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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January - 14 May, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate
EC Number:
642-902-2
Cas Number:
163802-53-7
Molecular formula:
C12H22O5Si
IUPAC Name:
3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate
Constituent 2
Reference substance name:
2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-
IUPAC Name:
2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-
Constituent 3
Reference substance name:
3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate
IUPAC Name:
3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate
Test material form:
liquid

Method

Target gene:
All Salmonella strains contain mutation, in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide
excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) gene. (Bacteria require biotin for growth).
The tester strains TA 98 and TA 100 contain the R·factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

The properties of the S. typhimurium and E. coli strains, with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are cheeked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9.
Test concentrations with justification for top dose:
Exposure Concentrations
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:

Experiment I (plate incorporation method):
31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II (pre-incubation method):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main Experiment I.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: miscible and stable in DMSO
Controls
Untreated negative controls:
yes
Remarks:
A. dest., BSL Bioservice Lot No, 120203, 120221
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, AppliChem Lot No. IP008076, I Y009099
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthacene
Remarks:
Pos. control substances assigned to different tester strains.
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9. S9 mix included 5% S9, with MgCl₂, KCl, glucose-6-phosphate and NADP. 500 μl of S9 mix were included in 2.7 ml agar, test solution and bacterial suspension, giving a final concentration of approximately 1% S9 in the plates.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period (second experiment): 60 minutes
- Exposure duration: at least 48 hours

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar

NUMBER OF REPLICATIONS: triplicate plates. The initial plate incorporation assay was repeated in an independent experiment using the pre-incubation method.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: clearing or diminution of background lawn or reduction in number of revertants to ≤ 0.5 relative to solvent control

Evaluation criteria:
A test item is considered mutagenic if a clear dose-related increase in the number of revertants occurs, and/or there is a biologically relevant response for at least on dose group in at least one strain with or without metabolic activation.
Statistics:
Not required.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction in background lawn was observed at concentrations of 1000 μg/plate and above. A reduction in the number of revertants to 50% of solvent control was observed in several strains. This was not considered biologically relevant.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results of Experiment 1 (plate incorporation) and Experiment 2 (pre-incubation). Revertants per plate (mean of three plates)

 

Experiment 1 (plate incorporation)

Concentration μg/plate (solvent or control substance)

TA98

TA100

TA1535

TA1537

E. coli WP2

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (distilled water)

22

27

101

115

5

7

9

10

41

50

0 (DMSO)

18

25

90

113

6

6

6

7

33

47

31.6

19

26

98

112

7

12

6

10

38

31

100

19

32

92

115

9

8

7

11

44

45

316

21

34

91

126

2

9

10

13

42

55

1000

23

42

80*

110

4*

10

7*

8

39*

58

2500

22

25

59*

104

4*

7

6*

9

38*

49

5000

22

28

70*

82

3*

8

3*

8

27*

47

10 (4-NOPD)

390

-

-

-

-

-

140

-

-

-

10 (NaN)

-

-

850

-

803

-

-

-

-

-

1 μl (MMS)

-

-

-

-

-

-

-

-

340

-

2.5 (2-AA)

-

2659

-

2075

-

126

-

213

-

133

 

Experiment 2 (pre-incubation)

Concentration μg/plate (solvent or control substance)

TA98

TA100

TA1535

TA1537

E. coli WP2

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (distilled water)

23

29

128

125

7

5

6

7

77

60

0 (DMSO)

19

24

107

110

5

9

4

7

51

49

10

23

34

113

93

8

8

7

3

46

55

31.6

20

25

114

110

9

6

5

5

55

56

100

23

28

109

98

5

4

7

6

53

63

316

21

28

85

108

6

7

7

3

50

46

1000

21

29

62*

106

2*

7

5*

6

52

56

2500

17

31

55*

102*

1*

8

4*

10

40

56

5000

19

28

67*

83*

3*

5*

4*

8*

38

49

10 (4-NOPD)

446

-

-

-

-

-

86

-

-

-

10 (NaN)

-

-

839

-

577

-

-

-

-

-

1 μl (MMS)

-

-

-

-

-

-

-

-

710

-

2.5 (2-AA)

-

1985

-

1543

-

57

-

185

-

161

* Reduction in background lawn

4-NOPD  4-nitro-o-phenylene-diamine

NaN      sodium azide

MMS      methylmethanesulfonate

2-AA      2-aminoanthacene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

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 frame shifts in the genome of the tester strains used. Appropriate positive, solvent and negative controls were included and gave expected results. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay under the conditions of the test.
Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. In two 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 of the test item were prepared and used in the experiments: Experiment I (plate incorporation method): 31.6, 100, 316, 1000, 2500 and 5000 μg/plate Experiment II (pre-incubation method): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in Experiment I and II (with and without metabolic activation). Cytotoxicity related to clearing or rather diminution of the background lawn and a reduction in the number of revertants down to a mutation factor equal and less than 0.5 in relation to the solvent control was noted in both experiments. Cytotoxicity was noted in four tester strains used in Experiment I and in three tester strains used in Experiment II. In Experiment I, reduced background lawn was observed in four tester strains (TA 100, TA 1535, TA 1537 and WP2 uvrA) at concentrations greater than or equal to 1000 μg/plate (without metabolic activation). In strain TA 1537, the mutation factor was 0.5 at 5000 μg/plate. One other strain TA1535 (without metabolic activation) had one concentration, 316 μg/plate, with a mutation factor less than 0.5. However, the finding is not considered to be biologically relevant, because it did not appear to be attributable to the test item. In Experiment II, reduced background lawn was observed in three tester strains (TA 100, TA 1535, and TA 1537) at concentrations of 1000 μg/plate and higher (without metabolic activation). With metabolic activation, two of same three strains (TA 1535, and TA 1537) had reduced background lawn at a concentration of 5000 μg/plate. The remaining strain (TA 100) had reduced background lawn concentrations greater than or equal to 2500 μg/plate. A mutation factor of 0.5 and less was noted at various concentrations for TA 100 (without metabolic activation), TA 1535 (with and without metabolic activation), and TA 1537 (with metabolic activation). However with the exception of strain TA 1535 (without metabolic activation) where the mutation factor was equal to or less than 0.5 at the three highest concentrations tested, the finding was not considered to be biologically relevant due to its sporadic incidence. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2,4 -hexadienoic acid, 3-(trimethoxysilyl) propyl ester at any concentration level, neither in the presence nor absence of metabolic activation in Experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. 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. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.