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Diss Factsheets

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:
17 January 2003 - 10 February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6,10-trimethylundec-9-enal
EC Number:
205-460-8
EC Name:
2,6,10-trimethylundec-9-enal
Cas Number:
141-13-9
Molecular formula:
C14H26O
IUPAC Name:
2,6,10-trimethylundec-9-enal
Test material form:
liquid
Specific details on test material used for the study:
Batch 9000484970
Expiry date 18 March 2003

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:
Dose range finding test (results used as part of the definitve test):
Strains TA100 and TA98; 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (with and without metabolic activation).

Definitive test - preincubation method:
Strains TA1535, TA1537 and TA102; 10, 33, 100, 333, 1000 and 3330 µg/plate (with and without metabolic activation).

Definitive test - direct plate method:
Strains TA1535, TA1537 and TA102; 3, 10, 33, 100, 333 and 1000 µg/plate (with and without metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cumene hydroperoxide
other: daunomycin; methylmethanesulfonate; 2-aminoanthracene; 1,8 dihydroxy anthraquinone
Details on test system and experimental conditions:
METHOD OF APPLICATION: First experiment, preincubation method; Second experiment, direct plate method.

DURATION
- Preincubation period: 30 minutes by 70 rpm at 37 °C.
- Exposure duration: 48 hours, incubated in the dark at 37 °C.

NUMBER OF REPLICATIONS: Each concentration was tested in triplicate.

NUMBER OF CELLS EVALUATED: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

DOSE RANGE FINDER
- Method: All doses were tested in triplicate in the preincubation and the direct plate assay.
- Results used for defining the definitive dosing range: The highest concentration of the test material used in the definitive mutation assay was the level at which the test material exhibited limited solubility. The dose range finding tests were reported as a part of the preincubation and direct plate assays for the definitive tests, respectively.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 2: Toxicity of the Test Material in the Dose Range Finding Study (preincubation assay)

Strain

Without S9-mix

With S9-mix

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

TA98

333-3330

5000

Slight

Slight

Slight

Moderate

5000

-1

-2

TA100

10-5000

Slight

-2

333-5000

Slight

-2

-1= No reduction of the bacterial background lawn

-2= No reduction in the number of revertant colonies.

Table 3: Toxicity to the Test Material in the Preincubation Assay (Definitive Test)

Strain

Without S9-mix

With S9-mix

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

Dose (µg/plate)

Bacterial Background Lawn

Revertant Colonies

TA1535

10

Slight

-1

333

Slight

-1

33-100

Slight

-2

333-1000

Moderate

-2

1000-3330

Moderate

-1

3330

Extreme

Microcolonies

TA1537

100

Slight

-1

333-3330

Slight

-1

 

333

Moderate

Extreme

1000

Moderate

Moderate

3330

Extreme

Microcolonies

TA102

333-1000

-3

-2

3330

-3

-1

3330

Slight

Moderate

 -1= No reduction in the number of revertant colonies

-2= Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range

-3= No reduction of the bacterial background lawn

Table 4: Mutagenic Response of the Test Material in the Preincubation Assay

Dose (µg/plate)

Mean number of revertant colonies/ 3 replicate plates (± SD)

TA1535

TA1537

TA98

TA100

TA102

Without S9-mix

Positive control

777 ± 42

75 ± 6

377 ± 38

440 ± 24

594 ± 23

Solvent control

11 ± 3

6 ± 3

15 ± 2

139 ± 11

210 ± 4

3

 

 

12 ± 3

127 ± 14

 

10

9 ± 11

6 ± 3

14 ± 4

151 ± 151

219 ± 21

33

8 ± 21

6 ± 3

19 ± 1

186 ± 131

206 ± 5

100

5 ± 21

7 ± 21

10 ± 1

195 ± 141

202 ± 22

333

8 ± 32

2 ± 12

10 ± 11

190 ± 131

163 ± 7

1000

7 ± 12

3 ± 12

10 ± 21

247 ± 101

140 ± 15

3330SP

MC3

MC3

9 ± 11

229 ± 271

109 ± 151

5000SP

 

 

8 ± 31

222 ± 41

 

 

With S9-mix

Positive control

99 ± 6

139 ± 24

344 ± 65

344 ± 21

693 ± 47

Solvent control

6 ± 2

6 ± 1

20 ± 3

137 ± 12

212 ± 14

3

 

 

20 ± 1

149 ± 25

 

10

9 ± 3

5 ± 2

17 ± 3

142 ± 12

268 ± 9

33

6 ± 4

5 ± 1

19 ± 2

138 ± 3

286 ± 44

100

9 ± 1

5 ± 2

18 ± 3

145 ± 18

230 ± 9

333

6 ± 21

6 ± 21

11 ± 2

159 ± 111

243 ± 12

1000

6 ± 22

6 ± 11

14 ± 2

159 ± 171

245 ± 19

3330SP

7 ± 42

5 ± 11

14 ± 3

212 ± 271

221 ± 16

5000SP

 

 

15 ± 2

180 ± 361

 

Solvent control: 0.1 mL DMSO

1 = Bacterial background lawn slightly reduced

2 = Bacterial background lawn moderately reduced

3 = Bacterial background lawn extremely reduced

SP = Slight Precipitate

MC = Microcolonies

 

Table 5: Mutagenic Response of the Test Material in the Direct Plate Assay

Dose (µg/plate)

Mean number of revertant colonies/ 3 replicate plates (± SD)

TA1535

TA1537

TA98

TA100

TA102

Without S9-mix

Positive control

1067 ± 54

605 ± 92

276 ± 20

998 ± 23

1403 ± 59

Solvent control

14 ± 3

6 ± 1

19 ± 2

140 ± 17

286 ± 22

3

12 ± 2

9 ± 3

20 ± 3

132 ± 6

184 ± 9

10

13 ± 2

7 ± 2

15 ± 3

131 ± 25

183 ± 16

33

14 ± 3

7 ± 2

17 ±3

123 ± 8

190 ± 7

100

12 ± 3

8 ± 3

18 ± 5

124 ± 16

239 ± 31

333

9 ± 1

8 ± 2

18 ± 4

114 ± 4

225 ± 6

1000SP

7 ± 3

9 ± 1

17 ± 6

119 ± 8

211 ± 13

3330SP

 

 

17 ± 2

124 ± 11

 

5000SP

 

 

14 ± 1

128 ± 6

 

 

With S9-mix

Positive control

281 ± 27

811 ± 57

1520 ± 39

1073 ± 61

1219 ± 48

Solvent control

13± 6

8 ± 3

22 ± 3

132 ± 10

269 ± 13

3

15 ± 5

9 ± 1

23 ± 6

149 ± 25

202 ± 12

10

15 ± 3

6 ± 2

24 ± 7

138 ± 25

212 ± 8

33

11 ± 2

9 ± 2

21 ± 3

147 ± 16

235 ± 15

100

12 ± 5

9 ± 6

22 ± 1

137 ± 5

205 ± 11

333

7 ± 4

7 ± 3

20 ± 3

123 ± 17

238 ± 19

1000SP

9 ± 5

6 ± 3

24 ± 2

117 ± 8

214 ± 14

3330SP

 

 

17 ± 4

99 ± 6

 

5000SP

 

 

21 ± 3

120 ± 6

 

Solvent control: 0.1 mL DMSO

SP = Slight Precipitate

Applicant's summary and conclusion

Conclusions:
Based on the test results of this study it is concluded that ADOXAL is not mutagenic in the Salmonella Typhimurium reverse mutation assay.
Executive summary:

ADOXAL was tested in the Salmonella typhimurium reverse mutation assay with five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9-mix). ADOXAL did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9 -metabolic activation. These results were confirmed in a separate experiment.