Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,1-Dimethoxyethane (CAS no. 534-15-6) was considered to be non-mutagenic in the tested species under the conditions of the test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 17 August 2017 Experimental completion date 08 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
Identification: 1,1-Dimethoxyethane (CAS no. 534-15-6)
Physical state/Appearance: Clear colourless liquid
Batch: 1706291650R
Purity: 99.92%
Expiry Date: 29 June 2019
Storage Conditions: Approximately 4 °C in the dark under nitrogen
Target gene:
Histidine locus in S. typhimurium and tryptophan locus in E.coli.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

Experiment 2 – Pre-Incubation Method: 15, 50, 150, 500, 1500, 5000 μg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum concentration in the second mutation test.
Vehicle:
The test item was miscible but considered viscous enough in sterile distilled water at 50 mg/mL to make dosing the test item difficult, however it was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2 μg/plate for WP2uvrA 3 μg/plate for TA100 5 μg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
80 μg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 μg/plate for TA98
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1 μg/plate for TA100 2 μg/plate for TA1535 and TA1537 10 μg/plate for WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
presence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 μg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
presence of S9-mix
Details on test system and conditions:
Test Item Preparation and Analysis
The highest concentration of the test item tested in this study was 50 mg/mL (final plate concentration was 5000 μg/plate). The test item was accurately weighed and diluted in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. The highest concentration in each test was diluted with dimethyl sulphoxide to produce a series of lower concentrations, separated by approximately half-log10 intervals. No correction was made for purity. Prior to use, the solvent (dimethyl sulphoxide) was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single manual count was required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
This study meets all items listed in the acceptability criteria outlined.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum concentration in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Experiment 1 – Without Metabolic Activation

Test Period

From: 31 August 2017

To: 03 September 2017

S9-Mix

(-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (DMSO)

67

68

64

(66)

2.1#

26

26

28

(27)

1.2

18

(20)

21

2.1 22

25

20

19

(21)

3.2

16

14

10

(13)

3.1

1.5 µg

63

73

64

(67)

5.5

24

19

20

(21)

2.6

23

(20)

17

3.1 21

22

23

14

(20)

4.9

11 7

17

(12)

5.0

5 µg

56

77

73

(69)

11.2

19

19

23

(20)

2.3

17

(19)

19

1.5 20

22

19

18

(20)

2.1

12

10

16

(13)

3.1

15 µg

62

64

65

(64)

1.5

24

21

20

(22)

2.1

24

(21)

18

3.0 21

19

22

22

(21)

1.7

16

13

10

(13)

3.0

50 µg

60

67

71

(66)

5.6

16

20

17

(18)

2.1

14

(19)

18

5.0 24

22

24

23

(23)

1.0

12

10

18

(13)

4.2

150 µg

71

76

60

(69)

8.2

22

16

15

(18)

3.8

24

(19)

17

4.0 17

25

13

21

(20)

6.1

12

11

12

(12)

0.6

500 µg

59

66

66

(64)

4.0

22

24

21

(22)

1.5

25

(16)

12

7.8 11

26

25

25

(25)

0.6

15

11

14

(13)

2.1

1500 µg

82

74

68

(75)

7.0

24

19

27

(23)

4.0

19

(17)

18

2.1 15

25

24

26

(25)

1.0

16

14

15

(15)

1.0

5000 µg

85

67

66

(73)

10.7

27

19

24

(23)

4.0

18

(24)

26

5.3 28

29

29

28

(29)

0.6

11

11

17

(13)

3.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

786

(627)

670

184.8

424

2073

(2144)

2186

61.8

2173

1347

(1224)

1206

115.1

1119

267

(258)

255

7.9 252

435

(403)

423

45.4

351

Experiment 1 – With Metabolic Activation

Test Period

From: 31 August 2017

To: 03 September 2017

S9-Mix

(+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (DMSO)

73

72

69

(71)

2.1#

38

33

29

(33)

4.5

25

(25)

29

4.0 21

32

24

23

(26)

4.9

14

18

22

(18)

4.0

1.5 µg

68

66

70

(68)

2.0

34

36

35

(35)

1.0

25

(27)

26

2.6 30

22

21

30

(24)

4.9

9

14

15

(13)

3.2

5 µg

62

74

76

(71)

7.6

33

29

31

(31)

2.0

21

(25)

26

4.0 29

29

23

22

(25)

3.8

13 8

12

(11)

2.6

15 µg

56

69

69

(65)

7.5

32

28

29

(30)

2.1

34

(27)

22

6.4 24

29

23

24

(25)

3.2

21

17

10

(16)

5.6

50 µg

57

66

68

(64)

5.9

37

33

29

(33)

4.0

19

(25)

29

5.1 26

27

27

25

(26)

1.2

13

20

13

(15)

4.0

150 µg

71

67

65

(68)

3.1

30

33

36

(33)

3.0

27

(20)

11

8.3 23

26

28

30

(28)

2.0

20

15

15

(17)

2.9

500 µg

67

65

84

(72)

10.4

38

31

40

(36)

4.7

22

(27)

29

4.7 31

38

23

21

(27)

9.3

14

18

15

(16)

2.1

1500 µg

61

62

82

(68)

11.8

38

31

31

(33)

4.0

24

(24)

23

0.6 24

35

22

30

(29)

6.6

22

13

15

(17)

4.7

5000 µg

77

65

56

(66)

10.5

36

35

37

(36)

1.0

34

(32)

21

9.7 40

22

29

29

(27)

4.0

11 7

14

(11)

3.5

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

 

2AA

2AA

 

BP

2AA

1 µg

 

2 µg

10 µg

5 µg

2 µg

1005

(1344)

1910

493.6

1116

419 505 436

(453) 45.5

320

(333)

379

41.1

300

207

(222)

252

25.7

208

540

(527)

537

19.4

505

Experiment 2 – Without Metabolic Activation

Test Period

From: 05 September 2017

To: 08 September 2017

S9-Mix

(-)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (DMSO)

80

67

129

(92)

32.7#

21

29

25

(25)

4.0

31

(30)

30

0.6 30

22

24

26

(24)

2.0

18

17

24

(20)

3.8

15 µg

79

100

73

(84)

14.2

18

29

21

(23)

5.7

28

(33)

37

4.6 34

21

34

21

(25)

7.5

11

18

21

(17)

5.1

50 µg

121

90

92

(101) 17.3

27

17

9

(18)

9.0

39

(35)

29

5.5 38

27

31

27

(28)

2.3

11

13

9

(11)

2.0

150 µg

101 112

87

(100) 12.5

24

31

22

(26)

4.7

28

(27)

25

1.5 27

27

26

29

(27)

1.5

18

8 8

(11)

5.8

500 µg

69

85

77

(77)

8.0

23

25

18

(22)

3.6

36

(31)

30

4.2 28

30

29

28

(29)

1.0

17

15

16

(16)

1.0

1500 µg

98

83

109

(97)

13.1

23

22

21

(22)

1.0

36

(33)

34

4.2 28

30

28

29

(29)

1.0

14

21

7

(14)

7.0

5000 µg

83

102

99

(95)

10.2

29

21

17

(22)

6.1

30

(30)

35

4.5 26

23

17

30

(23)

6.5

25

16

7

(16)

9.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1144

(974) 747

204.4 1030

1169

(1127)

1080

44.7 1131

179

(168)

178

17.6 148

365

(370)

367

7.6 379

238

(224)

244

29.0 191

Experiment 2 - With Metabolic Activation

Test Period

From: 05 September 2017

To: 08 September 2017

S9-Mix

(+)

Dose Level Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control (DMSO)

79

79

82

(80)

1.7#

17

18

20

(18)

1.5

28

(27)

26

1.2 26

37

30

42

(36)

6.0

25

14

9

(16)

8.2

15 µg

88

82

85

(85)

3.0

17

21

17

(18)

2.3

21

(29)

36

7.5 29

27

38

34

(33)

5.6

11

16

22

(16)

5.5

50 µg

83

67

78

(76)

8.2

24

21

28

(24)

3.5

32

(31)

26

4.2 34

42

19

37

(33)

12.1

24

11

9

(15)

8.1

150 µg

78

78

76

(77)

1.2

22

11

30

(21)

9.5

27

(27)

35

8.0 19

23

29

28

(27)

3.2

10 9

36

(18)

15.3

500 µg

82

80

62

(75)

11.0

32

21

26

(26)

5.5

21

(24)

30

4.9 22

35

27

23

(28)

6.1

12

10

21

(14)

5.9

1500 µg

65

90

82

(79)

12.8

19

18

18

(18)

0.6

29

(28)

22

6.0 34

40

28

27

(32)

7.2

24

23

10

(19)

7.8

5000 µg

86

96

85

(89)

6.1

16

18

19

(18)

1.5

22

(30)

25

11.9 44

39

27

30

(32)

6.2

9

10

9

(9)

0.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

 

2AA

2AA

 

BP

2AA

1 µg

 

2 µg

10 µg

5 µg

2 µg

828

(885)

930

51.9 896

214 235 250

(233) 18.1

165

(175)

169

13.4 190

156

(150)

123

25.0 172

343

(342)

364

22.0 320

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9 -Aminoacridine

#: Standard deviation

BP: Benzo(a)pyrene

Conclusions:
1,1-Dimethoxyethane (CAS no. 534-15-6) was considered to be non-mutagenic in the tested species under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (Experiment 2, pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum concentration in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Conclusion

1,1-Dimethoxyethane (CAS no. 534-15-6) was considered to be non-mutagenic in the tested species under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The test substance was considered to be non-mutagenic under the Ames study therefore the substance is not classified for Genetic toxicity.