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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:
The experimental phase of this study was performed between 13 May 2008 and 04 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 19-08-2008 Date of Signature on GLP certificate:04-03-2009
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Carbonic acid, compound with 2-aminoethanol (1:2)
EC Number:
244-600-2
EC Name:
Carbonic acid, compound with 2-aminoethanol (1:2)
Cas Number:
21829-52-7
Molecular formula:
C2H7NO.1/2CH2O3
IUPAC Name:
carbonic acid - 2-aminoethanol (1:2)
Constituent 2
Reference substance name:
MEA-carbamate
IUPAC Name:
MEA-carbamate
Test material form:
liquid: viscous
Details on test material:
-Sponsor's identification :MEA-carbamate
-Description : Pale amber coloured extremely viscous liquid
-Batch number :1314-206
-Date received : 14 April 2008
-Storage conditions :Room temperature in the dark
- Molecular formula (if other than submission substance): HOCH2CH2NH3+-O2C-NHCH2CH2OH
- Molecular weight (if other than submission substance): 166.17
- Analytical purity: 100%
- Purity test date: 12-05-2008
- Expiration date of the lot/batch: 21-11-2008
- Stability under test conditions: Stable under normal temperatures and pressure

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

120

86

90

107

98

136

111

107

118

102

100

+

TA100

93

103

99

118

101

108

106

103

114

96

99

-

WP2uvrA-

32

21

31

23

21

30

31

21

24

29

26

+

WP2uvrA-

37

40

36

31

25

26

21

21

18

22

24

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.

A history profile of vehicle and positive control values is presented in Appendix 4.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


Table1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

128

 

25

 

40

 

19

 

15

 

129

(132)

25

(23)

35

(41)

19

(20)

14

(15)

140

 

19

 

47

 

22

 

15

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

147

 

24

 

35

 

11

 

20

 

147

(149)

27

(28)

35

(31)

21

(18)

12

(14)†

154

 

34

 

24

 

22

 

11

 

 


Table2               Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From: 24 May 2008

To: 27 May 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

-

0

137

100

106

(114)

19.9#

24

22

20

(22)

2.0

34

30

30

(31)

2.3

16

18

19

(18)

1.5

12

10

12

(11)

1.2

-

50

111

114

109

(111)

2.5

23

18

18

(20)

2.9

43

30

27

(33)

8.5

22

13

20

(18)

4.7

21

13

11

(15)

5.3

-

150

121

114

114

(116)

4.0

23

29

23

(25)

3.5

25

24

37

(29)

7.2

18

19

14

(17)

2.6

11

10

13

(11)

1.5

-

500

123

109

111

(114)

7.6

26

29

25

(27)

2.1

36

29

35

(33)

3.8

19

19

20

(19)

0.6

14

14

10

(13)

2.3

-

1500

132

112

139

(128)

14.0

23

34

23

(27)

6.4

36

27

27

(30)

5.2

16

15

16

(16)

0.6

16

10

10

(12)

3.5

-

5000

96

109

110

(105)

7.8

25

19

29

(24)

5.0

32

34

31

(32)

1.5

20

22

20

(21)

1.2

7

13

11

(10)

3.1

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

1513

1127

1049

(1230)

248.5

114

119

120

(118)

3.2

651

660

647

(653)

6.7

115

115

114

(115)

0.6

2572

2606

2770

(2649)

105.9

 

 


Table3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 24 May 2008

To: 27 May 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

+

0

106

120

78

(101)

21.4#

16

11

12

(13)

2.6

36

36

30

(34)

3.5

35

22

40

(32)

9.3

29

24

13

(22)

8.2

+

50

102

82

103

(96)

11.8

8

9

18

(12)

5.5

49

33

35

(39)

8.7

26

42

29

(32)

8.5

23

21

19

(21)

2.0

+

150

114

101

101

(105)

7.5

14

13

20

(16)

3.8

41

41

33

(38)

4.6

22

29

25

(25)

3.5

16

18

19

(18)

1.5

+

500

115

115

120

(117)

2.9

18

15

20

(18)

2.5

56

38

38

(44)

10.4

30

30

35

(32)

2.9

19

13

12

(15)

3.8

+

1500

125

106

101

(111)

12.7

13

18

21

(17)

4.0

35

44

45

(41)

5.5

36

42

34

(37)

4.2

14

23

22

(20)

4.9

+

5000

86

91

96

(91)

5.0

15

16

23

(18)

4.4

43

45

44

(44)

1.0

29

36

38

(34)

4.7

25

16

12

(18)

6.7

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2370

2221

2043

(2211)

163.7

319

354

307

(327)

24.4

443

514

510

(489)

39.9

126

151

114

(130)

18.9

207

158

206

(190)

28.0

 

 


Table4               Test Results: Main Test– Without Metabolic Activation

Test Period

From: 31 May 2008

07 June 2008†

To: 03 June 2008

10 June 2008†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537†

-

0

126

132

146

(135)

10.3#

25

23

24

(24)

1.0

30

30

34

(31)

2.3

16

16

13

(15)

1.7

18

21

21

(20)

1.7

-

50

110

126

128

(121)

9.9

19

27

25

(24)

4.2

30

34

36

(33)

3.1

18

14

19

(17)

2.6

18

24

16

(19)

4.2

-

150

119

95

139

(118)

22.0

25

18

33

(25)

7.5

38

23

33

(31)

7.6

16

18

22

(19)

3.1

26

20

18

(21)

4.2

-

500

128

119

122

(123)

4.6

31

26

30

(29)

2.6

29

34

34

(32)

2.9

16

19

16

(17)

1.7

27

14

23

(21)

6.7

-

1500

141

120

150

(137)

15.4

20

19

31

(23)

6.7

40

34

25

(33)

7.5

22

15

13

(17)

4.7

16

16

20

(17)

2.3

-

5000

137

128

125

(130)

6.2

20

21

22

(21)

1.0

41

20

45

(35)

13.4

14

14

16

(15)

1.2

20

24

25

(23)

2.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

345

344

344

(344)

0.6

84

180

189

(151)

58.2

488

497

482

(489)

7.5

124

144

140

(136)

10.6

377

362

1651

(797)

739.9

 

 


Table5               Test Results: Main Test– With Metabolic Activation

Test Period

From: 31 May 2008

07 June 2008†

To: 03 June 2008

10 June 2008†

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537†

+

0

129

146

147

(141)

10.1#

11

12

12

(12)

0.6

44

30

33

(36)

7.4

26

20

23

(23)

3.0

18

18

19

(18)

0.6

+

50

129

133

120

(127)

6.7

8

7

7

(7)

0.6

35

32

32

(33)

1.7

24

26

21

(24)

2.5

12

13

13

(13)

0.6

+

150

115

110

133

(119)

12.1

10

9

9

(9)

0.6

21

31

29

(27)

5.3

23

26

22

(24)

2.1

20

23

16

(20)

3.5

+

500

123

123

132

(126)

5.2

8

10

14

(11)

3.1

46

22

36

(35)

12.1

22

20

29

(24)

4.7

18

15

21

(18)

3.0

+

1500

126

90

118

(111)

18.9

9

10

13

(11)

2.1

31

30

31

(31)

0.6

14

32

19

(22)

9.3

19

14

18

(17)

2.6

+

5000

135

109

139

(128)

16.3

3

8

9

(7)

3.2

42

41

32

(38)

5.5

23

19

20

(21)

2.1

27

18

12

(19)

7.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2915

2403

3244

(2854)

423.8

213

255

255

(241)

24.2

564

556

578

(566)

11.1

163

172

167

(167)

4.5

307

392

298

(332)

51.9

 

†            Experimental procedure repeated at a later date (presence and absence of S9) due to high solvent counts in original test

#            Standard deviation

PLEASE SEE ATTACHED 1) Figures1 -4 Dose-Response Curves. 2) Pages 23 -30 (Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

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