Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- negative: Ames test with S. typhimurium TA97a, TA98, TA100, TA102, and TA1535 (met. act.: with and without) (OECD TG 471; GLP); cytotoxicity: yes (TA98 and TA100: >/= 10 µg/plate without met. act., >/= 50 µg/plate with met. act.).
- negative: Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells (TK) (met. act.: with and without) (OECD TG 476; GLP); cytotoxicity: yes (> 4 µg/mL without met. act.; > 50 µg/mL with met. act.); read across from Stearic acid 3-(dimethylaminopropyl)amide
- negative: In vitro mammalian chromosome aberration test with cultured peripheral human lymphocytes (met. act.: with and without) (OECD TG 473; GLP); cytotoxicity: no (but tested up to precipitating concentrations); read across from Stearic acid 3-(dimethylaminopropyl)amide

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-04-26 to 1999-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102, and TA1535
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix
Test concentrations with justification for top dose:
range finding study: TA98 and TA100 with and without S9: 0.005, 0.0 1, 0.05, 0.1, 0.5, 1, and 5 mg/plateInitial and repeat plate incorporation assayall strains - S9: 0.0001, 0.0003, 0.0008, 0.00 1, 0.003, 0.008, and 0.01 mg/plateall strains + S9: 0.0008, 0.00 1, 0.003, 0.008, 0.01,0.03, and 0.05 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: solubility pretest
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene, Danthron - with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48-72 hNUMBER OF REPLICATIONS: 3 (test substance, positive controls), 9 negative controls in 2 independent assaysDETERMINATION OF CYTOTOXICITY- Method: decrease in the number of revertant colonies per plate and/or thinning or disappearance of the background bacterial lawn, or appearance of pinhead colonies in treated cultures.
Evaluation criteria:
A test substance producing no biologically relevant positive response in any one of the bacterial strains tested is considered to be non-mutagenic in this system.Test materials are considered to be mutagenic if at least one tester strain of Salmonella typhimurium shows at least a doubling in the mean number of revertans per plate over the appropriate control in tester strains TA97a, TA98, TA100, and TA102; in TA1535, a positive response is a threefold increase in revertant. A dose respons effect must also be observed over at least three concentrations of the test material in any tester strain.
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102, and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: no data- Effects of osmolality: no data- Evaporation from medium: no data- Precipitation: no precipitation described - Other confounding effects: noRANGE-FINDING/SCREENING STUDIES:A range-finding study was conducted in tester strains TA98 and TA100. COMPARISON WITH HISTORICAL CONTROL DATA:range finding study: Although the spontaneous mutation frequency of the medium control for tester strain TA98 was lower than the minimal acceptable level of 15 revertants/plate, the S9 control was in the acceptable range. All other positive and negative controls were within the acceptable ranges for tester strains TA98 and TAl00.ADDITIONAL INFORMATION ON CYTOTOXICITY:Toxicity was observed in tester strains TA98 and TA100 at 0.01 mg/plate in the absence of S9, and at 0.05 mg/plate in the presence of S9. Because of the variability in toxicity with S9, 0.01 mg/plate was selected as the highest dose level of test substance for definitive testing in the absence of S9, and 0.05 mg/plate was selected as the highest dose level for definitive testing in the presence of S9.
Conclusions:
C16 Alkylamidopropyltrimethylammonium Chloride was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA1535 in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to C16 Alkylamidopropyltrimethylammonium Chloride  in DMSO in concentrations of 0 (control), 0.1, 0.3, 0.8, 1, 3, 8, and 10 µg/plate in all strains in the absence of mammalian metabolic activation (rat liver S9 mix) and in concentrations of 0 (control), 0.8, 1, 3, 8, 10, 30, and 50 µg/plate in all strains in the presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Cytotoxic effects were noted in strains TA98 and TA100 starting at 10 µg/plate without metabolic activation, and at 50 µg/plate in with metabolic activation. Precipitation was not observed.The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA97a, TA98, TA 100, TA102, or TA1 535) examined at dose levels up to 10 µg/plate in the absence of a metabolic activation source (S9) or at dose levels up to 50 µg/plate in the presence of S9. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA97a, TA98, TA100, TA102, and TA 1535 under the conditions employed (plate incorporation assay).

There was no evidence of induced mutant colonies over background.

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-02 to 2012-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
not applicable, chromosome aberration assay
Species / strain / cell type:
lymphocytes: peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated foetal calf serum, 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin and 30 U/mL heparin
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test: 0, 1, 3, 10, 33, 100, 333 and 500 μg/mL
First experiment: without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL
Second experiment: without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (final concentration in culture medium: 0.5% (v/v))
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
TEST SYSTEMMETHOD OF APPLICATION: in medium
DURATION
first experiment, without and with S9-mix: 3 h exposure time, 24 h fixation time
second experiment, without S9-mix: 24 h and 48 h exposure time, 24 h and 48 h fixation time
second experiment, with S9-mix: 3 h exposure time, 48 h fixation time

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/mL)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: duplicates; 2 independent experiment

NUMBER OF CELLS EVALUATED: 1000 for mitotic index; 100 for chromosome aberrations

DETERMINATION OF CYTOTOXICITY- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
- The number of chromosome aberrations found in the solvent control cultures should be within the laboratory historical control data range.
- The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
- A homogeneous response between the replicate cultures is observed.
- A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
- A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
Chi-square test, one-sided
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: a concentration of 10 µg/mL already precipitated in the culture medium

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test a concentration of 33 µg/mL reduced the mitotic index to 0-10% of the control. Higher concentrations lead to cell lysis.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations, number of polyploid cells and cells with endoreduplicated chromosomes were within the laboratory historical control data range.

mitotic index

number of cell with aberrations (-gaps)

first experiment, without metabolic activation, 3 h exposure, 24 h fixation time

control

100

1

1

111

0

3

114

1

10

186

0

MMC 0.5 µg/mL

43

50

first experiment, with metabolic activation, 3 h exposure, 24 h fixation time

control

100

2

1

135

3

3

115

2

10

90

3

CP 10 µg/mL

30

52

second experiment, without metabolic activation, 24 h exposure, 24 h fixation time

control

100

2

3

87

4

6

73

1

10

57

3

15

26

not scored

20

7

not scored

25

4

not scored

MMC 0.2 µg/mL

30

35

second experiment, without metabolic activation, 48 h exposure, 48 h fixation time

control

100

2

3

92

2

6

79

1

10

55

2

15

13

not scored

20

4

not scored

25

3

not scored

MMC 0.1 µg/mL

54

49

second experiment, with metabolic activation, 3 h exposure, 48 h fixation time

control

100

0

1

87

2

3

64

0

10

59

2

CP, 10 µg/mL

**

38

** CP was fixed after 24 h, mitotic index was not calculated as % of control

Conclusions:
Based on the results of this study, it is concluded that Stearic acid 3-(dimethylaminopropyl)amide is not clastogenic in the in vitro mammalian chromosome aberration test.
Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, May 2008, peripheral human lymphocyte cultures were exposed to Stearic acid 3-(dimethylaminopropyl)amide in Ethanol at the following concentrations:

 

First experiment:

without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL

 

Second experiment:

without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL

with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Stearic acid 3-(dimethylaminopropyl)amide was tested up to precipitating concentrations (10 µg/mL).

Both in the absence and presence of S9-mix Stearic acid 3-(dimethylaminopropyl)amide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Stearic acid 3-(dimethylaminopropyl)amide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Stearic acid 3-(dimethylaminopropyl)amide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

There was no evidence of chromosome aberrations induced over background.

This study is classified as acceptable and satisfies the requirement for OECD Test Guideline 473 for in vitro cytogenetic mutagenicity data. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-08-15 to 2012-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Horse serum: inactivated by incubation at 56°C for at least 30 minutes. Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutaminGrowth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT) Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) - Properly maintained: yes - Periodically checked for Mycoplasma contamination: yes - Periodically checked for karyotype stability: no information given - Periodically "cleansed" against high spontaneous background: yes, prior to experiment
Additional strain / cell type characteristics:
other: thymidine kinase deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1
Without S9-mix: 0.003, 0.01, 0.03, 0.1, 0.3, 0.6, 1, 2.5, 5, 7.5 and 10 μg/mL exposure medium.
With 8% (v/v) S9-mix: 0.1, 0.6, 1, 5, 10, 20, 30, 40, 50, 60, 70 and 85 μg/mL exposure medium.

Experiment 2
Without S9-mix: 0.01, 0.03, 0.1, 0.3, 0.6, 1, 3, 4, 5, 6.5 and 7.5 μg/mL exposure medium.
With 12% (v/v) S9-mix: 0.1, 0.3, 1, 3, 10, 20, 30, 40, 50 and 60 μg/mL exposure medium.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Experiment 1:
Without S9-mix: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5 μg/mL exposure medium.
With S9-mix: 0.1, 0.6, 1, 5, 10, 20, 30 and 40 μg/mL exposure medium.

Experiment 2:
Without S9-mix: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3 μg/mL exposure medium.
With S9-mix: 0.1, 1, 3, 10, 30, 40, 50 and 60 μg/mL exposure medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol, final concentration of the solvent in the exposure medium was 0.4% (v/v)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION- Exposure duration: 3 h / 24 h (without metabolic activation); 3 h with 8 or 12 % S9 mix- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 (cloning efficiency) or 12 days (mutant frequency)
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments, 1 replicate each

NUMBER OF CELLS EVALUATED: 8E06 cells (1E06 cells/mL) for 3 hours treatment, 5E06 cells (1.25E05 cells/mL) for 24 hours treatment

DETERMINATION OF CYTOTOXICITY- Method: relative total growth
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (1E06) could be analysed for expression of the TK mutation. b
) The spontaneous mutation frequency in the solvent control is ≥ 50 per 1E06 survivors and ≤ 170 per 1E06 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 1E06 survivors, and for CP not below 700 per 1E06 survivors.
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range. The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126. b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix > 4 µg/mL, with S9 > 50 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- precipitation in the exposure medium at concentrations of 100 μg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 1000 μg/mL.

RANGE-FINDING/SCREENING STUDIES: After the 3 hours treatment, severe precipitate of the test substance was observed in the exposure medium at the highest dose levels of 333 and 1000 μg/mL. Therefore, no cell count was possible at these dose levels. In the absence of S9-mix, hardly any or no cell survival was observed at all test substance concentrations tested. In the presence of S9-mix, the relative suspension growth was 39% at the test substance concentration of 33 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test substance concentrations of 100 μg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range except in the absence of S9-mix, in which the mutation frequency of the solvent control cultures were just below the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: First mutagenicity test: In the presence of S9-mix, the dose levels of 50 to 85 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test substance concentration was reduced by 84% compared to the total growth of the solvent controls in the absence and presence of S9-mix. Second mutagenicity test:In the absence of S9-mix, the dose levels of 4 to 7.5 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 81% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 60% compared to the total growth of the solvent controls.

Experiment 1:

 

Mutation frequency per 1E06 survivors

Dose (µg/mL)

RSG (%)

CE day 2 (%)

RS day 2 (%)

RTG

Total

small

large

Without metabolic activation
3 hours treatment

SC1

100

131

100

100

48

15

32

SC2

123

48

16

32

0.003

101

93

73

74

61

23

36

0.01

110

101

79

87

64

19

43

0.03

119

118

93

110

59

22

35

0.1

118

95

75

89

58

22

34

0.3

114

104

82

93

51

19

30

1

89

135

106

95

48

15

31

2.5

74

78

61

45

84

33

49

5

23

88

69

16

56

28

27

MMS

80

51

40

32

840

192

578

With 8% (v/v) metabolic activation
3 hours treatment

SC1

100

104

100

100

82

22

57

SC2

95

85

23

59

0.1

94

94

94

89

89

26

60

0.6

97

110

110

108

74

24

47

1

102

83

83

85

97

34

60

5

47

95

96

45

88

42

42

10

79

74

74

59

119

42

72

20

57

104

104

59

69

30

36

30

44

86

87

38

101

26

72

40

19

84

84

16

107

32

71

CP

52

44

44

23

1502

395

892

 

 

Experiment 2:

 

Mutation frequency per 1E06 survivors

Dose (µg/ml)

RSG (%)

CE day 2 (%)

RS day 2 (%)

RTG

Total

small

large

Without metabolic activation
24 hours treatment

SC1

100

95

100

100

59

33

25

0.01

108

88

93

100

55

38

16

0.03

103

102

107

110

68

40

25

0.1

96

93

98

94

72

43

26

0.3

89

115

121

107

48

24

22

0.6

105

95

100

105

69

17

50

1

97

101

106

103

69

23

43

3

21

89

94

19

74

23

49

MMS

100

88

93

93

527

197

271

With 12% (v/v) metabolic activation
3 hours treatment

SC1

100

95

100

100

92

48

40

SC2

78

104

72

29

0.1

101

86

100

101

90

64

23

1

101

85

98

99

57

34

21

3

109

120

138

151

46

32

13

10

102

84

97

99

125

64

55

30

78

93

107

83

98

44

49

40

77

97

112

86

67

31

33

50

56

85

98

55

130

55

67

60

31

111

129

40

90

51

34

CP

58

50

58

34

1056

430

487

 

Note: all calculations were made without rounding off
RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

Conclusions:
It is concluded that Stearic acid 3-(dimethylaminopropyl)amide is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 467, adopted July 21, 1997 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to Stearic acid 3-(dimethylaminopropyl)amide (100% purity) in ethanol in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

First experiment

Without S9-mix, 3 h treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5 μg/mL

With 8% S9-mix, 3 h treatment: 0.1, 0.6, 1, 5, 10, 20, 30 and 40 μg/mL

Second experiment

Without S9-mix, 3 h treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3 μg/mL

With 12% S9-mix, 24 h treatment: 0.1, 1, 3, 10, 30, 40, 50 and 60 μg/mL

Stearic acid 3-(dimethylaminopropyl)amide was tested up to cyctotoxic concentrations.The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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

Additional information

A reverse gene mutation assay in bacteria according to OECD guideline 471 is available for the target substance C16 Alkylamidopropyltrimethylammonium Chloride. A mammalian cell gene mutation assay, and an in vitro mammalian chromosome aberration test are available for the structurally closely related source substance Stearic acid 3-(dimethylaminopropyl)amide. A justification for read-across is given below.

 

Summary

The target substance C16 Alkylamidopropyltrimethylammonium Chloride was not mutagenic in the Salmonella typhimurium reverse mutation assay. The source substance Stearic acid 3-(dimethylaminopropyl)amide is not mutagenic in the Salmonella typhimurium reverse mutation assay and the mammalian cell gene mutation assay (TK test) using mouse lymphoma L5178Y cells. In an in vitro mammalian cell cytogenetics assay Stearic acid 3-(dimethylaminopropyl)amide did not induce structural chromosomal aberrations in cultured peripheral human lymphocytes. Based on the available data, there was no evidence of genotoxicity for the target substance C16 Alkylamidopropyltrimethylammonium Chloride.

Based on the available reliable, relevant and adequate data, there was no evidence of genotoxicity for C16 Alkylamidopropyltrimethylammonium Chloride. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Bacterial reverse gene mutation assays

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to C16 Alkylamidopropyltrimethylammonium Chloride in DMSO in concentrations of 0 (control), 0.1, 0.3, 0.8, 1, 3, 8, and 10 µg/plate in all strains in the absence of mammalian metabolic activation (rat liver S9 mix) and in concentrations of 0 (control), 0.8, 1, 3, 8, 10, 30, and 50 µg/plate in all strains in the presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Cytotoxic effects were noted in strains TA98 and TA100 starting at 10 µg/plate without metabolic activation, and at 50 µg/plate in with metabolic activation. Precipitation was not observed.The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA97a, TA98, TA 100, TA102, or TA1 535) examined at dose levels up to 10 µg/plate in the absence of a metabolic activation source (S9) or at dose levels up to 50 µg/plate in the presence of S9. Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA97a, TA98, TA100, TA102, and TA 1535 under the conditions employed (plate incorporation assay).

There was no evidence of induced mutant colonies over background. Under the conditions of the study, the test substance was negative for mutagenic potential.

 

In a reverse gene mutation assay in bacteria equivalent to OECD guideline 471, strains ofS. Typhimurium(TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Stearic acid 3-(dimethylaminopropyl)amide (a.i. 85 %) for S. typhimurium at concentrations of 5, 10, 25, 50, and 75 µg/plate in the absence of mammalian metabolic activation, and at concentrations of 25, 50, 75, 100 and 250 µg/plate in the presence of mammalian metabolic activation. Strain E. coli WP2 uvrA was tested at concentrations of 10, 25, 50, 75, and 100 µg/plate in the absence and at 50, 75, 100, 250, and 500 µg/plate in the presence of metabolic mammalian activation.

Two tests were performed, the first test using the plate incorporation and the second test the preincubation method.

 In the first mutation assay (plate incorporation) Stearic acid 3-(dimethylaminopropyl)amide did not induce a significant dose-related increase in the number of revertant colonies in all five tested strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in the second mutation assay (preincubation method). No precipitation was observed.

Cytotoxic effects of the test substance were observed in the preincubation assay in allS. typhimuriumstrains, at a concentration of >/= 50 µg/plate in the absence of mammalian metabolic activation. Additionally at range finding test cytotoxic effects were shown at >/= 500 µg/plate in the E. coli WP2 uvr A strain. The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed.There was no evidence of induced mutant colonies over background.

 

Mammalian cell gene mutation assay

In a mammalian cell gene mutation assay according to OECD guideline 476, adopted July 21, 1997 (thymidine kinase (TK)), L5178Y mouse lymphoma cells cultured in vitro were exposed to Stearic acid 3-(dimethylaminopropyl)amide (100% purity) in ethanol in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

First experiment

Without S9-mix, 3 h treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2.5 and 5μg/mL

With 8% S9-mix, 3 h treatment: 0.1, 0.6, 1, 5, 10, 20, 30 and 40μg/mL

Second experiment

Without S9-mix, 3 h treatment: 0.01, 0.03, 0.1, 0.3, 0.6, 1 and 3μg/mL

With 12% S9-mix, 24 h treatment: 0.1, 1, 3, 10, 30, 40, 50 and 60μg/mL

Stearic acid 3-(dimethylaminopropyl)amide was tested up to cyctotoxic concentrations. The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

 

Mammalian cell cytogenetics assay

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, May 2008, peripheral human lymphocyte cultures were exposed to Stearic acid 3-(dimethylaminopropyl)amide in Ethanol at the following concentrations:

 First experiment: without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL

 Second experiment: without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL; with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Stearic acid 3-(dimethylaminopropyl)amide was tested up to precipitating concentrations (10 µg/mL).

Both in the absence and presence of S9-mix Stearic acid 3-(dimethylaminopropyl)amide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Stearic acid 3-(dimethylaminopropyl)amide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Stearic acid 3-(dimethylaminopropyl)amide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.There was no evidence of chromosome aberrations induced over background.

 

Justification for read-across

For details on substance identity, toxicokinetics and detailed toxicological profiles, please refer also to the general justification for read-across attached as pdf document to section 13 of the IUCLID file.

 

Structural similarity

a. Structural similarity and functional groups

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is manufactured from hexadecanoic acid and N,N-dimethyl-propylenediamine. Methyl chloride is used to quaternise the dimethylamino group of the fatty acid amidoamine.

The substance is composed of mainly C16 amides (ca. 92%) of DMAPA and small amounts of the C14 (ca. 2.5%) and C18 amide (ca. 5.5%).

 

The source substance Stearic acid 3-(dimethylaminopropyl)amide is manufactured from octadecanoic acid and N,N-dimethylpropylenediamine. It is composed of mainly C18 amides (> 89.8%) of DMAPA and small amounts of the C16 amide (<7%).

 

b. Common breakdown products

The metabolism that is expected to occur is for the target substance C16 Alkylamidopropyltrimethylammonium Chloride and the source substance Stearic acid 3-(dimethylaminopropyl)amide the hydrolysis of the amide-bond by amidases. Metabolism would result in free fatty acids and di- or trimethylaminopropylamine. The free fatty acids enter normal metabolic pathways (e.g. degradation by the mitochondrial beta-oxidation process) and are therefore indistinguishable from fatty acids from other sources including diet.

The amine compounds are not expected to be further metabolised, but excreted via the urine mainly unchanged. 

 

c. Differences

Chain length:

The slight differences in fatty acid chain length (higher percentage of C16 in the target substance vs. higher percentage C18 in the source substance Stearic acid 3-(dimethylaminopropyl)amide) are not considered to be of relevance for genotoxic potential.

 

Methylation/quaternation:

The target substance C16 Alkylamidopropyltrimethylammonium Chloride is methylated during the manufacturing process resulting in the quaternised ammonium ion.

Stearic acid 3-(dimethylaminopropyl)amide)on the other hand is not methylated during the manufacturing process. But based on physicochemical data (pKa) it is concluded that at physiological relevant pH, the substance is mostly protonated similarly resulting in a positively charged ammonium ion. This difference isnot considered to be of relevance for genotoxicity.

 

Overall, the provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of genotoxicity data

Genotoxicity data of the target substance C16 Alkylamidopropyltrimethylammonium Chloride and the source substance Stearic acid 3-(dimethylaminopropyl)amide

 

Endpoint

Target substance

C16 Alkylamidopropyltrimethylammonium Chloride 

Source substance

Stearic acid 3-(dimethylaminopropyl)amide

Genotoxicity in vitro:

 

a) bacterial reverse mutation test

Negative

 

OECD TG 471 (Ames test)
RL 1, GLP

 

S. typhimurium TA97a, TA98, TA100, TA102 and TA1535

 

Plate incorporation assay

 

negative for all strains (with and without metabolic activation)

 

Concentrations:

all strains - S9: 0.0001, 0.0003, 0.0008, 0.00 1, 0.003, 0.008, and 0.01 mg/plate

all strains + S9: 0.0008, 0.00 1, 0.003, 0.008, 0.01,0.03, and 0.05 mg/plate

 

cytotoxicity: yes

 

Negative

 

similar to OECD TG 471 (Ames test)
RL 1, GLP,
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 (met. act.: with and without)

 

Plate incorporation assay + pre-incubation assay

 

negative for all strains (with and without metabolic activation)

 

Concentrations:

S. typhimurium (w/o met act.): 5.0, 10, 25, 50, and 75 µg/plate

S. typhimurium (w met act.):

25, 50, 75, 100, and 250 µg/plate

E. coli (w/o met act.): 10, 25, 50, 75 and 100 µg/plate

E.coli (w met act.):

50, 75, 100, 250 and 500 µg/plate

 

cytotoxicity: yes 

Genotoxicity in vitro:

 

b) Mammalian gene mutation test

No data, read-across

Negative

 

OECD TG 476 (Mammalian cell gene mutation, MLA), RL 2, GLP

 

negative for mouse lymphoma L5178Y cells (with and without metabolic activation)

 

cytotoxicity: yes

Genotoxicity in vitro:

 

c) Chromosome aberration assay

No data, read-across

Negative

 

OECD TG 473 (In vitro mammalian chromosome aberration), RL 1, GLP

 

negative for cultured peripheral human lymphocytes (with and without metabolic activation)

 

cytotoxicity: yes

Sensitisation

Negative (not sensitising)

 

Comparable to OECD TG 406 (Buehler Test), RL 2, GLP

 

Induction and challenge exposures with 100% test substance (97.8% a.i.)

 

Negative (not sensitising)

 

OECD TG 406 (GPMT), RL 1, GLP

 

Intradermal induction: 2.5% in paraffinum perliquidum

Dermal induction: 1% in paraffinum perliquidum

Challenge: 2% in paraffinum perliquidum

 

C16 Alkylamidopropyltrimethylammonium Chloride was not mutagenic in a bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102, and TA1535 in the presence and absence of rat liver S9 mix.

The source substance Stearic acid 3-(dimethylaminopropyl)amide was also not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

These comparable results in addition to the structural similarity justify the read-across approach by extrapolation from the source substance for the mammalian gene mutation assay and the mammalian chromosome assay.

Stearic acid 3-(dimethylaminopropyl)amide was not mutagenic in the mouse lymphoma L5178Y test and not clastogenic in the in vitro mammalian chromosome aberration test.

 

Certain endpoints such as skin sensitisation and genotoxicity are characterised by covalent binding as a rate determining step or MIE (molecular initiating event). The consistency across endpoints - both, source substances and target substance were not sensitising - also helps to increase confidence in the read-across approach especially when MIEs are common for example, skin sensitisation and genotoxicity are underpinned by electrophilicity.

Overall, the available data are comparable for source and target substance, supporting the validity of the grouping approach.

 

Quality of the experimental data of the analogues:

The target substance C16 Alkylamidopropyltrimethylammonium Chloride has been tested in a reliable OECD TG 471 test.

The source substance Stearic acid 3-(dimethylaminopropyl)amide has been tested in reliable OECD TG 471, OECD TG 476 and OECD TG 473 test. All tests have been conducted according to GLP criteria. Therefore this data have no uncertainties and can be used in an analogue approach.

 

Conclusion

The structural similarities between the source and the target substances and the similarities in their breakdown products presented above support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances and their subsequent degradation products have similar toxicity profiles as demonstrated in detail in the general justification for read-across.

The negative results from the bacterial reverse mutation assay, which is available for the source substances and the target substance, justify this read-across approach.

Further support is given by the lacking skin sensitisation potential for both, source and target substances. The endpoint sensitisation is – similar to the endpoint genotoxicity – based on covalent binding of the substance itself or reactive metabolites to cellular macromolecules as rate determining step. The consistency across the endpoints increases the confidence in the conclusion that there is no concern for reactive metabolites. 

The negative outcome of the complete testing battery of in vitro genotoxicity tests for the source substance Stearic acid 3-(dimethylaminopropyl)amide is considered to be relevant also for the target substance C16 Alkylamidopropyltrimethylammonium Chloride. No classification for genotoxic properties is required. 

 

Justification for classification or non-classification

Based on reliable, relevant and adequate data on the target substance C16 Alkylamidopropyltrimethylammonium Chloride as well as from the closely related source substance Stearic acid 3-(dimethylaminopropyl)amide, the target substance C16 Alkylamidopropyltrimethylammonium Chloride is considered to be not mutagenic and not clastogenic. According to Regulation EC No 1272/2008 no classification and labelling for mutagenicity is required.