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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28th November 2017 - March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(hydroxyethyl)methyloleylammonium chloride
EC Number:
242-332-0
EC Name:
Bis(hydroxyethyl)methyloleylammonium chloride
Cas Number:
18448-65-2
Molecular formula:
C23H48NO2.Cl
IUPAC Name:
bis(hydroxyethyl)methyloleylammonium chloride
Test material form:
solid
Specific details on test material used for the study:
Bis (2-hydroxyethyl) oleyl methyl ammonium chloride (CAS number 18448-65-2; EC number 242-332-0), batch number SC171117, was a yellow/amber/brown waxy solid. It was received on 06 December 2017 and stored at 15-25°C, protected from light and dessicated. Purity was stated as ca. 96%, mono-constituent substance and the expiry date was given as November 2018, see Certificate of Analysis.

Method

Target gene:
Histidine (in S. Tryhimurium); Tryptophan (in E-coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver metabolising system (S-9).
Test concentrations with justification for top dose:
Experiment 1 : Ttreatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bis (2-hydroxyethyl) oleyl methyl ammonium chloride at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity ranging from a slight thinning of the background bacterial lawn, with or without a marked reduction in revertant numbers, to a complete killing of the background bacteria was observed at 16 or 50 µg/plate and above (depending on the strain) in all strains in the absence of S-9, and at 50 µg/plate or above (depending on the strain) in all strains in the presence of S-9 except strain WP2 uvrA where it was observed at 500 µg/plate and above.

Experiment 2 : Ttreatments of all the tester strains were performed in the absence and in the presence of S-9. For all strains the maximum test concentration was reduced based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.05-50 µg/plate (TA98, TA100, TA1535 and TA1537 in the absence of S-9), 0.16-160 µg/plate (WP2 uvrA in the absence of S-9 and TA98, TA100, TA1535 and TA1537 in the presence of S-9) or 0.5–500 µg/plate (WP2 uvrA in the presence of S-9), in order to examine more closely those concentrations of Bis (2-hydroxyethyl) oleyl methyl ammonium chloride approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity
Vehicle / solvent:
Anhydrous analytical grade dimethyl sulphoxide (DMSO).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 1-aminoanthracene
Details on test system and experimental conditions:
Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.

Mutation Experiments
Bis (2-hydroxyethyl) oleyl methyl ammonium chloride was tested for mutation (and toxicity) in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) in two separate experiments at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted, incubated at 37±1°C and protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4).
As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or split in agar or a very thin bacterial lawn affected the accuracy of the automated counter.

Evaluation criteria:
Evaluation Criteria

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3 fold (in strains TA1535 and TA1537) the concurrent vehicle control values
2. The positive trends/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if either of the above criteria were met.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

 

 

 Raw Plate Counts and calculated Mutagenicity Data, Experiment 1, without S-9

Strain

Compound

Conc

Level (µg/plate)

Mean

Standard

Deviation

Fold Increase

Revertant Number Per Plate

TA98

DMSO

-

26.3

9.5

-

26, 36, 17

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

22.3

2.5

0.8

20, 25, 22

 

16

18.3

6.7

0.7

14 S, 26 S, 15 S

 

50

-

-

-

- T, - T, - T

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

2NF

5

908.7

22.5

34.5

932, 907, 887

 

 

 

 

 

 

 

 

TA100

DMSO

-

83.7

5.0

-

79, 83, 89

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

87.3

9.0

1.0

92, 77, 93

 

16

70.0

5.3

0.8

74 S, 72 S, 64 S

 

50

-

-

-

- T, - T, - T

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

NaN3

2

627.3

58.8

7.5

673, 648, 561

 

 

 

 

 

 

 

 

TA1535

DMSO

-

8.0

2.6

-

5, 9, 10

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

12.7

3.8

1.6

10, 17, 11

 

16

8.3

1.2

1.0

9 S, 9 S, 7 S

 

50

-

-

-

- T, - T, - T

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

NaN3

2

454.7

7.0

56.8

462, 448, 454

 

 

 

 

 

 

 

 

TA1537

DMSO

-

18.0

1.7

-

19, 16, 19

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

16.0

1.0

0.9

17, 16, 15

 

16

14.0

6.2

0.8

21 S, 12 S, 9 S

 

50

-

-

-

- T, - T, - T

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

AAC

50

493.0

69.1

27.4

569, 476, 434

 

 

 

 

 

 

 

 

WP2uvrA

DMSO

-

21.7

4.9

-

16, 25, 24

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

20.0

3.5

0.9

22, 22, 16

 

16

15.0

4.0

0.7

19, 15, 11

 

50

7.3

4.2

0.3

6 S, 12 S, 4 S

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

NQO

2

363.3

88.7

16.8

418, 411, 261

 

 

 

 

 

 

 

 

 

 Raw Plate Counts and Calculated Mutagenicity Data, Experiment 1, with S-9

Strain

Compound

Conc

Level (µg/plate)

Mean

Standard

Deviation

Fold Increase

Revertant Number Per Plate

TA98

DMSO

-

37.3

8.4

-

47, 33, 32

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

38.7

8.3

1.0

36, 48, 32

 

16

27.7

2.5

0.7

25, 30, 28

 

50

42.0

4.6

1.1

47, 38, 41

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

B[a]P

10

323.7

14.6

8.7

340, 312, 319

 

 

 

 

 

 

 

 

TA100

DMSO

-

128.3

12.3

-

125, 118, 142

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

108.7

16.0

0.8

92, 110, 124

 

16

96.3

24.5

0.8

111, 110, 68

 

50

84.7

14.4

0.7

79 S, 101 S, 74 S

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

AAN

5

1690.0

141.4

13.2

1763, 1780, 1527

 

 

 

 

 

 

 

 

TA1535

DMSO

-

11.7

4.5

-

12, 16, 7

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

12.7

3.1

1.1

12, 10, 16

 

16

17.3

2.9

1.5

19, 19, 14

 

50

9.3

2.5

0.8

7 S, 12 S, 9 S

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

AAN

5

182.0

1.0

15.6

181, 182, 183

 

 

 

 

 

 

 

 

TA1537

DMSO

-

12.0

2.0

-

14, 10, 12

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

16.7

3.8

1.4

15, 21, 14

 

16

11.7

4.9

1.0

15, 14, 6

 

50

9.7

4.6

0.8

7 S, 7 S, 15 S

 

160

-

-

-

- T, - T, - T

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

AAN

5

359.7

21.7

30.0

376, 368, 335

 

 

 

 

 

 

 

 

WP2uvrA

DMSO

-

25.3

3.1

-

26, 28, 22

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

5

23.0

9.5

0.9

22, 14, 33

 

16

22.7

1.2

0.9

24, 22, 22

 

50

25.3

2.3

1.0

24, 24, 28

 

160

26.3

2.1

1.0

24, 27, 28

 

500

-

-

-

- T, - T, - T

 

 

1600

-

-

-

- T, - T, - T

 

 

5000

-

-

-

- T, - T, - T

 

AAN

15

227.3

7.6

9.0

224, 222, 236

 

 

 

 

 

 

 

 

   

 Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, without S-9

Strain

Compound

Conc

Level (µg/plate)

Mean

Standard

Deviation

Fold Increase

Revertant Number Per Plate

TA98

DMSO

-

19.3

6.8

-

27, 17, 14

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.05

20.0

10.0

1.0

10, 30, 20

 

0.16

19.7

4.0

1.0

16, 19, 24

 

0.5

21.7

9.5

1.1

31, 12, 22

 

1.6

17.3

3.5

0.9

17, 21, 14

 

5

21.7

3.8

1.1

20, 26, 19

 

 

16

24.0

6.6

1.2

30, 17, 25

 

 

50

-

-

-

- T, - T, - T

 

2NF

5

1000.3

66.8

51.7

955, 969, 1077

 

 

 

 

 

 

 

 

TA100

DMSO

-

94.0

11.4

-

89, 86, 107

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.05

107.0

4.6

1.1

111, 102, 108

 

0.16

97.0

15.1

1.0

81, 111, 99

 

0.5

108.7

5.0

1.2

114, 104, 108

 

1.6

106.3

12.5

1.1

106, 119, 94

 

5

89.3

17.6

1.0

108, 87, 73

 

 

16

84.0

13.9

0.9

93 S, 68 S, 91 S

 

 

50

-

-

-

- T, - T, - T

 

NaN3

2

645.7

44.7

6.9

594, 672, 671

 

 

 

 

 

 

 

 

TA1535

DMSO

-

10.7

2.9

-

9, 9, 14

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.05

15.7

3.5

1.5

16, 19, 12

 

0.16

13.7

3.5

1.3

10, 14, 17

 

0.5

10.3

1.2

1.0

9, 11, 11

 

1.6

10.0

0.0

0.9

10, 10, 10

 

5

10.0

5.6

0.9

15, 4, 11

 

 

16

12.3

4.6

1.2

15 S, 7 S, 15 S

 

 

50

-

-

-

- T, - T, - T

 

NaN3

2

518.7

57.8

48.6

455, 533, 568

 

 

 

 

 

 

 

 

TA1537

DMSO

-

9.3

3.8

-

11, 5, 12

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.05

7.7

1.2

0.8

7, 9, 7

 

0.16

12.7

2.3

1.4

14, 14, 10

 

0.5

16.3

3.2

1.8

20, 15, 14

 

1.6

11.3

0.6

1.2

12, 11, 11

 

5

6.3

2.5

0.7

4, 9, 6

 

 

16

12.0

4.4

1.3

10 S, 17 S, 9 S

 

 

50

-

-

-

- T, - T, - T

 

AAC

50

353.3

51.3

37.9

310, 410, 340

 

 

 

 

 

 

 

 

WP2uvrA

DMSO

-

18.0

4.4

-

15, 16, 23 M B

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.16

17.3

1.5

1.0

17, 19, 16

 

0.5

20.7

6.0

1.1

27, 20, 15

 

1.6

20.0

7.9

1.1

29, 17, 14

 

5

19.0

5.3

1.1

15, 17, 25

 

16

25.0

9.5

1.4

35, 16, 24

 

 

50

9.7

2.5

0.5

12, 7, 10

 

 

160

-

-

-

- T, - T, - T

 

NQO

2

437.7

58.3

24.3

505, 404, 404

 

 

 

 

 

 

 

 

  Raw Plate Counts and Calculated Mutagenicity Data, Experiment 2, with S-9

Strain

Compound

Conc

Level (µg/plate)

Mean

Standard

Deviation

Fold Increase

Revertant Number Per Plate

TA98

DMSO

-

22.3

11.8

-

36, 16, 15

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.16

31.7

8.0

1.4

24, 40, 31

 

0.5

27.3

11.9

1.2

41, 22, 19

 

1.6

20.3

5.8

0.9

17, 27, 17

 

5

31.3

9.0

1.4

40, 32, 22

 

16

30.0

4.6

1.3

29, 35, 26

 

 

50

28.7

4.7

1.3

25, 27, 34

 

 

160

27.3

2.3

1.2

30 S, 26 S, 26 S

 

B[a]P

10

433.3

60.9

19.4

412, 502, 386

 

 

 

 

 

 

 

 

TA100

DMSO

-

99.0

8.9

-

92, 96, 109

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.16

100.3

5.8

1.0

107, 97, 97

 

0.5

114.0

12.1

1.2

112, 127, 103

 

1.6

98.3

4.0

1.0

94, 99, 102

 

5

118.3

16.2

1.2

137, 109, 109

 

16

106.3

20.5

1.1

89, 101, 129

 

 

50

101.0

10.4

1.0

96, 113, 94

 

 

160

-

-

-

- T, - T, - T

 

AAN

5

1739.7

42.6

17.6

1758, 1691, 1770

 

 

 

 

 

 

 

 

TA1535

DMSO

-

11.7

3.8

-

9, 10, 16

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.16

12.7

2.1

1.1

12, 11, 15

 

0.5

9.7

2.5

0.8

7, 10, 12

 

1.6

13.7

1.5

1.2

12, 14, 15

 

5

16.3

4.5

1.4

21, 12, 16

 

16

15.0

4.6

1.3

11, 14, 20

 

 

50

10.3

0.6

0.9

11, 10, 10

 

 

160

7.3

2.1

0.6

9 M V, 8 M V, 5 M V

 

AAN

5

165.0

13.5

14.1

179, 164, 152

 

 

 

 

 

 

 

 

TA1537

DMSO

-

13.0

1.7

-

14, 14, 11

 

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.16

15.0

4.0

1.2

11, 15, 19

 

0.5

15.3

1.5

1.2

17, 14, 15

 

1.6

11.3

2.5

0.9

14, 9, 11

 

5

11.3

5.0

0.9

6, 16, 12

 

16

10.0

1.7

0.8

9, 9, 12

 

 

50

12.0

6.1

0.9

5, 16, 15

 

 

160

5.0

1.7

0.4

3 M V, 6 M V, 6 M V

 

AAN

5

157.7

15.6

12.1

143, 174, 156

 

 

 

 

 

 

 

 

WP2uvrA

DMSO

-

28.0

3.5

-

30, 24, 30

Bis (2-hydroxyethyl) oleyl methyl ammonium chloride

0.5

29.3

4.5

1.0

29, 34, 25

 

1.6

25.0

9.8

0.9

22, 36, 17

 

5

45.3

18.4

1.6

50, 25, 61

 

16

32.3

2.3

1.2

31, 35, 31

 

50

43.3

6.4

1.5

48, 36, 46

 

 

160

32.7

5.1

1.2

37, 27, 34

 

 

500

-

-

-

- T, - T, - T

 

AAN

15

124.3

13.5

4.4

111, 124, 138

 

Applicant's summary and conclusion

Conclusions:
Bis(2-hydroxyethyl)methyloleylammonium chloride, CAS No. 18448-65-2, EC No. 242-332-0, did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a rat liver metabolic activation system (S-9) in the OECD 471, Bacterial Reverse Phase Mutation study : Negative.
Executive summary:

A study was performed to evaluate the mutagenic potential of the test material (Bis(2-hydroxyethyl)methyloleylammonium chloride, CAS No. 18448-65-2, EC No. 242-332-0).

The study was conducted according to OECD guideline 471 and in accordance with GLP.

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

These test conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) and a toxic concentration, in the absence and in the presence of a rat liver metabolic activation system (S-9) in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

Under the conditions of the study it was concluded that Bis(2-hydroxyethyl)methyloleylammonium chloride did not induce mutation in four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA).

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