Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 - 21 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor induced rats
Test concentrations with justification for top dose:
5000 μg, 1667 μg, 556 μg, 185 μg and 62 μg. No justification for the top dose provided.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was used for the suspension. The solvent THF was tested, but resulted in a polymer-like precipitation on mixing with the bacterial suspension.
Controls
Untreated negative controls:
yes
Remarks:
Control cultures were treated with solvent
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other:
Remarks:
Concentrations of the positive control susbtances detailed in Table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: In the confirmation test only, for 20 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 per concentration per test

DETERMINATION OF CYTOTOXICITY
- Method: The number of colonies per plate was counted
- Any supplementary information relevant to cytotoxicity: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.

- OTHER:
Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. The highest concentration of test item and a sample of the S9 mix were added respectively to top agar preheated at about 45°C and poured over minimal agar medium plates. The plates were incubated for about 72 hours at about 37°C. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix respectively.
Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility.
Rationale for test conditions:
According to guideline
Evaluation criteria:
Several criteria are used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result was considered positive whenever the number of revertants is increased compared to the vehicle-control to at least 2 fold of the vehicle control for TA 98, 100, and 102 strains, and 3-fold of the solvent control for TA 1535 and 1537 strains. However biological relevance of the results was considered first.

Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains. Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system.
Statistics:
Mean and standard deviation results were calculated. Additionally, the R ratio was calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None reported
- Effects of osmolality: None reported
- Evaporation from medium: None reported
- Water solubility: Solubility was assessed assessed as precipitation in the final mixture under test conditions. No observations of precipitation were reported.
- Precipitation: None reported
- Other confounding effects: No appropriate solvent was found for the test item thus, a suspension in water was used.

RANGE-FINDING/SCREENING STUDIES: None reported

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) :
Strain TA 98 without metabolic activation, CDI+: Mean = 284, Standard Deviation = 62; Range = 150 - 373 (n = 36)
Strain TA 98 without metabolic activation, CDI-: Mean = 12, Standard Deviation = 5; Range = 5 - 30 (n = 36)
Strain TA 98 without metabolic activation, R = 23.4
Strain TA 98 without metabolic activation, CDI-: Mean = 927, Standard Deviation = 218; Range = 250 - 1212 (n = 36)
Strain TA 98 without metabolic activation, CDI+: Mean = 9, Standard Deviation = 3; Range = 4 - 15 (n = 36)
Strain TA 98 without metabolic activation, R = 100.5

Strain TA 98 with metabolic activation, CDI+: Mean = 507, Standard Deviation = 166; Range = 291 - 1025 (n = 36)
Strain TA 98 with metabolic activation, CDI-: Mean = 15, Standard Deviation = 3; Range = 9 - 21 (n = 36)
Strain TA 98 with metabolic activation, R = 34.7
Strain TA 98 with metabolic activation, CDI-: Mean = 951, Standard Deviation = 148; Range = 656 - 1257 (n = 36)
Strain TA 98 with metabolic activation, CDI+: Mean = 22, Standard Deviation = 7; Range = 7 - 35 (n = 36)
Strain TA 98 with metabolic activation, R = 43.4

Strain TA 100 without metabolic activation, CDI+: Mean = 444, Standard Deviation = 59; Range = 308 - 583(n = 36)
Strain TA 100 without metabolic activation, CDI-: Mean = 65, Standard Deviation = 9; Range = 46 - 85(n = 36)
Strain TA 100 without metabolic activation, R = 6.8
Strain TA 100 without metabolic activation, CDI-: Mean = 396, Standard Deviation = 81; Range = 267 - 587 (n = 36)
Strain TA 100 without metabolic activation, CDI+: Mean = 71, Standard Deviation = 21; Range = 125 - 43 (n = 36)
Strain TA 100 without metabolic activation, R = 5.6

Strain TA 100 with metabolic activation, CDI+: Mean = 501, Standard Deviation = 92; Range = 286 - 742 (n = 36)
Strain TA 100 with metabolic activation, CDI-: Mean = 102, Standard Deviation = 23; Range = 46 - 138 (n = 36)
Strain TA 100 with metabolic activation, R = 4.9
Strain TA 100 with metabolic activation, CDI-: Mean = 676, Standard Deviation = 166; Range = 425 - 1121 (n = 36)
Strain TA 100 with metabolic activation, CDI+: Mean = 112, Standard Deviation = 31; Range = 42 - 149 (n = 36)
Strain TA 100 with metabolic activation, R = 6.0

Strain TA 102 without metabolic activation, CDI+: Mean = 1094, Standard Deviation = 133; Range = 865 - 1297 (n = 36)
Strain TA 102 without metabolic activation, CDI-: Mean = 198, Standard Deviation = 33; Range = 114 - 244(n = 36)
Strain TA 102 without metabolic activation, R = 5.5
Strain TA 102 without metabolic activation, CDI-: Mean = 490, Standard Deviation = 208; Range = 218 - 1077(n = 36)
Strain TA 102 without metabolic activation, CDI+: Mean = 199, Standard Deviation = 38; Range = 144 - 309 (n = 36)
Strain TA 102 without metabolic activation, R = 2.5

Strain TA 102 with metabolic activation, CDI+: Mean = 1112, Standard Deviation = 184; Range = 527 - 1369 (n = 36)
Strain TA 102 with metabolic activation, CDI-: Mean = 271, Standard Deviation = 33; Range = 199 - 328 (n = 36)
Strain TA 102 with metabolic activation, R = 4.1
Strain TA 102 with metabolic activation, CDI-: Mean = 591, Standard Deviation = 114; Range = 412 - 841 (n = 36)
Strain TA 102 with metabolic activation, CDI+: Mean = 271, Standard Deviation = 42; Range = 202 - 390 (n = 36)
Strain TA 102 with metabolic activation, R = 2.2

Strain TA 1535 without metabolic activation, CDI+: Mean = 364, Standard Deviation = 74; Range = 135 - 448 (n = 36)
Strain TA 1535 without metabolic activation, CDI-: Mean = 13, Standard Deviation = 3; Range = 6 - 18 (n = 36)
Strain TA 1535 without metabolic activation, R = 27.9
Strain TA 1535 without metabolic activation, CDI-: Mean = 186, Standard Deviation = 62; Range = 112 - 421(n = 36)
Strain TA 1535 without metabolic activation, CDI+: Mean = 11, Standard Deviation = 4; Range = 4 - 23 (n = 36)
Strain TA 1535 without metabolic activation, R = 17.2

Strain TA 1535 with metabolic activation, CDI+: Mean = 558, Standard Deviation = 142; Range = 144 - 729 (n = 36)
Strain TA 1535 with metabolic activation, CDI-: Mean = 27, Standard Deviation = 5; Range = 15 - 35 (n = 36)
Strain TA 1535 with metabolic activation, R = 20.3
Strain TA 1535 with metabolic activation, CDI-: Mean = 483, Standard Deviation = 162; Range = 194 - 922 (n = 36)
Strain TA 1535 with metabolic activation, CDI+: Mean = 25, Standard Deviation = 7; Range = 11 - 39 (n = 36)
Strain TA 1535 with metabolic activation, R = 19.5

Strain TA 1537 with metabolic activation, CDI+: Mean = 15, Standard Deviation = 4; Range = 7 - 25 (n = 36)
Strain TA 1537 with metabolic activation, CDI-: Mean = 6, Standard Deviation = 2; Range = 2 - 9 (n = 36)
Strain TA 1537 with metabolic activation, R = 2.5
Strain TA 1537 with metabolic activation, CDI-: Mean = 459, Standard Deviation = 122; Range = 213 - 753 (n = 36)
Strain TA 1537 with metabolic activation, CDI+: Mean = 8, Standard Deviation = 4; Range = 2 - 18 (n = 36)
Strain TA 1537 with metabolic activation, R = 56.1

Strain TA 1537 without metabolic activation, CDI+: Mean = 73, Standard Deviation = 29; Range = 22 - 131 (n = 36)
Strain TA 1537 without metabolic activation, CDI-: Mean = 9, Standard Deviation = 3; Range = 2 - 18 (n = 36)
Strain TA 1537 without metabolic activation, R = 8.6
Strain TA 1537 without metabolic activation, CDI-: Mean = 26, Standard Deviation = 14; Range = 11 - 74(n = 36)
Strain TA 1537 without metabolic activation, CDI+: Mean = 7, Standard Deviation = 3; Range = 3 - 15 (n = 36)
Strain TA 1537 without metabolic activation, R = 3.5

Where; CDI+ = Positive control direct incorporation method; CPI+ = Positive Control preincubation method; CDI- = Negative control direct incorporation method; CPI- = Negative control preincubation method; n = number of replicates; R = ratio of the number revertant colonies in the positive control divided by the number revertant colonies in the negative control. Note; 95 % confidence intervals for historic data not reported in the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.
- Other observations when applicable: None reported

Any other information on results incl. tables

Table 4: -S9 direct incorporation (main test); Strain Salmonella typhimurium TA98

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 17   19  15  17.0  2.0  -
 Positive Control; 2-Nitrofluorene  5  295  366  309  323.3  37.6  19.0
 Test item  5000  13  20  22  18.3  4.7  1.1
 1667  14  29  18  20.3  7.8

1.2

 556

 16

 19

 17

 17.3

 1.5

 1.0

 185

 20

 22

 17

 19.7

 2.5

 1.2

 62

 25

 16

 15

 18.7

 5.5

 1.1

Table 5:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA100

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  69  78  90

 79.0

 10.5

 -

 Positive Control; Sodium Azide

 2.5

 459

 545

 559

 521.0

 54.1

 6.6

 Test item

 5000

 78

 87

 78

 81.0

 5.2

 1.0

 1667

 64

 67

 98

 76.3

 18.8

1.0

556

 87

 88

 88

 87.7

 0.6

 1.1

 185

108

 90

 86

 94.7

 11.7

 1.2

 62

 78

 98

 78

 84.7

 11.5

 1.1

Table 6:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA102

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 247   257  276

 260.0

 14.7

 -

 Positive Control; Mitomycin C

 0.75

 947

 920

 1026

 964.3

 55.1

 3.7

 Test item

 5000

 301

 272

 298

 290.3

 15.9

 1.1

 1667

 284

 306

 303

 297.7

 11.9

1.1

556

 253

 262

 330

 281.7

 42.1

 1.1

 185

282

 306

 262

 283.3

 22.0

 1.1

 62

 295

 346

 237

 292.7

 54.5

 1.1

Table 7:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA1535

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 13   16  21

 16.7

 4.0

 -

 Positive Control; Sodium Azide

 2.5

 465

 493

 767

 575.0

 166.9

 34.5

 Test item

 5000

 16

 23

 14

 17.7

 4.7

 1.1

 1667

 15

 23

 17

 18.3

 4.2

1.1

556

 18

 22

 19

 19.7

 2.1

 1.2

185

25

20

 18

 21.0

 3.6

 1.3

 62

 24

 15

 17

 18.7

 4.7

 1.1

Table 8:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA1537

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  7  9

 7.0

 2.0

 -

 Positive Control; 9 -Aminoacridine

 30

 101

 140

 138

 126.3

 22.0

 18.0

 Test item

 5000

 7

 3

 8

 6.0

 2.6

 0.9

 1667

 8

 8

 10

 8.7

 1.2

1.2

556

 4

 7

 7

 6.0

 1.7

 0.9

 185

9

 8

 2

 6.3

 3.8

 0.9

 62

 6

 11

 8

 8.3

 2.5

 1.2

Table 9: -S9 preincubation (confirmation test); Strain Salmonella typhimurium TA98

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  12  15  18

 15.0

 3.0

 -

 Positive Control; 2 -Nitrofluorene

 5

 650

 388

 612

 550.0

 141.6

 36.7

 Test item

 5000

 19

 15

 21

 18.3

 3.1

 1.2

 1667

 15

 18

 13

 15.3

 2.5

1.0

556

 21

 13

 21

 18.3

 4.6

 1.2

 185

16

 14

 20

 16.7

 3.1

 1.1

 62

 12

 15

 26

 17.7

 7.4

 1.2

Table 10:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA100

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 79   99  84

 87.3

 10.4

 -

 Positive Control; Sodium Azide

 2.5

 348

 365

 589

 434.0

 134.5

 5.0

 Test item

 5000

 119

 91

 108

 106.0

 14.1

 1.2

 1667

 75

 93

 70

 79.3

 12.1

0.9

 556

 101

 106

 99

 102.0

 3.6

 1.2

 185

113

 118

 106

 112.3

 6.0

 1.3

 62

 115

 115

 94

 108.0

 12.1

 1.2

Table 11:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA102

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 185   266  221

 224.0

 40.6

 -

 Positive Control; Mitomycin C

 0.75

 1172

 985

 1136

 1097.7

 99.2

 4.9

 Test item

 5000

 249

 293

 259

 267.0

 23.1

 1.2

 1667

 268

 250

 273

 263.7

 12.1

1.2

556

 220

 266

 256

 247.3

 24.2

 1.1

 185

221

 251

 289

 253.7

 34.1

 1.1

 62

 248

 254

 257

 253.0

 4.6

 1.1

Table 12:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA1535

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 11   12  16

 13.0

 2.6

 -

 Positive Control; Socium Azide

 2.5

 201

 265

 312

 259.3

 55.7

 19.9

 Test item

 5000

 13

 13

 12

 12.7

 0.6

 1.0

 1667

 19

 16

 13

 16.0

 3.0

1.2

556

 12

 14

 16

 14.0

 2.0

 1.1

 185

13

 15

 15

 14.3

 1.2

 1.1

 62

 15

 14

 18

 15.7

 2.1

 1.2

Table 13:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA1537

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 10   5  8

 7.7

 2.5

 -

 Positive Control; 9 -Aminoacridine

 30

 84

 98

 135

 105.7

 26.4

 13.8

 Test item

 5000

 9

 7

 9

 8.3

 1.2

 1.1

 1667

 7

 6

 5

 6.0

 1.0

0.8

556

 6

 8

 6

 6.7

 1.2

 0.9

 185

7

 5

 6

 6.0

 1.0

 0.8

 62

 11

 7

 6

 8.0

 2.6

 1.0

Table 14:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA98

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  26  11  22

 19.7

 7.8

 -

 Positive Control; 2 -Aminoanthracene

 1.5

 465

 564

 645

 558.0

 90.1

 28.4

 Test item

 5000

 36

 12

 17

 21.7

 12.7

 1.1

 1667

 12

 17

 16

 15.0

 2.6

0.8

556

 16

 15

 19

 16.7

 2.1

 0.8

 185

11

 19

 17

 15.7

 4.2

 0.8

 62

 17

 14

 12

 14.3

 2.5

 0.7

Table 15:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA100

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  91  80  69

 80.0

 11.0

 -

 Positive Control; 2 -Aminoanthracene

 2.5

 599

 382

 292

 424.3

 157.8

 5.3

 Test item

 5000

 93

 79

 68

 80.0

 12.5

 1.0

 1667

 72

 74

 87

 77.7

 8.1

1.0

556

 85

 79

 76

 80.0

 4.6

 1.0

 185

80

 68

 80

 76.0

 6.9

 1.0

 62

 87

 75

 75

 79.0

 6.9

 1.0

Table 16:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA102

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  235  355  255

 281.7

 64.3

 -

 Positive Control; 2 -Aminoanthracene

 30

 1020

 897

 923

 946.7

 64.8

 3.4

 Test item

 5000

 327

 302

 309

 312.7

 12.9

 1.1

 1667

 339

 314

 256

 303.0

 42.6

1.1

556

 317

 299

 284

 300.0

 16.5

 1.1

 185

312

 347

 292

 317.0

 27.8

 1.1

 62

 327

 324

 291

 314.0

 20.0

 1.1

Table 17:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA1535

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 16   13  25

 18.0

 6.2

 -

 Positive Control; 2 -Aminoanthracene

 30

 456

 254

 357

 355.7

 101.0

 19.8

 Test item

 5000

 12

 16

 17

 15.0

 2.6

 0.8

 1667

 12

 16

 18

 15.3

 3.1

0.9

556

 9

 15

 15

 13.0

 3.5

 0.7

 185

13

 23

 13

 16.3

 5.8

 0.9

 62

 17

 22

 17

 18.7

 2.9

 1.0

Table 18:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA1537

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  8  8

 8.3

 0.6

 -

 Positive Control; 2 -Aminoanthracene

 10

 95

 65

 45

 68.3

 25.2

 8.2

 Test item

 5000

 7

 11

 13

 10.3

 3.1

 1.2

 1667

 7

 5

 2

 4.7

 2.5

0.6

556

 2

 5

 4

 3.7

 1.5

 0.4

 185

9

 8

 6

 7.7

 1.5

 0.9

 62

 14

 5

 7

 8.7

 4.7

 1.0

Table 19:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA98

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 27   18  22

 22.3

 4.5

 -

 Positive Control; 2 -Aminoanthracene

1.5 

 976

 541

 941

 819.3

 241.7

 36.7

 Test item

 5000

 23

 18

 23

 21.3

 2.9

 1.0

 1667

 18

 22

 28

 22.7

 5.0

1.0

556

 22

 14

 21

 19.0

 4.4

 0.9

 185

17

 18

 21

 18.7

 2.1

 0.8

 62

 21

 20

 19

 20.0

 1.0

 0.9

Table 20:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA100

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  126  96  102

 108.0

 15.9

 -

 Positive Control; 2 -Aminoanthracene

 2.5

 690

 684

 927

 767.0

 138.6

 7.1

 Test item

 5000

 128

 119

 99

 115.3

 14.8

 1.1

 1667

 112

 116

 108

 112.0

 4.0

1.0

556

 100

 133

 99

 110.7

 19.3

 1.0

 185

114

 113

 115

 114.0

 1.0

 1.1

 62

 116

 122

 107

 115.0

 7.5

 1.1

Table 21:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA102

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  - 274   250  284

 269.3

 17.5

 -

 Positive Control; 2 -Aminoanthracene

 30

 933

 815

 875

 874.3

 59.0

 3.2

 Test item

 5000

 314

 334

 315

 321.0

 11.3

 1.2

 1667

 350

 268

 303

 307.0

 41.1

1.1

556

 316

 312

 311

 313.0

 2.6

 1.2

 185

323

 294

 339

 318.7

 22.8

 1.2

 62

 310

 310

 306

 308.7

 2.3

 1.1

Table 22:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA1535

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  25  21  32

 26.0

 5.6

 -

 Positive Control; 2 -Aminoanthracene

 30

 457

 446

 648

 517.0

 113.6

 19.9

 Test item

 5000

 26

 29

 26

 27.0

 1.7

 1.0

 1667

 25

 28

 24

 25.7

 2.1

1.0

556

 26

 27

 23

 25.3

 2.1

 1.0

 185

29

 21

 20

 23.3

 4.9

 0.9

 62

 26

 25

 30

 27.0

 2.6

 1.0

Table 23:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA1537

 Substance  Dose / plate (µg)  Replicate  Mean  S.D.  R
 1  2  3
 Negative Control  -  7  8

 8.0

 1.0

 -

 Positive Control; 2 -Aminoanthracene

 10

 69

 91

 49

 69.7

 21.0

 8.7

 Test item

 5000

 8

 7

 9

 8.0

 1.0

 1.0

 1667

 7

 7

 4

 6.0

 1.7

0.8

556

 6

 8

 4

 6.0

 2.0

 0.8

 185

6

 2

 12

 6.7

 5.0

 0.8

 62

 8

 4

 11

 7.7

 3.5

 1.0

Applicant's summary and conclusion

Conclusions:
The mutagenic and cytotoxic potential of the test item was investigated in an Ames test, and was found to non mutagenic and non pro-mutagenic under the test conditions and no evidence of cytoxicity was observed.
Executive summary:

The present bacterial reverse mutation test (Ames test) was performed in order to evaluate the mutagenic potential of the test item. Suspensions of 5 amino-acid requiring strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537) were exposed by the direct plate incorporation method to five doses of the test item ranging from 5000 μg to 62 μg per plate in the presence and in the absence of an exogenous metabolic activation system. Both tests were repeated with the pre-incubation method. Revertant bacteria due to point or frameshift-mutations at specific locus are able to grow, forming colonies. These colonies were counted and compared to the number of spontaneous revertant colonies on the solvent control plate (negative control). Similarly, specific standard mutagens were tested and used as positive controls.

Based on the results obtained in this study, the test item was found to be non-mutagenic and non-pro-mutagenic under the test conditions.

The study is a GLP compliant guideline experimental study and is acceptable without restriction for assessment of this endpoint.