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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January 2006 to 02 February 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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:
other: Commission Directive 2000/32/EC L1362000 Annex 4D
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:
Ethyl 4-dimethylaminobenzoate
EC Number:
233-634-3
EC Name:
Ethyl 4-dimethylaminobenzoate
Cas Number:
10287-53-3
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl 4-(dimethylamino)benzoate
Test material form:
solid
Details on test material:
- Appearance: White solid
- Storage conditions of test material: Room temperature

Method

Target gene:
- S. typhimurium: Histidine locus
- E. coli: Tryptophan locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 ML ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Obtained from Trinova Biochem GmbH (35394 Gießen, Germany)
- Method of culture: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

MEDIA USED
- Type and identity of media for pre-cultures: From the thawed ampoules of the strains, 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.
- Properly maintained: Yes. Regular checking of the properties of the strains regarding the membrane permeability as well as spontaneous mutation rates is performed.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment I) and pre-incubation (experiment II)
The following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent or positive control), 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 µL bacteria suspension and 2000 µL overlay agar.
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: The study was performed in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of the background lawn
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test material and reduced background growth, the revertant colonies were partly counted manually.
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data
- The positive control substances should produce a significant increase in mutant colony frequencies

EVALUATION OF RESULTS
A test material 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 WP2 uvrA) 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 was not carried out

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
Remarks:
5000 µg/plate +S9 mix
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:
cytotoxicity
Remarks:
2500 and 5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix
Vehicle controls validity:
valid
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
Remarks:
5000 µg/plate -S9mix; 2500 and 5000 µg/plate +S9mix
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:
cytotoxicity
Remarks:
5000 µg/plate -S9mix; 1000 to 5000 µg/plate +S9mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 to 5000 µg/plate +S9mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
The historical control range was slightly exceeded in the untreated WP2 uvrA with/without activation in experiment I. This deviation is based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Positive controls validity:
valid
Additional information on results:
No visible reduction of the background growth was observed with or without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (µg/plate):
TA 1535: 5000 µg/plate with S9 mix (Experiment II)
TA 1537: 5000 µg/plate with and without S9 mix (Experiment I); 2500 and 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
TA 98: 5000 µg/plate with and without S9 mix (Experiment I); 2500, 5000 µg/plate with S9 mix (Experiment II)
TA 100: 5000 µg/plate without S9 mix, 1000 - 5000 µg/plate with S9 mix (Experiment II)
WP2 uvrA: 5000 µg/plate with S9 mix (Experiment I); 1000 - 5000 µg/plate with S9 mix (Experiment II).
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
The laboratory´s historical control range was slightly exceeded in the untreated control of strain WP2 uvrA with and without metabolic activation in experiment I. This deviation is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.
Under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Any other information on results incl. tables

Table 1: Summary of Pre-Experiment and Experiment I

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

DMSO

Untreated

3

10

33

100

333

1000

2500

5000

114

121

110

123

112

113

95

112

101P

85P

21

16

19

19

23

19

16

22

23P

20P

47

50

56

51

55

61

45

41

39P

30P

27

28

27

25

30

26

28

29

28P

11P

13

14

8

12

11

13

10

6

7P

6P

+

DMSO

Untreated

3

10

33

100

333

1000

2500

5000

128

123

115

125

131

130

111

110

98P

74P

26

21

25

18

25

26

17

24

20P

16P

63

68

69

64

64

69

61

45

38P

13P

30

29

28

31

29

28

25

28

26P

8P

15

16

19

15

12

13

19

20

18P

5P

Positive Controls

-

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration (µg/plate)

10

10

4.0 µL

10

50

Mean no. colonies/plate

2013

1236

1435

357

109

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

2.5

2.5

10

2.5

2.5

Mean no. colonies/plate

1749

286

294

1364

134

P = Precipitate

NaN3 = sodium azide

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

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Table 2: Summary of Experiment II

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

DMSO

Untreated

10

33

100

333

1000

2500

5000

115

164

139

147

129

104

117

61P

44P

16

17

19

18

19

15

18

10P

9P

57

66

50

53

53

49

47

31P

28P

24

24

28

26

27

18

16

16P

11P

9

13

8

7

8

5

4

2P

2P

+

DMSO

Untreated

10

33

100

333

1000

2500

5000

163

198

172

158

155

129

62

50P

53P

21

28

30

26

29

15

12

10P

9P

68

72

77

69

82

64

28

18P

10P

38

36

29

29

33

34

26

12P

12P

12

8

12

9

14

16

3

3P

2P

Positive Controls

-

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentration (µg/plate)

10

10

4.0 µL

10

50

Mean no. colonies/plate

1865

1347

416

390

95

+

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

2.5

2.5

10

2.5

2.5

Mean no. colonies/plate

3476

521

272

4953

143

P = Precipitate

NaN3 = sodium azide

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

MMS = methyl methane sulfonate

2AA = 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was determined to be non-mutagenic in both the presence and absence of metabolic activation.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and Commission Directive 2000/32/EC L1362000 Annex 4D under GLP conditions using the bacterial reverse mutation assay.

The study was performed to investigate the potential of the test material to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test material was tested at the following concentrations: Pre-Experiment /Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

No visible reduction of the background growth was observed with and without metabolic activation in experiment I. In experiment II, reduced background growth was observed at higher concentrations with and without metabolic activation in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at 5000 µg/plate in strains TA 1537 and TA 98 with and without metabolic activation and in strain WP2 uvrA with metabolic activation in experiment I. In experiment II, toxic effects were observed at higher concentrations with metabolic activation in all strains and without metabolic activation in strains TA 1537 and TA 100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Appropriate untreated and solvent controls were also used and the study was considered to be valid.

During the described mutagenicity test and under the experimental conditions reported, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Under the conditions of this study, the test material is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

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