### Thefollowingcriteria determined theacceptance ofan 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 historicaldata (see Chapter 8)
. . 2
. The positive controls had to show sufficient effects, as defined bythe 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
. Thefollowing 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 (onlyTA 100) 10 (only TA1537) 0.5 TA 

### 0 .2 (oniyTA110e) 50 (onlyTA 1023) 3

' onlyin plate Incorporation trials 3only In proincubation trials 

# "hUBLIC COPY

### Test 21 

# Cd
formulated in deionized waterand formed black suspensions
. 49 "nw%w W vasdIssoIved in deionized water. The other positive controls were dis-~ solved In DMSO

. 

### Theformutatkm was performed according to the following proce ure: were placed in a wide-necked 100ml Erfmeyerflask
. 30ml 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 upto 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 15minutes at about 25 " C
. During this time watertemperature 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 priorto 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 preincubatkmexperiments
. 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 fromour own experience (see Chapter 8), Indicating that this solvent had no Influence on the spontaneous mutant counts ofthe 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, TA100 and TA98 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 scientificjudgment
. , 

### 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 xosomeTest 22

4.6 . Study Guidelines 

### Thestudywas performedaccording at least to thefolbwing guidelines: Commission Directive 200013?JECof 19 May2000

, Official Journal ofthe European Communities of 8.6 .2000, L 136/57- L136/64 B .13114. Mutagenidty - Reverse Mutation Test Bacteria OECDGuidelines forTesting ofChemicals No
. 471 "Bacterial Reverse Mutation Test" Adopted: 21stJuly 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 ofTest and Stu 

### Salmonella/Mkxosome 4 .7.2
. Responsibilities Head ofToxicology Section Head Genetic Toxicology Study Director Senior Technlciart Head ofArchives Quality Assurance Analyst(s) 

# U-BLIC COPS
. 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 bacteriotoxiceffect t doses of up to and Including 5000pg 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 ofthe negative controls
. This applied both to the tests with and without S9 mix (Tables 1 to 5) and was confirmed by the results ofthe preincubadon trials, ables 6to 10)
.

Summary of the Results in the Salmonella/M S9mbc TA 1535 TA100 TA 1537 TA98 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 S9mix
. 

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5.2
. Tabulated Summary of Data 24

Summa of Mean Values Without S9 Mix Table and Strain Group TA 1535 TA 100 TA1537 TA98 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 

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### 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 

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### 8
. Assessment test, employing doses of upto 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 respectto relevant assessment parameters (dose effect, reproducibility)

-revealed no biologically relevant variations fromthe respectlve negative controls
. . In spite of the lowdoses 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 .typhimuriumstrains used
. 

### Dueto 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: Asimple test system combining liver homogenates'for activation and bacteria fordetection 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 forthe 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 : Methodsfor detecting carcinogens and mutagenswith the Salmonellalmammalian-micxosome mutagenicity test Mutation Res
. 31, 347-364,1975 5. Andrews, A

.W ., L.H
. Thibault and W

. Lijinsky: 

### Therelationship between cardnogenidty and mutagenicity of some polynudear hydrocarbons Mutation Res
. 51, 311-318, 1978 8
. Clayson, D' .B
.: ICPEMCworking paper 2/1
. Comparison between In vitro and In vivo tests forcarcinogenicity
. An overview Mutation Res
. 75,205-213, 1980 7. Glatt, H.R

., H
. Schwind, F
. Zajdela, A

. Crolsy, P.C
. Jacquignon and F
. 8sch: Mutagenicityof 43structurallyrelated 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 zumLebemnikrosomen-Test, einem Systemzum 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 ofS
. 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: 

### Anew Salmonella tester strain (TA 102) with A -T base pairsat the site of mutation detectsoxidative mutagens Proc
. Nag
. Aced
. Sd
. (USA) 79, 7445-7449,1982 . 

### 12
. Maron, D

.M . and B.N
. Ames: Revised methods forthe Salmonella mutagenicity test

, Mutation Res
. 113,173-215,1983 13. McCann, J ., E
. Choi, E
. Yamasaki and B .N
. Ames: Detection ofcarcinogens as mutagena in the Salmonellalmlcrosome Test: I
. Assay of 300chemicals 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 strainswith Rfactor plasmids Proc
. nat. Aced
. Scl
. (USA) 72, 979-983,1975b 

# PUBLIC COPY29

10=0

cxosome Test ~D 15 . McCann, J
. and B

.N
. Ames: Detection ofcarcinogens as mutagens In the Salmonellalmicrosome test: Assay of 300chemicals: 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 termtests for detecting organic chemical carcinogens and recommendations fortheir 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 termtests for detecdhg organic chemical carcinogens ' Brit
. J
. Cancer37, 873-959, 1978 18
. Rinkus, S.J
. and M

.S
. Legator
. Chemical characterization of 485known,or suspected carcinogens and their correlation with mutagenk ; activity in the Salmonella typhimuriumsystem Cancer Res
. 39, 3289-3318,1979 19
. Zeiger, E:: 

### Cardnogenidtyof mutagens: Predictive capability oftheSalmonella mutagenesis assayfor rodent cardnogenicity Cancer Res
. 47,1287-1296,1987 

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# -

8
. Historical Controls 8 .1 . Plate incorporation Method



### Summaryof Historical Negative and Positive Controls Using Mean Values Presentedas Medians (Z) andSeml-O Range (OR)

1996 Compound Strain I and TA1 535 TA 100 TA 1537 TA98 TA 102 S9mix 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 

# COPYSummary 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 TA100 TA1 537 TA98 TA102 S9Mix 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 TA1 535 TA 100 TA 1537 TA98 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



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### 32
Summaryof Historical Negative and Positive Controls

Using Mean Values Presented as Medians (Z) and Semi-Q Range (QR)



### 1999 Compound Strain and TA1535 TA 100 TA1 537 TA98 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 477 791 

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### Summaryof Historical Negative and Positive Controls ' using meanvalues Presented as Medians (Z) andSemi-O Range (OR) Compound Strain and TA 1535 TA 100 TA1 537 TA98 TA102 S9Mix 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 TA1 537 TA98 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



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### 34
Summary ofHistorical 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 TA1537 TA98 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 

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### Summaryof Historical Negative and Positive Controls Using MeanValues Presentedas Medians (Z) and Semi-QRange (OR)

2003 Compound Strain and TA1 535 TA 100 TA1537 TA98 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 138 721 114 1 

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Summary of Historical Negative and Positive Controls

Using Mean Values Presented as 

### Medians (Z) andSemi-Q Range (QR)

2004 Compound Strain and TA1 535 TA 100 TA1 537 TA98 TA 102 S9Mix 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 

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### Summaryof Historical Negative and Positive Controls Using Mean Values Presented as. Medians (Z) and Semi-O Range (4R) Compound Strain and TA1 535 TA 100 TA 1537 TA98 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 1 718 991 2005



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### 8

.2
. Preincubation Method 38

Summary ofHistorical Negative and Positive Controls

Using Mean Values Presented as Medians (Z) and Seml-QRange (OR) Compound Strain and TA1 535 TA 100 TA1537 TA98 TA 102 S9Mix 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`,SummaryofHistorical Negative and Posidve Controls Using Mean Values Presented as Medians (Z) and Semi-Q
. Range (OR) 1997 Compound Strain and TA 1535 TA 100 TA 1537 TA98 TA102 

### 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 TA100 TA1537 TA98 TA102 S9Mix 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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### - & -~ 

### ll c 

### ro s o 

### rne

### T e 

### st 40 ------



### . Summaryof Historical Negative and Positive Controls

Using Mean Values Presented as Medians (Z) and Semi-QRange (QR)

1999 Compound Strain and TA1 535 TA100 TA1 537 TA98 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



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### Summaryof Historical Negative and Positive Controls Using Mean Values Presented as . Medians (Z) and Semi-QRange (OR) Compound Strain and . TA 1535 TA 100 TA1 537 TA98 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 TA1 537 TA98 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



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3 S7

### Summaryof Historical Negative and Positive Controls Using Mean Values Presented as Medians (Z) and Semi-QRange (OR) Compound .Strain and TA1 535 TA100 TA1 537 TA98 TA 102 S9Mix 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



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### 43
Summary ofHistorical Negative and Positive Controls

Using Mean Values Presented as Medians (Z) and Semi-Q Range (OR)



### 2003 Compound Strain
. -and TA1535 TA 100 TA1 537 TA98 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 

### Curflene - 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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### lmonela rosome Test 44

Summary of Historical Negative and Positive Controls
.

Using Mean Values Presented as 

### Medians (Z) and Semi-QRange (OR)

2004 Compound ~ Strain and TA 1 535 TA 100 TA1 537 TA98 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& 

### Summaryof Historical Negative and Positive Controls Using Mean Values Presented as Medians (Z) and Semi-Q Range (OR) Compound Strain and TA1535 TA100 TA1537 TA98 ' TA 102 

### S9Mix 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



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9
. Statement of GLPCompliance

Alr Umwelttlfid Nstiu~l ~_ Landwirtschaft und V~ !

t doLande 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 '+~twwwer»o~oem malowuw

aet~alKlt
. - -"-

Pt011+rqm

nech K-apollgn Area ofeNOWsm 1. "rae.wrv,ovata8oroo,rrry p ewqayaaoo,~rr.l Kalp'"»: erpetp: 11"Mom SurIlstueieaM

evtairisplwra bdtb smu

a.roryi rW .q.nW s. .~.~.~, er~e- rw1 " afta nNuINgeft .wr., 1% rnaA1en On vtYOwallInrpp . ~y11 sawlpaho11a0m awwAIMdeG

~Tsiambpac Ip~ePwar, trst~uudod laio.eta, *MOM, neaeOwrarlr; oftna.rk,.p"on Dal
.awe,p.mno~ . ao .Au"a.03
.s.q.IneK2W4 onaoAmM ulwras.pwrb

.rzao4 

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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 COPY10
. 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 effectp. 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.typhimuriumTA 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 COPYp. 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 COPYp. 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 COPYTable : 5 Study Number Study Director Technician M Date ~ 30, 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.typhimuriumTA 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 .typhimuriumTA 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 Date D~.T, ~006 w- -L 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 COPYTable : 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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