Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No significant genotoxicity could be derived for the registration substance by use of read-across appoach. The read-across supporting substance Bis (2-hydroxyethyl) coco alkylamine, CAS 71786 -60 -2 did not show any genotoxic acitivity in three in-vitro tests (Ames test, Chromosome aberration test, and Mouse lymphoma assay).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Justification for type of information:
The registration substance (target) and the source substance are members of Primary Fatty Amine Ethoxylates Category (see the category justification provided in Chapter 13).
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Conclusions:
Negative in three in-vitro genotoxicity test; not mutagenic and not clastogenic
Executive summary:

The genotoxicity of the registration substance is derived by use of data on the read-across supporting substance Bis (2-hydroxyethyl) coco alkylamine CAS 71786 -60 -2

Bis (2-hydroxyethyl) coco alkylamine was investigated for its genotoxicity according to the OECD Guideline 471 (bacterial reverse mutation assay ), OECD 473 (chromosome aberration test) and OECD 476 (mouse lymphoma assay). No significant gentoxicity was found.

Likewise, no significant genotoxicity is expected for the registration substance.

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 09 September 2009 and 11 October 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
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
Experiment one: Salmonella strains (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Experiment two: Salmonella strains (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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:
Dimethyl sulphoxide
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
Dunnett's Linear Regression Analysis
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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:
cytotoxicity
Remarks:
In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively. 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: In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 RESULTS

Preliminary Toxicity Test

The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively.  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

87

69

71

71

84

84

71

0V

0T

0T

0TP

+

TA100

73

86

95

77

80

62

83

77

0V

0T

0TP

-

WP2uvrA-

32

33

24

28

31

25

25

17

 

0V

0T

0TP

+

WP2uvrA-

30

33

28

26

27

21

20

31

15S

0T

0TP

S         Sparse bacterial background lawn
V         Very weak bacterial background lawn
T         Toxic, no bacterial background lawn

P         Precipitate

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 inTable 1 and 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.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically 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. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

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.

 

Table 1               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

123

 

11

 

22

 

26

 

10

 

98

(105)

15

(15)

18

(19)

16

(22)

12

(11)

95

 

18

 

18

 

24

 

11

 

Main Test

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

128

 

15

 

21

 

22

 

14

 

100

(108)

16

(16)

22

(22)

22

(22)

12

(12)

95

 

18

 

22

 

22

 

11

 

 



Table 2               Test Results: Range-Finding Test– Without Metabolic Activation

 

Test Period

From: 27 September 2009

To: 30 September 2009

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

109

97

89

(98)

10.1#

14

14

18

(15)

2.3

25

22

21

(23)

2.1

17

22

21

(20)

2.6

10

10

8

(9)

1.2

-

0.5

96

102

85

(94)

8.6

13

21

15

(16)

4.2

N/T

22

18

16

(19)

3.1

13

13

8

(11)

2.9

-

1.5

85

99

106

(97)

10.7

15

14

14

(14)

0.6

24

19

16

(20)

4.0

17

17

18

(17)

0.6

9

12

11

(11)

1.5

-

5

114

98

106

(106)

8.0

15

15

17

(16)

1.2

19

19

22

(20)

1.7

22

20

25

(22)

2.5

9

9

10

(9)

0.6

-

15

104

92

110

(102)

9.2

17

16

12

(15)

2.6

19

19

22

(20)

1.7

22

20

19

(20)

1.5

15

9

10

(11)

3.2

-

50

136

105

90

(110)

23.5

13

19

16

(16)

3.0

19

20

21

(20)

1.0

19

18

18

(18)

0.6

7

8

7

(7)

0.6

-

150

92 V

49 V

48 V

(63)

25.1

7 V

6 V

15 V

(9)

4.9

20

17

17

(18)

1.7

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

-

500

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

-

1500

N/T

N/T

0 TP

0 TP

0 TP

(0)

0.0

N/T

N/T

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

383

293

381

(352)

51.4

205

276

243

(241)

35.5

719

761

668

(716)

46.6

191

106

126

(141)

44.4

1011

1003

913

(976)

54.4

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Table 3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 27 September 2009

To: 30 September 2009

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

80

84

85

(83)

2.6#

15

11

12

(13)

2.1

22

19

20

(20)

1.5

24

24

24

(24)

0.0

8

7

11

(9)

2.1

+

1.5

82

90

91

(88)

4.9

11

10

11

(11)

0.6

21

21

20

(21)

0.6

25

24

25

(25)

0.6

13

10

13

(12)

1.7

+

5

101

101

90

$$$

(97)

6.4

10

8

12

(10)

2.0

22

19

19

(20)

1.7

21

25

18

(21)

3.5

7

12

7

(9)

2.9

+

15

82

90

81

(84)

4.9

11

7

10

(9)

2.1

20

21

24

(22)

2.1

21

17

17

(18)

2.3

10

9

9

(9)

0.6

+

50

89

88

80

(86)

4.9

8

9

8

(8)

0.6

19

19

22

(20)

1.7

24

27

22

(24)

2.5

9

8

8

(8)

0.6

+

150

80

79

80

(80)

0.6

8

9

8

(8)

0.6

17

18

20

(18)

1.5

21

20

15

(19)

3.2

12

11

10

(11)

1.0

+

500

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

12 S

9 S

11 S

(11)

1.5

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

+

1500

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1510

2079

2379

(1989)

441.4

112

156

183

(150)

35.8

371

340

348

(353)

16.1

215

251

208

(225)

23.1

313

292

314

(306)

12.4

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

$$$     p<0.005

#        Standard deviation

PLEASE SEE OVERALL REMARKS,) Tables 4 and 5 (Experiment 2)

Conclusions:
Negative in Ames test
Executive summary:

The genotoxicity of the registration substance is derived by use of data on the read-across supporting substance Bis (2-hydroxyethyl) coco alkylamine CAS 71786 -60 -2

Bis (2-hydroxyethyl) coco alkylamine was investigated for its genotoxicity according to the OECD Guideline 471. No significant gentoxicity was found.

Likewise, no significant genotoxicity is expected for the registration substance.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 02 September 2009 and 14 December 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine ) (µg/ml)
4(20)-hour without S9 0*, 1.5, 3, 6*, 9*, 12*, 18, MMC 0.4*
4(20)-hour with S9 0*, 3, 6*, 12*, 24*, 36, 48, CP 5*

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
24-hour without S9 0*, 1.5*, 3*, 6*, 12, 18, 24, MMC 0.2*
4(20)-hour with S9 0*, 3*, 6*, 12*, 24, 36, 48, CP 5*
* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required
concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 Migrated to IUCLID6: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in medium


DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9.
Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells
with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted
according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with
chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results in attached tables.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in DMSO
- Precipitation:
Premlinary toxictiy test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 100 µg/ml in the 4(20)-hour without S9 exposure group and in the continuous exposure group. In the 4(20)-hour with S9 exposure group precipitate was seen at and above 25 µg/ml at the end of exposure. Haemolysis was also seen at the end of exposure at and above 100 µg/ml in the 4(20)-hour
exposure group without S9 and the continuous exposure group. In the 4(20)-hour exposure group in the presence of S9 haemolysis was seen at and
above 200 µg/ml.
Experiment 1: No precipitate of the test material was observed at the end of exposure in either exposure group.
Experiment 2: No precipitate of the test material was observed at the end of exposure in either exposure group.

See attached tables 1 to 8 for results

RESULTS
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.13 to 800 µg/ml. The maximum dose selected was 800 µg/ml and was limited by the toxicity
seen in the Mouse Lymphoma Assay on the same test material (Harlan Laboratories Ltd Project No. 0142/0415). A precipitate of the test material was
observed in the parallel blood-free cultures at the end of the exposure, at and above 100 µg/ml in the 4(20)-hour without S9 exposure group and in
the continuous exposure group. In the 4(20)-hour with S9 exposure group precipitate was seen at and above 25 µg/ml at the end of exposure.
Haemolysis was also seen at the end of exposure at and above 100 µg/ml in the 4(20)-hour exposure group without S9 and the continuous exposure
group. In the 4(20)-hour exposure group in the presence of S9 haemolysis was seen at and above 200 µg/ml. Microscopic assessment of the slides
prepared from the exposed cultures showed that metaphase cells were present up to 25 µg/ml in the 4(20)-hour exposure group in the absence of
metabolic activation (S9) and in the continuous exposure group. The maximum dose with metaphases present in the 4(20)-hour exposure group in
the presence of metabolic activation (S9) was 50 µg/ml. The mitotic index data are presented in the attached Table 1. The test material induced dose
related toxicity in all three of the exposure groups.
The selection of the maximum dose level was based on toxicity and was 18 and 48 µg/ml for the 4(20)-hour exposure groups in both the absence and presence of S9 respectively, and was 24 µg/ml for the continuous exposure group used in Experiment 2.

Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
4(20)-hour without S9 0*, 1.5, 3, 6*, 9*, 12*, 18, MMC 0.4*
4(20)-hour with S9 0*, 3, 6*, 12*, 24*, 36, 48, CP 5*
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were
metaphases suitable for scoring present at 18 µg/ml and 36 µg/ml in the absence and presence of S9 respectively. No precipitate of the test material
was observed at the end of exposure in either exposure group.
The mitotic index data are given in the attached Table 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index
was observed, and that 81% and 42% mitotic inhibition was achieved at 18 and 12 µg/ml respectively in the absence of S9. In the presence of S9 73%
and 51% mitotic inhibition was achieved at 36 and 24 µg/ml respectively.
The maximum dose level selected for metaphase analysis was 12 µg/ml and 24 µg/ml in the absence and presence of S9 respectively, selected on the
basis of toxicity.
The chromosome aberration data are given in the attached Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The polyploid cell frequency data are given in the attached Table 8. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) (µg/ml)
24-hour without S9 0*, 1.5*, 3*, 6*, 12, 18, 24, MMC 0.2*
4(20)-hour with S9 0*, 3*, 6*, 12*, 24, 36, 48, CP 5*
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 36 µg/ml in the presence of S9, however due to the obvious toxicity at this dose level it was not selected for a mitotic index or metaphase analysis. In the absence of S9 the maximum test
material dose level with metaphases suitable for scoring was 12 µg/ml. No precipitate of the test material was observed at the end of exposure in
either exposure group.
The mitotic index data are given in the attahed Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 79% and 44% mitotic inhibition was achieved at 24 and 12 µg/ml respectively in the presence of S9. In the continuous exposure
group 64% and 40% mitotic inhibition was achieved at 6 and 3 µg/ml respectively.
The maximum dose level selected for metaphase analysis was based on toxicity and was 6 µg/ml for the continuous exposure group and 12 µg/ml for
the 4(20)-hour exposure group in the presence of S9.
The chromosome aberration data are given in the attached Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or
presence of metabolic activation.
The polyploid cell frequency data are given in the attached Table 8. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Not clastogenic in in-vitro chromosome aberration test
Executive summary:

The genotoxicity of the registration substance is derived by use of data on the read-across supporting substance.

Bis (2-hydroxyethyl) coco alkylamine CAS 71786 -60 -2 was investigated for its genotoxicity according to the OECD Guideline 473. No significant gentoxicity was found.

Likewise, no significant genotoxicity is expected for the registration substance.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 08 September 2009 and 26 October 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 1419706 15108051 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used:
Vehicle (DMSO) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.
The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.31 to 15 µg/ml in the absence of metabolic activation and 0.63 to 25 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.08 to 5 µg/ml in the absence of metabolic activation, and 2.5 to 25 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 0.31 to 80 µg/ml for both of the 4-hour exposure groups, and 0.16 to 40 µg/ml for the repeat of the 24‑hour exposure group.

In all three of the exposure groups there were marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three of the exposure groups. Precipitate of the test material was not observed at any of the dose levels. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity.

Mutagenicity Test

Experiment 1

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSGand RTG values. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred. Near optimum levels of toxicity were achieved in the absence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the very sharp onset of toxicity, despite using a very narrow dose interval. However, with no evidence of a response in either the absence or presence of metabolic activation in this experiment, or in the second experiment where optimum levels of toxicity were achieved, and the subsequent dose level to the upper surviving dose levels exhibiting excessive levels of toxicity, the test material was considered to have been adequately tested (Tables 3 and 6). The excessive toxicity observed at 15 µg/ml in the absence of metabolic activation, resulted in this dose not being plated for viability or 5-TFT resistance. The toxicity observed at 25 µg/ml in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). No precipitate of test material was observed at any of the dose levels.

Experiment 2

As was seen previously, there was evidence of a marked dose-related reduction in % RSGand RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. On this occasion optimum levels of test material-induced toxicity were achieved in both the absence and presence of metabolic activation . The excessive toxicity observed at and above 20 µg/ml in the presence of metabolic activation resulted in these doses not being plated for viability or 5-TFT resistance. The toxicity observed at 5 µg/ml in the absence of metabolic activation, and at 17.5 µg/ml in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from the statistical analysis. Both positive controls induced acceptable levels of toxicity
The 24-hour exposure without metabolic activation demonstrated that the extended time point had an effect on the toxicity of the test material. It was also noted that the lowering of the S9 concentration to 1% S9 in this second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). No precipitate of test material was observed at any of the dose levels.


Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.
Conclusions:
Not mutagenic in Mouse Lymphoa Assay
Executive summary:

The genotoxicity of the registration substance is derived by use of data on the read-across supporting substance Bis (2-hydroxyethyl) coco alkylamine CAS 71786 -60 -2.

Bis (2-hydroxyethyl) coco alkylamine was investigated for its genotoxicity according to the OECD Guideline 476. No significant gentoxicity was found.

Likewise, no significant genotoxicity is expected for the registration substance. 

 

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

Additional information

Justification for classification or non-classification

No significant genotoxicity could be derived for the registration substance by use of read-across appoach. The read-across supporting substance Bis (2-hydroxyethyl) coco alkylamine, CAS 71786 -60 -2 did not show any genotoxic acitivity in three in-vitro tests (Ames test, Chromosome aberration test, and Mouse lymphoma assay).

No classification is warranted.