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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:
2005-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
448-300-4
EC Name:
-
Cas Number:
88642-03-9
Molecular formula:
C16H28O
IUPAC Name:
(6E)-cyclohexadec-6-en-1-one; (7Z)-cyclohexadec-7-en-1-one; (8E)-cyclohexadec-8-en-1-one; (8Z)-cyclohexadec-8-en-1-one

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:
Type and composition of metabolic activation system:
- source of S9 : Phenobarbital/ß-Naphthoflavone induced rat liver

- method of preparation of S9 mix: prepared from 8 - 12 weeks old male Wistar Hanlbm rats (approx. 220 - 320 g) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and ß-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week. The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404) in the pre-experiment and experiment I (strains TA 98 and TA 100), and 32.8 mg/mL (lot no. R 151004) in experiment I and II.

- concentration or volume of S9 mix and S9 in the final culture medium: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Test concentrations with justification for top dose:
Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Concentrations applied in the main experiment (I, II and IIa) werer based on the results of the Pre-Experiment.
Vehicle / solvent:
- Vehicle used: ethanol

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, experiment I) and preincubation (experiment II and IIa)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
Rationale for test conditions:
according to the test guidelines
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.

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:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated weakly at different concentrations in the overlay agar. The undissolved particles of the test item had no influence on the data recording.


Ames test:
- Signs of toxicity : cytotoxicity was observed

HISTORICAL CONTROL DATA
- Positive historical control data: Please refer to "Any other information on results"
- Negative (solvent/vehicle) historical control data: Please refer to "Any other information on results"

Any other information on results incl. tables

Table 1 Summary of Results Pre-Experiment and Experiment I

 

Metabolic Activation

Test Item

Dose Level (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 98

TA 100

Without Activation

Ethanol

 

36 ± 6

92 ± 14

Untreated

 

26 ± 6

99 ± 6

Aurelione

3 µg

26 ± 3

91 ± 25

10 µg

28 ± 4

83 ± 3

33 µg

29 ± 7

74 ± 12

100 µg

31 ± 7

86 ± 12

333 µg

33 ± 6

70 ± 6

1000 µg

33 ± 4

78 ± 11

2500 µg

34 ± 7

79 ± 5

5000 µg

31 ± 3

64 ± 7

4-NOPD

10 µg

376 ± 5

 

Sodium azide

10 µg

 

2211 ± 80

With Activation

Ethanol

 

49 ± 3

118 ± 10

Untreated

 

37 ± 6

114 ± 5

Aurelione

3 µg

49 ± 6

93 ± 11

10 µg

44 ± 6

104 ± 6

33 µg

51 ± 5

112 ± 30

100 µg

41 ± 4

124 ± 3

333 µg

43 ± 9

96 ± 9

1000 µg

37 ± 7

44 ± 2

2500 µg

30 ± 5

45 ± 5

5000 µg

32 ± 8

48 ± 3

2-AA

2.5 µg

2505 ± 191

2282 ± 179

 

4-NOPD      4-nitro-o-phenylene-diamine

2-AA            2-aminoanthracene

 

Table 2 Summary of Results Experiment I

 

Metabolic Activation

Test Item

Dose Level (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 102

Without Activation

Ethanol

 

30±7

17±6

450±15

Untreated

 

31±2

12±3

416±27

Aurelione

33 µg

33±8

20±7

439±24

 

100 µg

31±9

15±6

453±28

 

333 µg

39±6

8±3

365±62

 

1000 µg

32±5

13±5

241±47

 

2500 µg

30±15

10±2

167±25

 

5000 µg

21±4

18±3

156±23

Sodium azide

10 µg

1228±152

 

 

4-NOPD

50 µg

 

94±12

 

MMS

4.0 µL

 

 

5696±196

With Activation

Ethanol

 

33±8

17±3

554±37

Untreated

 

32±2

17±5

482±69

Aurelione

33 µg

35±8

18±1

520±76

 

100 µg

32±4

18±3

529±69

 

333 µg

32±2

9±4

498±10

 

1000 µg

27±6

9±1

263±13

 

2500 µg

30±12

14±3

189±13

 

5000 µg

28±3

9±3

165±18

2-AA

2.5 µg

270±12

219±33

 

2-AA

10.0 µg

 

 

4004±238

 

4-NOPD      4-nitro-o-phenylene-diamine

MMS           methyl methane sulfonate

2-AA            2-aminoanthracene

 

 

 

Table 3 Summary of Results Experiment II

 

Metabolic Activation

Test Item

Dose Level (µg/plate)

Revertant Colony Counts (Mean ± SD)

TA 1535

TA 1537

TA 98

TA 100

TA 102

II

IIa

Without Activation

Ethanol

 

24 ± 2

11 ± 1

34 ± 3

21 ± 2

132 ± 4

301 ± 3

Untreated

 

22 ± 6

12 ± 3

35 ± 11

27 ± 6

144 ± 22

292 ± 14

Aurelione

10 µg

24 ± 8

9 ± 2

31 ± 4

26 ± 4

125 ± 14

325 ± 32

33 µg

29 ± 6

11 ± 2

32 ± 12

24 ± 2

122 ± 7

327 ± 20

100 µg

24 ± 7

8 ± 1

32 ± 6

21 ± 4

113 ± 13

305 ± 19

333 µg

31 ± 2

11 ± 3

43 ± 5

7 ± 5

77 ± 5

243 ± 48

1000 µg

23 ± 4

15 ± 8

52 ± 8

14 ± 5

72 ± 13

140 ± 3

2500 µg

30 ± 6

9 ± 3

101 ± 13

n.a.

63 ± 12

84 ± 9

5000 µg

27 ± 9

12 ± 2

80 ± 4

n.a.

56 ± 1

26 ± 5

Sodium azide

10 µg

1291 ± 46

 

 

 

1773 ± 102

 

4-NOPD

10 µg

 

 

458 ± 61

 

 

 

4-NOPD

50 µg

 

100 ± 7

 

354 ± 14

 

 

MMS

4.0 µL

 

 

 

 

 

1593 ± 130

With Activation

Ethanol

 

36 ± 7

31 ± 8

50 ± 1

 

151 ± 10

317 ± 16

Untreated

 

35 ± 4

36 ± 5

60 ± 2

 

160 ± 17

402 ± 5

Aurelione

10 µg

37 ± 7

34 ± 3

53 ± 6

 

138 ± 3

360 ± 12

33 µg

30 ± 3

31 ± 4

58 ± 10

 

142 ± 16

213 ± 32

100 µg

31 ± 2

33 ± 3

49 ± 3

 

129 ± 5

141 ± 22

333 µg

39 ± 3

41 ± 5

61 ± 2

 

114 ± 12

57 ± 13

1000 µg

37 ± 8

31 ± 2

56 ± 6

 

85 ± 17

32 ± 13

2500 µg

35 ± 5

4 ± 1

35 ± 5

 

98 ± 20

19 ± 7

5000 µg

27 ± 4

6 ± 2

33 ± 3

 

77 ± 8

16 ± 2

2-AA

2.5 µg

376 ± 34

166 ± 19

1417 ± 81

 

1462 ± 127

 

2-AA

10.0 µg

 

 

 

 

 

1628 ± 128

 

4-NOPD      4-nitro-o-phenylene-diamine

MMS           methyl methane sulfonate

2-AA            2-aminoanthracene

n.a.              not analysable due to reduced background growth

 

 


 

Table 4 Historical Control Data

 

Strain

 

Without S9 mix

With S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

Negative control

Positive control

20

18

3042

6

5

756

9

10

1003

30

29

3618

18

18

357

10

10

111

7

9

172

39

38

476

TA 1537

Solvent control

Negative control

Positive control

11

12

97

7

6

21

4

5

52

29

29

191

18

18

141

9

6

47

6

8

94

31

29

380

TA 98

Solvent control

Negative control

Positive control

24

26

379

9

10

98

14

15

137

58

52

976

37

43

1239

13

15

510

21

17

229

57

64

5466

TA 100

Solvent control

Negative control

Positive control

121

141

2089

29

23

408

91

101

1262

198

189

2872

149

147

921

36

43

346

109

13

546

281

254

2589

TA 102

Solvent control

Negative control

Positive control

338

326

2764

77

53

1479

242

242

1220

430

390

5593

426

450

2104

70

60

752

332

280

872

514

556

3052

 

Mean = mean value of revertants/plate

SD = standard deviation

Min = minimal value

Max = maximal value

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102, according to OECD 471. The assay was performed in two independent experiments both with and without liver microsomal activation. An additional experiment was performed as pre-incubation with strain TA 98 without S9, only (reported as exp. IIa). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment 1: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II, Ila: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background background growth in almost all strains with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants, occurred in strains TA 100 (with S9 mix) and TA 102 (with and without S9 mix) in experiment I and in strains TA 98 (IIa), 100, and TA 102 without S9 mix and in strains TA 1537 and TA 102 with S9 mix in experiment II. A seemingly dose dependent increase in revertant colony numbers was observed following treatment with the test item in strain TA 98 without metabolic activation in experiment II. The number of colonies reached or exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 2500 µg/plate and above in the absence of metabolic activation. To verify the results an additional experiment was performed as pre-incubation test with TA 98 without S9 mix (exp. IIa). In this experiment an increase of the revertant colonies was not observed. However, reduced background growth was observed from 333 µg/plate up to 5000 µg/plate. Therefore, the observed large number of very small colonies in experiment II were judged to be based upon toxicity rather than indicating a possible mutagenic potential. A major reduction of the background growth results in less bacteria competing for the traces of histidine introduced by the top agar. The traces of histidine are sufficient to allow the surviving bacteria to grow into very small colonies until the histidine is depleted. All other strains did not show any mutagenic effect of the test item up to the maximal concentration applied. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.