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Toxicological information

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:
2014-03-18 to 2014-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
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-[(3-{[(3-acetoxy-2,2-dimethylpropylidene)amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate
EC Number:
805-722-7
Cas Number:
1064082-81-0
Molecular formula:
C24H42N2O4
IUPAC Name:
3-[(3-{[(3-acetoxy-2,2-dimethylpropylidene)amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate
Test material form:
liquid

Method

Target gene:
Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. Main DNA target GC.
Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
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:
other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver induced by Phenobarbital (PB) and β-naphthoflavone (BNF)
Test concentrations with justification for top dose:
5000; 1581, 500, 158, 50 and 15.8 μg/plate
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Test item is good soluble in DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
E.coli and all of Salmonella strains with S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535 without S9-mix
Untreated negative controls:
yes
Remarks:
untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Salmonella TA98 without S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation
The Experiment I (Initial Mutation Test) of the main study the plate incorporation method was used. In the Experiment II (Confirmatory Mutation Test) the pre-incubation method was applied and the concentrations examined were the same as investigated in the Experiment I.

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth; Toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment.
Evaluation criteria:
Evaluation of Experimental Data

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least


Conditions for the Validity of the Test

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
Statistics:
None

Results and discussion

Test results
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:
In the Confirmatory Mutation the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1535 and TA1537 at 5000 μg/plate (-S9 Mix) and in S. typhimurium TA1535 at 1581 μg/plate (-S9 Mix).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicity

The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA100, in the whole examined concentration range of 5000-5 μg/plate, with addition of metabolic activation (+S9 Mix).
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at the concentrations of 50 μg/plate, with addition of metabolic activation (+S9 Mix) and in TA100 in the concentration range of 5000-5 μg/plate, without metabolic activation (-S9 Mix).

COMPARISON WITH HISTORICAL CONTROL DATA: The obtained changes, slightly lower or higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.

Any other information on results incl. tables

Table 1 summary of results of initial mutation test

 

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

13.7

0.84

23.7

1.09

87.3

1.10

135.3

1.24

5.7

0.74

10.7

1.03

8.7

1.00

7.0

0.91

25.3

1.01

25.7

0.94

DMSO Control

16.3

1.00

21.7

1.00

79.7

1.00

109.0

1.00

7.7

1.00

10.3

1.00

8.7

1.00

7.7

1.00

25.0

1.00

27.3

1.00

Ultrapure Water Control

90.3

1.00

7.0

1.00

30.0

1.00

5000

11.3

0.69

21.0

0.97

114.0

1.43

132.7

1.22

10.0

1.30

9.3

0.90

12.3

1.42

10.3

1.35

20.7

0.83

26.3

0.96

1581

13.7

0.84

19.3

0.89

102.7

1.29

124.0

1.14

7.3

0.96

12.3

1.19

7.3

0.85

9.7

1.26

25.3

1.01

28.0

1.02

500

15.3

0.94

20.0

0.92

88.0

1.10

122.0

1.12

8.7

1.13

11.3

1.10

8.0

0.92

9.0

1.17

21.7

0.87

29.0

1.06

158

13.0

0.80

22.3

1.03

85.0

1.07

117.0

1.07

6.0

0.78

9.0

0.87

7.0

0.81

7.0

0.91

25.7

1.03

33.0

1.21

50

11.3

0.69

21.0

0.97

90.0

1.13

120.7

1.11

7.3

0.96

9.0

0.87

9.0

1.04

6.3

0.83

23.0

0.92

29.7

1.09

15.8

13.3

0.82

22.0

1.02

87.7

1.10

114.0

1.05

6.3

0.83

10.3

1.00

8.7

1.00

8.7

1.13

27.7

1.11

26.0

0.95

NPD (4mg)

173.3

10.61

SAZ (2mg)

948.0

10.49

729.3

104.19

9AA (50mg)

452.7

52.23

MMS (2mL)

826.0

27.53

2AA (2mg)

1725.3

79.63

1856.0

17.03

209.3

20.26

175.0

22.83

2AA (50mg)

284.3

10.40

 

Table 2 Summary of results of confirmatory mutation test

 

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

16.7

1.09

23.3

1.06

108.7

1.32

139.3

1.23

9.3

0.93

17.7

1.20

6.3

0.79

7.3

0.79

31.3

1.12

42.0

1.09

DMSO Control

15.3

1.00

22.0

1.00

82.3

1.00

113.7

1.00

10.0

1.00

14.7

1.00

8.0

1.00

9.3

1.00

28.0

1.00

38.7

1.00

Ultrapure Water Control

78.7

1.00

9.3

1.00

40.0

1.00

5000

5.3

0.35

14.3

0.65

44.7

0.54

100.0

0.88

0.0

0.00

12.7

0.86

0.0

0.00

11.3

1.21

23.3

0.83

29.7

0.77

1581

13.0

0.85

20.7

0.94

82.7

1.00

112.7

0.99

5.7

0.57

14.3

0.98

7.3

0.92

9.0

0.96

32.7

1.17

39.7

1.03

500

13.7

0.89

20.0

0.91

95.3

1.16

114.3

1.01

10.3

1.03

13.0

0.89

6.3

0.79

7.0

0.75

35.0

1.25

43.7

1.13

158

14.3

0.93

23.0

1.05

89.7

1.09

115.7

1.02

10.0

1.00

11.0

0.75

6.7

0.83

8.3

0.89

31.3

1.12

41.0

1.06

50

13.7

0.89

20.3

0.92

84.7

1.03

127.3

1.12

10.3

1.03

13.3

0.91

7.7

0.96

7.0

0.75

28.0

1.00

43.3

1.12

15.8

14.3

0.93

24.3

1.11

87.3

1.06

122.3

1.08

11.7

1.17

14.0

0.95

8.7

1.08

8.0

0.86

24.0

0.86

40.0

1.03

NPD (4mg)

178.0

11.61

SAZ (2mg)

1230.0

15.64

497.3

53.29

9AA (50mg)

365.0

45.63

MMS (2mL)

1096.0

27.40

2AA (2mg)

709.3

32.24

1310.7

11.53

120.3

8.20

111.7

11.96

2AA (50mg)

261.7

6.77

 

Table Historical control data for Revertants/Plate (for the period of 2008-2013)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

22.6

110.1

10.1

7.5

24.7

SD

3.3

31.0

1.4

2.7

4.2

Minimum

11

66

2

2

11

Maximum

40

162

23

19

41

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

29.6

117.7

12.0

8.7

34.2

SD

3.6

22.9

1.6

2.3

3.5

Minimum

13

76

4

2

20

Maximum

48

170

24

20

54

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

22.1

106.0

9.9

7.4

23.8

SD

3.1

29.3

1.2

2.8

3.5

Minimum

11

66

3

2

10

Maximum

40

156

23

18

42

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

28.6

117.4

12.1

8.4

34.2

SD

3.4

22.1

1.6

2.2

3.8

Minimum

17

73

3

2

17

Maximum

49

168

25

18

55

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

23.8

110.3

9.9

6.5

25.2

SD

6.8

30.9

1.4

3.4

3.8

Minimum

9

70

2

1

12

Maximum

43

159

24

16

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

29.8

118.8

11.8

7.8

35.1

SD

7.4

23.0

1.5

3.4

4.9

Minimum

10

81

3

2

19

Maximum

48

174

24

20

55

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

199.6

904.8

792.5

457.5

700.3

SD

35.1

107.9

106.0

111.8

58.9

Minimum

165

477

332

110

341

Maximum

248

1953

1278

1439

1236

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

199.6

1340.5

173.3

152.1

280.8

SD

35.1

347.7

37.6

23.3

80.0

Minimum

248

491

87

68

144

Maximum

165

2869

603

465

520

 

Applicant's summary and conclusion

Conclusions:
The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471 and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled.

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix).

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.