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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 2017 - 14 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
revised 31 March 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid
Details on test material:
Storage: ambient (5 °C – 30 °C), dark, dry
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared in purified water, diluted prior to treatment and was used within 2 hours of preparation.
- Preliminary purification step (if any): The test item was prepared in purified water.
- Final dilution of a dissolved solid, stock liquid or gel: 50 mg/L
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A

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 Mix
Test concentrations with justification for top dose:
The test item was tested in the pre-experiment with the following concentrations:
2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (referring to the main constituent)
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (referring to the test item).
The following concentrations (referring to the main constituent) of the test item were prepared and used in the experiments:
Pre-Experiment (Plate-incorporation Test): 2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (TA 98 and TA 100)
Experiment I (Plate-incorporation Test): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II (Pre-incubation Test): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102)
These concentrations correspond to the following concentrations of the test item: Pre-Experiment (Plate-incorporation Test): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98 andTA 100)
Experiment I (Plate-incorporation Test): 12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 1535, TA 1537 and TA 102)
Experiment II (Pre-incubation Test): 12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102).
5000 µg/plate (concentration of main constituent) was selected as the top dose in the main experiment following the results of the pre-experiment, where it was found that toxic effects occurred at 5000 µg/plate (with and without metabolic activation). It is also the guideline-recommended maximum dose.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest. (negative controls) or DMSO (positive controls)
- Justification for choice of solvent/vehicle: The chosen solvent A. dest. was compatible with the survival of the bacteria and the S9 activity. DMSO is a guideline recommendation.
Controls
Untreated negative controls:
yes
Remarks:
A. dest. (Eurofins Munich, Lot No. 170228 & 170515
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD: 4-nitro-o-phenylene-diamine; 2-AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar - plate incorporation (Pre-experiment and experiment I) & preincubation (experiment II)

DURATION
- Preincubation period: 60 min
- Exposure duration (plate incorporation and pre-incubation method): At least 48 h
- Expression time (cells in growth medium): 12 h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Three plates at each of the eight concentrations tested.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by Eurofins Munich.
Vogel-Bonner-salts contain per litre:
10 g MgSO4 x 7 H2O
100 g citric acid
175 g NaNH4HPO4 x 4 H2O
500 g K2HPO4
Sterilisation was performed for 20 min at 121 °C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solution (40%)
Sterilisation was performed for 20 min at 121 °C in an autoclave.
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilisation was performed for 20 min at 121 °C in an autoclave.

Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10^9 cells/mL). The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCl
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

NUMBER OF CELLS EVALUATED: N/A

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Any supplementary information relevant to cytotoxicity: N/A

OTHER EXAMINATIONS:
N/A
Rationale for test conditions:
The OECD Guideline for Testing of Chemicals, Section 4, No. 471 - Bacterial Reverse Mutation Test - recommends using a combination of S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.
Evaluation criteria:
Criteria of Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2014 -2016)):
- S9 + S9
min max min max
TA 98 11 58 15 59
TA 100 49 155 62 160
TA 1535 4 41 3 38
TA 1537 3 35 3 36
TA 102 141 472 157 586
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results - a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not examined
- Effects of osmolality: Not examined
- Evaporation from medium: Not examined
- Water solubility: Not examined
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
- Definition of acceptable cells for analysis: N/A
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data range outliend by Eurofins
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES: N/A

NUMBER OF CELLS WITH MICRONUCLEI : N/A

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) without S9 (-S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance 4-NOPD NaN3 NaN3 4-NOPD MMS
Conc./plate 10 µg 10 µg 10 µg 40 µg 1 µL - 1.3 mg
Mean 430.7 612.1 792.0 94.5 1729.2
SD 155.5 220.0 299.5 22.7 518.8
Min 141 132 38 35 272
Max 1830 1423 1854 273 3321
RSD [%] 36.1 35.9 37.8 24.0 30.0
n 971 1188 931 929 682

Historical Laboratory Control Data of the Positive Control (in 2014 - 2016) with S9 (+S9)
TA 98 TA 100 TA 1535 TA 1537 TA 102
Substance 2-AA 2-AA 2-AA 2-AA MMS
Mean 1880.5 1727.7 133.9 234.1 801.2
SD 708.5 522.0 134.9 101.4 223.7
Min 70 169 22 26 137
Max 3606 3132 1954 682 3588
RSD [%] 37.7 30.2 100.8 43.3 27.9
n 966 1184 927 925 678

- Negative (solvent/vehicle) historical control data:
Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 24.2 90.7 13.8 8.2 270.4
SD 6.7 15.6 6.7 2.9 55.0
Min 11 49 4 3 141
Max 58 155 41 35 472
RSD [%] 27.7 17.2 48.6 35.3 20.3
n 972 1191 929 931 682

Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9):
TA 98 TA 100 TA 1535 TA 1537 TA 102
Mean 29.0 96.4 10.5 8.3 339.7
SD 6.8 14.1 4.5 3.1 71.3
Min 15 62 3 3 157
Max 59 160 38 36 586
RSD [%] 23.4 14.6 42.7 37.4 21.0
n 967 1189 925 926 676

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were noted in tester strain TA 102 up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
Toxic effects of the test item were noted in all other tester strains evaluated in the pre-experiment, experiment I and II.
In the pre-experiment toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2000 µg/plate and higher (without metabolic activation) and at a concentration of 4000 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 800 µg/plate and higher (without metabolic activation) and at a concentration of 4000 µg/plate (with metabolic activation).
In experiment I toxic effects of the test item were seen in tester strain TA 1535 at a concentration of 5000 µg/plate (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at a concentration of 5000 µg/plate (with and without metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 µg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (with and without metabolic activation).

Any other information on results incl. tables

Table 1: Pre-experimental Results (Plate-incorporation Test)

Treatment

Dose (µg/plate)

Mutation factor (toxicity)

 

 

Without S9

 

 

With S9

TA 98

Test item

2.53

1.1

1.1

8.00

1.0

0.8

25.3

1.2

1.1

80

1.4

0.8

253

1.2

0.6

800

1.5

0.8

2000

0.4 [B]

0.7

4000

0.6 [B]

0.5

A dest.

-

1.0

1.0

4-NOPD

10

21.2

-

NaN3

10

-

-

2-AA

2.5

-

54.4

TA 100

Test item

2.53

1.0

0.8

8.00

1.1

1.0

25.3

1.2

0.9

80

1.0

0.9

253

0.8

0.8

800

0.6 [B]

1.0

2000

0.6 [B]

1.0

4000

0.4 [B]

0.8 [B]

A dest.

-

1.0

1.0

4-NOPD

10

-

-

NaN3

10

7.2

-

2-AA

2.5

-

24.1

*(toxicity parameter): B = Background lawn reduced; N = No background lawn

Dose refers to concentration of main constituent.

Table 2: Experiment I Results (Plate-incorporation Test)

Treatment

 Dose

(µg/plate)

Revertant colonies per plate

Mutation factor

Without S9 (mean)

SD

With S9 (mean)

SD

-S9

+S9

TA 98

Test item

2.53

25

2.6

28

9.1

1.1

1.1

8.00

23

6.8

22

6.7

1.0

0.8

25.3

27

4.6

27

0.6

1.2

1.1

80

31

4.4

21

7.5

1.4

0.8

253

26

1.2

15

4.0

1.2

0.6

800

33

2.1

22

7.2

1.5

0.8

2000

9 [B]

8.5

18

7.9

0.4

0.7

4000

13 [B]

2.5

13

6.7

0.6

0.5

A dest.

-

22

8.5

26

4.0

1.0

1.0

4-NOPD

10

466

77.5

/

/

21.2

/

2-AA

2.5

/

/

1415

333.4

/

54.4

TA 100

Test item

2.53

116

15.7

69

5.5

1.0

0.8

8.00

133

2.5

86

3.5

1.1

1.0

25.3

141

12.5

83

17.9

1.2

0.9

80

126

8.6

77

13.1

1.0

0.9

253

94

5.0

73

4.6

0.8

0.8

800

68 [B]

18.8

90

17.9

0.6

1.0

2000

72 [B]

5.5

91

12.9

0.6

1.0

4000

47 [B]

2.5

75 [B]

3.1

0.4

0.8

A dest.

-

121

4.0

89

19.6

1.0

1.0

NaN3

10

874

81.1

/

/

7.2

/

2-AA

2.5

/

/

2152

201.3

/

24.1

TA 1535

Test item

10

19

5.1

17

1.7

1.1

1.1

31.6

16

2.9

16

1.0

0.9

1.0

100

18

6.1

15

5.9

1.1

1.0

316

22

3.5

13

3.1

1.3

0.8

1000

17

0.0

12

3.5

1.0

0.8

2500

17

2.6

14

4.0

1.9

0.9

5000

4 [B]

1.0

14

4.7

0.2

0.9

lA dest.

-

17

46.5

16

4.4

1.0

1.0

NaN3

10

1010

46.5

/

/

60.6

/

2-AA

2.5

/

/

110

13.9

/

6.9

TA 1537

Test item

10

11

1.5

9

1.2

1.0

1.0

31.6

10

0.6

9

0.6

0.9

1.0

100

12

0.6

8

1.2

1.1

0.9

316

13

1.2

8

0.6

1.1

1.0

1000

12

1.0

8

1.2

1.0

1.0

2500

11

1.0

6

0.6

0.9

0.7

5000

2 [B]

1.5

1 [B]

1.0

0.2

0.1

A dest.

-

12

0.6

9

0.6

1.0

1.0

4-NOPD

40

100

15.4

/

8.6

8.6

0

2-AA

2.5

/

/

268

/

/

31.0

TA 102

Test item

10

303

13.0

353

43.7

0.9

0.9

31.6

319

11.9

366

5.1

0.9

0.9

100

332

16.8

352

31.4

1.0

0.9

316

313

44.3

355

47.2

0.9

0.9

1000

349

45.7

394

13.2

1.0

1.0

2500

356

44.0

400

54.2

1.0

1.0

5000

294

17.6

369

11.1

0.9

1.0

A dest.

-

340

28.6

388

35.0

1.0

1.0

MMS

1.3 (mg/plate)

1342

62.6

/

/

3.9

/

2-AA

10

/

/

972

62.1

/

2.5

SD = standard deviation; P = precipitation; B = background lawn reduced; N = no background lawn; C contamination

Mutation factor = mean revertants (test item) / mean revertants (vehicle control)

Table 3: Experiment II Results (Pre-incubation Test)

Treatment

 Dose

(µg/plate)

Revertant colonies per plate

Mutation factor

Without S9 (mean)

SD

With S9 (mean)

SD

-S9

+S9

TA 98

Test item

10

23

6.4

20

0.0

0.9

0.7

31.6

21

1.2

29

8.7

0.9

1.0

100

25

9.1

26

7.2

1.0

1.0

316

28

4.4

25

2.1

1.1

0.9

1000

23

6.1

25

2.3

0.9

0.9

2500

18

2.1

26

4.5

0.7

1.0

5000

5 [B]

2.5

13 [B]

3.1

0.2

0.5

A dest.

-

25

6.0

27

4.5

1.0

1.0

4-NOPD

10

441

94.5

/

/

17.9

/

2-AA

2.5

/

 

1027

 

/

37.6

TA 100

Test item

10

83

11.0

79

6.1

0.9

0.8

31.6

95

4.9

89

5.1

1.0

0.9

100

94

16.0

81

6.4

1.0

0.9

316

92

10.1

93

9.0

1.0

1.0

1000

81

8.5

72

3.2

0.9

0.8

2500

42 [B]

4.9

68

7.8

0.4

0.7

5000

20 [B]

16.1

48 [B]

18.4

0.2

0.5

A dest.

-

94

3.157.8

95

13.1

1.0

1.0

NaN3

10

587

57.8

/

/

6.3

/

2-AA

2.5

/

/

814

142.8

/

8.6

TA 1535

Test item

10

22

0.6

13

2.0

1.1

0.8

31.6

22

2.3

14

0.6

1.2

0.9

100

20

2.1

14

2.0

1.0

0.9

316

19

2.1

15

0.6

1.0

0.9

1000

23

3.2

15

2.1

1.2

0.9

2500

3 [B]

1.5

4 [B]

1.0

0.1

0.3

5000

2 [B]

0.6

1 [B]

0.6

0.1

0.1

A dest.

-

19

1.7

16

 

1.0

1.0

NaN3

10

250

13.1

/

/

13.2

/

2-AA

2.5

/

/

684

85.9

/

42.8

TA 1537

Test item

10

11

1.2

8

1.0

0.9

1.0

31.6

13

1.5

8

1.5

1.1

1.0

100

12

0.6

8

2.3

1.0

1.0

316

12

2.6

8

0.6

1.0

1.0

1000

11

0.6

9

0.6

0.9

1.1

2500

5 [B]

0.6

4 [B]

3.1

0.4

0.5

5000

2 [B]

1.0

0 [B]

1.5

0.2

0.0

A dest.

-

12

2.0

8

0.6

1.0

1.0

4-NOPD

40

111

8.6

/

/

9.3

/

2-AA

2.5

/

/

87

4.0

/

10.9

 

TA 102

Test item

10

268

5.5

359

39.2

1.0

0.9

31.6

281

23.9

379

12/1

1.0

1.0

100

262

17.4

359

6.7

1.0

0.9

316

243

14.0

329

31.0

0.9

0.8

1000

308

25.4

354

30.1

1.1

0.9

2500

308

16.3

391

21.0

1.1

1.0

5000

193

12.1

385

16.5

0.7

1.0

A dest.

-

275

27.5

389

2.5

1.0

1.0

MMS

1.3 (mg/plate)

744

78.4

/

/

2.7

/

2-AA

10

/

/

866

95.6

/

2.2

SD = standard deviation; P = precipitation; B = background lawn reduced; N = no background lawn; C contamination

Mutation factor = mean revertants (test item) / mean revertants (vehicle control)

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met. The negative control plates (A. dest.) with and without S9 mix are within the historical control data range.The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Applicant's summary and conclusion

Conclusions:
The test item is considered to be non-mutagenic under the experimental conditions of this bacterial reverse mutation assay.
The substance does not meet the criteria for classfication in accordance with GHS or Regulation (EC) No 1272/2008 (CLP).
Executive summary:

In accordance with OECD 471, the test item sodium octane-1-sulphonate was tested for it's potential to induce gene mutations. A plate incorporation test and pre-incubation test was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In three independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.

The following concentrations referring to the main constituent of the test item were prepared and used in the experiments:

Pre-Experiment (Part of Experiment I):

2.53, 8.00, 25.3, 80.0, 253, 800, 2000 and 4000 µg/plate (TA 98 and TA 100)

Experiment I:

10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535, TA 1537 and TA 102)

Experiment II:

10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102)

These concentrations correspond to the following concentrations of the test item:

Pre-Experiment (Part of Experiment I):

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 98 and TA 100)

Experiment I:

12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 1535, TA 1537 and TA 102)

Experiment II:

12.5, 39.5, 125, 395, 1250, 3125 and 6250 µg/plate (TA 98, TA 100, TA 1535, TA 1537 and TA 102).

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

No toxic effects of the test item were noted in tester strain TA 102 up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.

Toxic effects of the test item were noted in all other tester strains evaluated in the pre-experiment, experiment I and II.

In the pre-experiment toxic effects of the test item were observed in tester strain TA 98 at concentrations of 2000 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 800 μg/plate and higher (without metabolic activation) and at a concentration of 4000 μg/plate (with metabolic activation).

In experiment I toxic effects of the test item were seen in tester strain TA 1535 at a concentration of 5000 μg/plate (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at a concentration of 5000 μg/plate (with and without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 μg/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were seen at concentrations of 2500 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 μg/plate and higher (with and without metabolic activation).

No biologically relevant increases in revertant colony numbers in any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. All criteria of validity were met. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.