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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:
October 22, 2007 - December 21, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
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-[1-(2-{4-[2-(5,5-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl)piperidin-4-yl]-1H-1,3-benzodiazole
EC Number:
813-966-0
Cas Number:
202189-81-9
Molecular formula:
C28H36N4O
IUPAC Name:
2-[1-(2-{4-[2-(5,5-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl)piperidin-4-yl]-1H-1,3-benzodiazole
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: sponsor
- Lot: 5BM

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in darkness.
- Solubility and stability of the test substance in the solvent/vehicle: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. A stock concentration of 100 mg/ml was prepared in DMSO from which 1:5 dilutions were made.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The top concentration of the test item was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16; and 0.032 mg/ml.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Solvent is compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cumene hydroperoxide
other: 2-aminoantracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation
Each point of the two series of tubes (with and without S9) was tested in duplicate and with the following composition: phosphate buffer (or S9 mixture), 2E9 cell/ml bacterial culture and the solvent (negative control), the test item (each of the five concentrations) or the reference item (positive controls). The tubes were placed in a water bath at 37 ºC for 45 minutes. Then 2 mL of surface agar supplemented with histidine/biotin 0.5 mM was added to each tube and poured out onto a minimum agar plate. The plates were left to set for 1 hour and they were then placed in the incubator at 37 ºC for 48-72 hours.

DURATION
- Preincubation period: 45 minutes
- Exposure duration:48 -72 hours

SELECTION AGENT (mutation assays): The lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize an essential amino acid.

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
- Method: visual observation of the colonies.

OTHER EXAMINATIONS:
Phenotype and sterility controls were also performed.

- OTHER:
Solutions preparation: Sodium phosphate buffer, 200mM, pH=7.4, was used as the vehicle to prepare the item concentrations. In all cases, these concentrations were prepared on the day they were used. A stock concentration of 100mg/ml was prepared in DMSO from which 1:5 dilutions were carried out.

Test system: Prior to the study, the master plates of each strain were prepared. The strains were plated out in minimum agar plates enriched with Biotin 0.5 mM and Histidine 0.1 M. In the case of strains TA98 and TA100 the plates also contained ampicillin 8 mg/ml and in the case of strain TA102 they contaided tetracycline 8 mg/ml, in addition to Histidine, Biotin and Ampicillin. The plates were cultivated for 48 hous at 37 ºC.
Rationale for test conditions:
The top concentration of the test item, 100 mg/ml, was toxic for Salmonella typhimurium so, the following concentrations were tested: 20; 4; 0.8; 0.16 and 0.032 mg/ml.
Evaluation criteria:
Criteria conclusion: the result of the test is considered as positive if the test item induce an increase of colonies with respect to non-treated plates, dependent on the concentration of one, or several of the 5 strains, without and/or with metabolic activation.

Results and discussion

Test resultsopen allclose all
Key result
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
Key result
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
Key result
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
Key result
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
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:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No


Any other information on results incl. tables

The conditions listed below indicate that the tests are acceptable:

1. The plates show a firm, uniform lawn, which demonstrates that there is no toxicity in the concentrations that were taken as a reference to evaluate the mutagenic power.

2. The number of colonies in the spontaneous mutation plates is within the normal range for each strain.

3. The positive controls induce a clear increase in the number of revertants in all cases.

4. The phenotype control plates show the expected results for each strain.

From the results expressed on the tables below it can be deduced that the test item does not induce an increase in colonies in any of the strains used in this study, neither in the presence of S9 nor in its absence.

Calculation of the mutation index (MI)

MI = nº. of mut. in a dose / nº. of mut. in the control

Strain TA98

 

-S9

+S9

 

No. Col.

Average

MI

No. Col.

Average

MI

Sp. Mut.

22/20

21.0

--

20/17

18.5

--

0mg/ml

18/20

19.0

--

15/16

15.5

--

32mg/ml

17/17

17.0

0.895

18/14

16.0

1.032

160mg/ml

27/25

26.0

1.368

20/13

16.5

1.065

800mg/ml

16/22

19.0

1.000

24/15

19.5

1.258

4000mg/ml

16/19

17.5

0.921

18/24

21.0

1.355

2000mg/ml

26/24

25.0

1.316

27/15

21.0

1.355

Control +

>2000/>2000

>2000

>105.263

>2000/>2000

>2000

129.032

 

Strain TA100

 

-S9

+S9

 

No. Col.

Average

MI

No. Col.

Average

MI

Sp. Mut.

173/179

176.0

--

200/184

192.0

--

0mg/ml

180/176

178.0

--

191/197

194.0

--

32mg/ml

172/174

173.0

0.972

182/175

178.5

0.920

160mg/ml

181/173

177.0

0.994

190/194

192.0

0.990

800mg/ml

168/174

171.0

0.961

181/176

178.5

0.920

400mg/ml

190/176

183.0

1.028

198/179

188.5

0.972

20000mg/ml

184/191

187.5

1.053

207/210

208.5

1.075

Control +

>2000/>2000

>2000

>11.236

>2000/>2000

>2000

>10.309

 

Strain TA102

 

-S9

+S9

 

No. Col.

Average

MI

No. Col.

Average

MI

Sp. Mut.

330/324

327.0

--

420/416

418.0

--

0mg/ml

321/324

322.5

--

380/410

395.0

--

32mg/ml

330/326

328.0

1.017

426/430

428.0

1.084

160mg/ml

321/337

329.0

1.020

412/400

406.0

1.028

800mg/ml

320/326

323.0

1.002

430/422

426.0

1.078

4000mg/ml

329/334

331.5

1.028

416/420

418.0

1.058

20000mg/ml

323/329

326.0

1.011

410/420

415.0

1.021

Control +

>2000/>2000

>2000

>6.202

880/900

890.0

2.253

 

Strain TA1535

 

-S9

+S9

 

No. Col.

Average

MI

No. Col.

Average

MI

Sp. Mut.

6/7

6.5

--

13/15

14.0

--

0mg/ml

8/6

7.0

--

16/17

16.5

--

32mg/ml

4/5

4.5

0.643

18/18

18.0

1.091

160mg/ml

7/8

7.5

1.071

16/18

17.0

1.030

800mg/ml

7/5

6.0

0.857

22/15

18.5

1.121

4000mg/ml

6/5

5.5

0.786

24/17

20.5

1.242

20000mg/ml

5/7

6.0

0.857

21/20

20.5

1.242

Control +

>1500/>1500

>1500

>214.286

245/257

251.0

15.212

  

Strain TA1537

 

-S9

+S9

 

No. Col.

Average

MI

No. Col.

Average

MI

Sp. Mut.

4/4

4.0

--

10/7

8.5

--

0mg/ml

7/6

6.5

--

6/8

7.0

--

32mg/ml

7/5

6.0

0.923

6/8

7.0

1.000

160mg/ml

5/4

4.5

0.692

4/8

6.0

0.857

800mg/ml

2/5

3.5

0.538

13/10

11.5

1.643

4000mg/ml

5/4

4.5

0.692

12/7

9.5

1.357

20000mg/ml

7/5

6.0

0.923

9/5

7.0

1.000

Control +

164/171

167.5

25.769

180/191

185.5

26.500

--: It was not possible to count colonies

 

Results of the phenotype control

 

TA98

TA100

TA1535

TA1537

TA102

Ampicilyne

Resistant

Resistant

Sensitive

Sensitive

Resistant

Violet Crystal

Sensitive

Sensitive

Sensitive

Sensitive

Sensitive

UV light

Sensitive

Sensitive

Sensitive

Sensitive

Sensitive

Tetracycline

n.t

n.t

n.t

n.t

Resistant

n.t.: not tested

Applicant's summary and conclusion

Conclusions:
The test item does not induce a dose-dependent increase in Salmonella typhimurium strains. Therefore, it was considered as non-mutagenic under test conditions.
Executive summary:

A Bacterial reverse mutation test was performed according OECD guideline 471 with GLP. Based on a previous toxicity test, 1-2E9 cell/mL of Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 were exposed to 0.032, 0.16, 0.8, 4 and 20 mg/mL test item, solvent and positive controls with and without metabolic activation (two replicates each). The incubation mixtures were pre-incubated at 37 ºC for 45 minutes and incubated at 37 ºC for 48 -72 hours. Then, the revertant colonies were counted. Phenotype and sterility controls were also performed. The plates showed a firm, uniform lawn, which demonstrates that there was no toxicity. The number of colonies in the spontaneous mutation plates was within the normal range for each strain. The positive controls induced a clear increase in the number of revertants in all cases and the phenotype control plates show the expected results for each strain. The test item does not induce a dose-dependent increase in any of the Salmonella typhimurium. Therefore, the test item was determined to be non-mutagenic under test conditions.

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