date ov . , 2006 

Strain : S.typhimurium TA 98 

------------------------------------------------------ 

REVERTANTS PER PLATE TITER QUOTIENT 

PUBLIC COPY 
Table : 5 Study Number 

Study Director 

Technician 

Date ~ 30, M 2006 

Strain : S .typhimurium TA 102 

--------------------------------------------- 

REVERTANTS PER PLATE TITER QUOTIENT 

Dose/Plate 10$ ' Dilution per ml 

(jig/Plate) -S9 M SD +S9 M SD 10-` 10'° -S9 ~ +S9 

------------------------------------=--------------------------------------------

Water 210 196 15 308 292 35 214 22 .4 1 .0 1 .0 

199 251 234 

180 316 

50 213 204 20 279 263 14 ~ / 1 .0 0 .9 181 256 

218 253 

158 242 P 240 38 224 P 203 19 6 / 1 .2 0 .7 277 P 198 P 

201 P 188 P 

500 219 P 207 22 241 P 256 15 1 .1 0.9 220 P 256 P 

182 P 271 P 

1581 177 P 224 41 250 P 272 19 1 / 1 .1 0 .9 246 P 283 P 

249 P 282 P 

5000 234 P 212 19 241 P R28 30 ~ / 1 .1 0 .8 199 P 249 P 

202 P 193 P . 

MMC , 481 460 49 2 .3* / 0 .2 404 

496 

2-AA 662 641 23 2,2* 

3 617 

645 

---------------------------------------------------------------------------------- 
Table : 6 Study Number 

, Study Director 

. Technician 

Date : Dec . 7, 2006 INCUBATION aith Strain : S.typhimurium TA 1535 

------------------ --- - - ------------------------------------------------------ 

REVERTANTS PER PLATE TITER QUOTIENT 

Dose/Tube 104 Dilution per ml 

(pq/Tube) -S9 M SD +S9 . M SD 10-° 10'8 -S9 . +S9 

---------------------------------------------------------------------------------

Water 13 14 1 11 12 2 214 19 .4 1.0 1 .0 

14 11 173 

14 14 

50 14 15 3 16 .14 3 $ / 1 .1 1 .1 

18 14 

13 11 

158 .12 P 12 2 21 P 15 6 +6 / 0 .9 1 .2 

14 P 11 P 

10 P 12 P 

500 12 P '13 2 10 P 11 1 $ / 0.9 0.9 

15 P 12 P 

11 P 11 P 

1581 12 P 12 2 , 12 P 12 1 $ / 0 .9 1 .0 

10 P 13 P 

14 P 12 P 

5000 12 P 12 1 14 P 11 4 $ / 0 .9 0 .9 11 P 11 P 

12 P 7 P 

Na-azide 728 748 17 % / 54 .7* / 10 760 

756 , 

2-AA 155 161 18 13 .4* 

3 181 

146 

-----------------------------------------=--------------------------------------- 
p. 36 

55 

Table : 7 

Dose/Tube 101 Dilution per ml (pg/Tube) -S9 M SD +S9 M SD 10-` 10`° -S9 +S9 

--------------------------------------------------------------------------------- 

Water 139 132 10 ~ 185 188 21 120 12 .6 1 .0 1 .0 137 169 131 

120 210 

50 136 133 6 183 188 10 $ / 1 .0 1 .0 126 181 

138 199 

158 113 P 126 19 188 P 190 6 $ / 1 .0 1 .0 116 P 185 P 

148 P 197 P 

500 134 P 130 5 158 P 182 21 $ / 1 .0 1 .0 125 P ' 193 P 

131 P 196 P 

1581 119 P 128 8 179 P 175 7 ~ / 1 .0 0.9 135 P 178 P 

131 P 167 P 

5000 116 P 123 6 168 P 173 9 ~ / 0 .9 0.9 128 P 167 P . 

124 P 183 P 

NF 472 489 24 3 .7" / 0.2 478 

517 

2-A)( . 1149 1161 11 3 / / 6,2r 

3 1163 

1171 

--------------------------------------------------------------------------- 

Strain : S .typhimurium TA 100 

Date k"'46-c . 

Study Number 

Study,Director 

Technician 

----------------------------------------- 

REVERTANTS PER PLATE TITER QUOTIENT 

LIC Cop, 
i 

I 

I 

i 

Table : 8 Study Number 

F 

Study Director 

Technician 

mmzmw~~Mmmmw Date c. 7, 2006 

CUBATION with Strain : S .typhimurium TA 1537 

--------------------- - ~~- ----------------------------------------- 

REVERTANTS PER PLATE TITER QUOTIENT 

Dose/Tube 103 Dilution per ml 

(jig/Tube) -S9 M SD +S9 M SD 10-6, 10`1 -S9 +S9 

--------------------------------------------------------------------------------- 

Water 7 7 1 13 11 3 50 5 .4 1 .0 1 .0 

7 8 57 

6 12 

! 50 6 7 1 13 10 4 ~ / 1 .0 0 .9 

6 11 

8 5 

158 7 P 6 1 4 P 9 4 $ / 0 .9 0.8 

5 P 11 P 

5 P 12 P 

500 4 P 5 1 10 P 10 2 $ / 0 .8 0 .9 

6 P 8 P 

5 P 11 P 

1581 5 P 3 2 4 P 5 2 $ / 0 .5 0.5 

2 P 7 P 

2 .P 4 P , 

5000 4 P 3 1 4 P 3 1 $ / 0.5 0 .3 

4 P 3 P 

2 P 2 P 

4-NPDA 108 109 27 16 .3* / 

10 82 

136 

2-AA 

3 

'b / / 173 192 23 17 .5* 

218 . 

186 , 

" -------------------------------------------------------------------------T------- 
p. 38 

57 

Table : 9 Study Number 

Study Director 

Technician 

w- -L Date D~.T, ~006 

ON wit Strain : S.typhimurium 1'A 98 

-------------------------- ----------------

REVERTANTS PER PLATE TITER QUOTIENT 

Do3e/Tube 101 . Dilution per ml 

(pq/Tube) -S9 , M SD +S9 M SD 10-s 10`8 -S9 +S9 

--------------------------------------------------------------------------------- 

Water 14 . 14 3 23 25 2 166 16 .1 1 .0 1 .0 

12 27 155 

17 25 

50 16 15 2 24 21 4 $ / 1 .0 0 .8 

13 22 

16 ' 17 ' 

158 12 P 13 2 12 P 15 4 ~ % 0 .9 0 .6 

15 P 19 P 

12 P 14 P 

500 11 P 12 2 14 P 14 1 0.8 0 .6 14 P 14 P 

11 P 15 P 

1581 7P 9 2 " 12P 13 2 ~ / 0 .7 0.5 11 P 12 P 

10 P 16 P 

5000 9 P 1 9 3 14 P 12 2 3 / 0 .7 0 .5 12P 11P ' 

7 P 12 P 

4-NPDA 126 127 8 8 .9* / 0 .5 120 

135 

2-AA 988 995 42 39 .8* 

3 ' 1040 . ' 

957 

PUBLIC COPY 
Table : 10 Study Number 

Study Director 

Technician 

Date 

Strain: S . typhieaurium T1~ 102 

------------------------------------------- 

REVERTIi1NTS PER PLATE TITER QUOTIENT Dose/Tube 10" Dilution per ml (pq/Tube) -S9 M SD +S9 I~! SD 10'4 10`8 -S9 +S9 

------------------------------------------------------------------- 9fater 269 244 22 256 284 31 250 22 .5 1 .0 1 .0 234 278 199 

318-- -- - - - - -- - 

50 252 258 26 313 266 64 8 / 1,1 0,9 236 292 

286 193 

158 188 P 211 21 263 P 249 15 " / 0.9 0.9 230 P 233 P 

215 P 252 P 

500 265 P 245 22 308 P 292 20 " / 1 .0 1.0 248 P 299 P 

221 P 270 P 

1581 239 P 242 16 306 P 286 32 " / 1 .0 1 .0 227 P 303 e 

259 P 249 P 

5000 214 P 213 ~ 22 195 P 207 24 0 / 0,9 0 .7 191 P 235 P 

235 P 191 P 

Cue~ne 513 496 23 8 2.0, / 

50 505 

469 ' 

2-~ 461 462 8 1 . / / 1 .6+ 3 471 

. 455 

-------------------------------------------------------------------------~------- 

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