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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data is available from an Ames study on N-(hydrogenated tallow alkyl) trimethylenediamine itself and from in vitro mammalian chromosomal aberration and mutagenicity studies based on N-oleyl trimethylenediamine. All studies were performed under GLP according to current guidelines. Cross-reading from this substance is acceptable on the basis of similarities of structure with same functional groups, properties leading to common biological activity, and common metabolic degradation. The higher level of unsaturation in oleyl-alkyl chains can be considered a worst case representation.

Link to relevant study records

Referenceopen allclose all

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:
2008-05-14 - 2008-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
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
Target gene:
histidine operon (his)
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver rat S9-mix (induced with ß-naphthoflavone and phenobarbital)
Test concentrations with justification for top dose:
Experiment 1:
0.316, 0.1, 3.16, 10.0, 31.6, 100 and 316 µg/plate (TA 98, TA 1535, TA 1537)
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (TA 100, E. coli WP2 uvrA)

Experiment 2:
0.158, 0.50, 1.58, 5.0, 15.8, 50 and 158 µg/plate (TA 98, TA 100, TA 1535, TA 1537)
1.58, 5.0, 15.8, 50, 158, 500 and 1580 µg/plate ( E. coli WP2 uvrA)
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 10 µg/plate sodium azide (NaN3) (TA 100, TA 1535); 10 µg/plate (TA 98) and 40 µg/plate (TA 1537) 4-nitro-o-phenylene-diamine (4-NOPD); 1 µL/plate methylmethanesulfonate (MMS) (E. coli); 2.5 µg/plate for TA 98, 100, 1535, 1537 and 10 µg/plate for E. coli
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in 2 experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

ADDITIONAL INFORMATION
- Data recording:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation criteria:
Criteria of validity:
A test is considered acceptable if for each strain:
1. the bacteria demonstrate their typical response to ampicillin (TA 98, TA 100)
2. the control plates with and without metabolic activation are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
TA 98: 18 - 54 (-S9) and 18 - 71 (+S9)
TA 100: 75 - 167 (-S9) and 81 - 168 (+S9)
TA 1535: 5 - 29 (-S9) and 6 - 31 (+S9)
TA 1537: 5 - 30 (-S9) and 6 - 36 (+S9)
E. coli WP2 uvrA: 35 - 92 (-S9) and 37 - 101 (+S9)
3. corresponding background growth on both negative control and test plates is observed
4. the positive controls show a distinct enhancement of revertant rates over the control plate

Evaluation of mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
1. a clear and dose-related increase in the number of revertants occurs and/or
2. a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
1. if in tester strains Ta 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
2. if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
Statistics:
no data
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.00316 - 0.1 µL/plate (details in "Additional information on results")
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in all tester strains used in experiment I and II without S9 and in some tester strains used in experiment I and II with S9. In exp. I precipitation was found at 31.6µg/plate and higherwithout S9 and at a dose of 1000 µg/plate and higher with S9. In exp. II precipitation was found at 50 µg/plate and higher without S9 and at a dose of 500 µg/plate and higher with S9.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strain TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as for the main experiment.
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduetion in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µL/plate
Based on the pre-experiment results the following concentrations were used for the main experiments:
Experiment 1: 0.316 - 316 gL/plate (TA 98, TA 1535, TA 1537) and 3.16 - 2500 gL/plate (TA 100, E. coli WP2 uvrA)
Experiment 2: 0.158 - 158 gL/plate (TA 98, TA 100, TA 1535, TA 1537) and 1.58 - 1580 gL/plate ( E. coli WP2 uvrA)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects in experiment 1:
at 10 µg/plate (-S9) and 100 µg/plate (+S9) (TA 98);
at 31.6 µg/plate (-S9) and 316 µg/plate (+S9) (TA 100);
at 10 µg/plate (-S9) and 316 µg/plate (+S9) (TA 1535);
at 31.6 µg/plate (-S9) and 100 µg/plate (+S9) (TA 1537);
at 316 µL/plate (-S9 and +S9) (E. coli WP2 uvrA)

Cytotoxic effects in experiment 2:
at 50 µg/plate (-S9 and +S9) (TA 98, TA 1537);
at 1580 µg/plate (-S9 and +S9) (TA 100, TA 1535);
at 500 µg/plate (-S9 and +S9) (E. coli WP2 uvrA)
Remarks on result:
other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Remarks:
Migrated from field 'Test system'.

Table 1: Ames Test Results - Experiment 1

With or without S9-Mix

Test substance concentration

(dose/plate)

Mean number of revertant colonies per plate (triplicates)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

-

A. dest.

108

23

51

25

7

-

Vehicle control (EtOH)

98

24

41

24

6

-

0.316 µg

12

25

6

-

1.00 µg

21

31

6

-

3.16 µg

113

21

39

24

9

-

10 µg

99

15

39

24

8

-

31.6 µg

84

11

40

19

6

-

100 µg

64

3

37

0

2

-

316 µg

6

0

36

0

0

-

1000 µg

0

0

-

2500 µg

0

0

Positive

controls

- S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10 µg

10 µg

1 µL

10 µg

40 µg

Number of colonies/plate

968

1403

382

478

104

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

+

A. dest.

100

9

54

43

8

+

Vehicle control (EtOH)

110

26

59

33

12

+

0.316 µg

26

31

7

+

1.00 µg

30

42

12

+

3.16 µg

112

24

53

39

13

+

10 µg

123

29

54

36

12

+

31.6 µg

126

26

63

33

11

+

100 µg

97

17

59

23

4

+

316 µg

34

10

49

0

0

+

1000 µg

0

0

+

2500 µg

0

0

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5 µg

2.5 µg

10 µg

2.5 µg

2.5 µg

Number of colonies/plate

2019

281

208

2937

372

Table 2: Ames Test Results - Experiment 2

With or without S9-Mix

Test substance concentration

(dose/plate)

Mean number of revertant colonies per plate (triplicates)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

-

A. dest.

125

22

55

22

7

-

Vehicle control (EtOH)

120

19

45

27

9

-

0.158 µg

118

19

22

13

-

0.5 µg

128

26

23

8

-

1.58 µg

129

27

45

23

5

-

5.0 µg

135

19

50

20

6

-

15.8 µg

110

19

42

24

9

-

50 µg

101

20

56

13

4

-

158 µg

71

16

55

8

0

-

 500 µg

44

-

 1580 µg

28

Positive

controls

- S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10 µg

10 µg

1 µL

10 µg

40 µg

Number of colonies/plate

1087

1333

393

622

101

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

+

A. dest.

117

13

56

34

8

+

Vehicle control (EtOH)

114

22

60

34

10

+

0.158 µg

124

26

39

8

+

0.5 µg

128

18

39

8

+

1.58 µg

149

22

59

39

7

+

5.0 µg

155

22

61

43

8

+

15.8 µg

157

26

54

40

12

+

50 µg

147

34

63

24

3

+

158 µg

44

6

48

5

2

+

 500 µg

29

+

 1580 µg

5

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5 µg

2.5 µg

10 µg

2.5 µg

2.5 µg

Number of colonies/plate

1923

240

227

2649

251

 

NaN3= Sodium azide

MMS = Methyl methane sulfonate

4-NOPD = 4-Nitro-o-phenylene-diamine

2-AA = 2-Aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N-(Hydrogenated tallow)-1,3- diaminopropane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, N-(Hydrogenated tallow)-1,3-diaminopropane is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item N-(Hydrogenated tallow)-1,3-diaminopropane was investigated for its potential to induce gene mutations according to the OECD guideline 471.

In two independent plate incorporation tests using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicates. The following concentrations of the test item were prepared and used in the experiments:

 

Experiment 1:

0.316, 0.1, 3.16, 10.0, 31.6, 100 and 316 µL/plate (TA 98, TA 1535, TA 1537)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µL/plate (TA 100, E. coli WP2 uvrA)

Experiment 2:

0.158, 0.50, 1.58, 5.0, 15.8, 50 and 158 µL/plate (TA 98, TA 100, TA 1535, TA 1537)

1.58, 5.0, 15.8, 50, 158, 500 and 1580 µL/plate ( E. coli WP2 uvrA)

 

Precipitation of the test item was observed in all tester strains used in experiment I and II without metabolic activation and in some tester strains used in experiment I and II with metabolic activation. In experiment I precipitation was found at 31.6 µg/plate and higher without metabolic activation and at a dose of 1000 µg/plate and higher with metabolic activation. In experiment II precipitation was found at 50 µg/plate and higher without metabolic activation and at a dose of 500 µg/plate and higher with metabolic activation.

 

Toxic effects of the test item were noted in all tester strains evaluated in experiment 1 and 2.

In experiment 1 toxic effects of the test item were observed in tester strain TA 98 al doses of 10 µg/plate and higher (without metabolic activation) and al doses of 100 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at doses of 31.6 µg/plate and higher (without metabolic activation) and at doses of 316 µg/plate and higher (with metabolic activation), In tester strain TA 1535 toxic effects of the test item were found at doses of 10 µg/plate and higher (without metabolic activation) and at doses of 316 µg/plate (with metabolic activation).In tester strain TA 1537 toxic effects of the test item were observed at doses of 31.6 µg/plate and higher (without metabolic activation) and at doses of 100 µg/plate and higher (with metabolic activation). In tester strainE,coli WP2 uvrA toxic effects of the test item were noted at doses of 316 µg/plate and higher (with and without metabolic activation).

 

In experiment 2 toxic effects of the test item were noted in tester strains TA 98 and TA 1537 at doses of 50 µg/plate and higher (with and without metabolic activation). In tester strain TA 100 and 1535 toxic effects of the test item were observed at doses of 158 µg/plate and higher (with and without metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were seen at doses of 500 µg/plate and higher (with and without metabolic activation).

 

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-(Hydrogenated tallow)-1,3-diaminopropane at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-05-14 - 2008-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
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
Target gene:
histidine operon (his)
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver rat S9-mix (induced with ß-naphthoflavone and phenobarbital)
Test concentrations with justification for top dose:
Experiment 1:
0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate
Experiment 2:
0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA)
0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158 and 0.05 µL/plate (TA 1535)
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 10 µg/plate sodium azide (NaN3) (TA 100, TA 1535); 10 µg/plate (TA 98) and 40 µg/plate (TA 1537) 4-nitro-o-phenylene-diamine (4-NOPD); 1 µL/plate methylmethanesulfonate (MMS) (E. coli); 2.5 µg/plate for TA 98, 100, 1535, 1537 and 10 µg/plate for E. coli
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates in 2 experiments

DETERMINATION OF CYTOTOXICITY
- Method: background lawn or reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

ADDITIONAL INFORMATION
- Data recording:
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH. If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation criteria:
Criteria of validity:
A test is considered acceptable if for each strain:
1. the bacteria demonstrate their typical response to ampicillin (TA 98, TA 100)
2. the control plates with and without metabolic activation are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
TA 98: 18 - 54 (-S9) and 18 - 71 (+S9)
TA 100: 75 - 167 (-S9) and 81 - 168 (+S9)
TA 1535: 5 - 29 (-S9) and 6 - 31 (+S9)
TA 1537: 5 - 30 (-S9) and 6 - 36 (+S9)
E. coli WP2 uvrA: 35 - 92 (-S9) and 37 - 101 (+S9)
3. corresponding background growth on both negative control and test plates is observed
4. the positive controls show a distinct enhancement of revertant rates over the control plate

Evaluation of mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
1. a clear and dose-related increase in the number of revertants occurs and/or
2. a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
1. if in tester strains Ta 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
2. if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
Statistics:
no data
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 0.00316 - 0.1 µL/plate (details in "Additional information on results")
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any of the 5 tester strains used in experiment 1 and 2 with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strain TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as for the main experiment.
Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduetion in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Based on the pre-experiment results the following concentrations were used for the main experiments:
Experiment 1: 0.0001 - 0.1 µL/plate and Experiment 2: 0.000158 - 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA) and 0.00005 - 0.05 µL/plate (TA 1535)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects in experiment 1:
at 0.00316 µL/plate (-S9) and 0.0316 µL/plate (+S9) (TA 98 and TA 100);
at 0.010 µL/plate (-S9) and 0.1 µL/plate (+S9) (TA 1535);
at 0.01 µL/plate (-S9) and 0.0316 µL/plate (+S9) (TA 1537);
at 0.0316 µL/plate (-S9) and 0.1 µL/plate (+S9) (E. coli WP2 uvrA)

Cytotoxic effects in experiment 2:
at 0.0050 µL/plate (-S9) and 0.05 µL/plate (+S9) (TA 98, TA 100, TA 1535, TA 1537);
at 0.0158 µL/plate (-S9) and 0.1 µL/plate (+S9) (E. coli WP2 uvrA)
Remarks on result:
other: strain/cell type: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Remarks:
Migrated from field 'Test system'.

Table 1: Ames Test Results - Experiment 1

With or without S9-Mix

Test substance concentration

(dose/plate)

Mean number of revertant colonies per plate (triplicates)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

-

A. dest.

95

20

53

28

6

-

Vehicle control (EtOH)

102

23

46

24

6

-

0.000100µL

96

17

42

29

9

-

0.000316 µL

67

16

45

23

10

-

0.00100 µL

94

26

46

26

11

-

0.00316 µL

67

24

46

24

7

-

0.0100 µL

41

13

53

15

4

-

0.0316 µL

0

0

0

0

0

-

0.1 µL

0

0

0

0

0

Positive

controls

- S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10 µg

10 µg

1 µL

10 µg

40 µg

Number of colonies/plate

1257

1145

407

395

99

 

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

+

A. dest.

99

19

56

33

10

+

Vehicle control (EtOH)

97

23

57

38

11

+

0.000100µL

108

29

56

40

9

+

0.000316 µL

103

23

46

36

7

+

0.00100 µL

112

25

63

44

8

+

0.00316 µL

114

26

62

43

11

+

0.0100 µL

129

25

69

41

13

+

0.0316 µL

62

13

66

21

6

+

0.1 µL

0

0

15

0

0

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5 µg

2.5 µg

10 µg

2.5 µg

2.5 µg

Number of colonies/plate

2209

243

204

3114

211

Table 2: Ames Test Results - Experiment 2

With or without S9-Mix

Test substance concentration

(dose/plate)

Mean number of revertant colonies per plate (triplicates)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

-

A. dest.

134

26

54

21

7

-

Vehicle control (EtOH)

134

31

51

30

7

-

0.000050µL

-

32

-

-

-

-

0.000158µL

126

24

56

29

9

-

0.0005 µL

124

28

53

23

7

-

0.00158 µL

117

19

50

22

12

-

0.005 µL

75

15

50

26

8

-

0.0158 µL

13

0

35

3

0

-

0.05 µL

0

0

0

0

0

-

0.1 µL

0

-

0

0

0

Positive

controls

- S9

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

(μg/plate)

10 µg

10 µg

1 µL

10 µg

40 µg

Number of colonies/plate

1519

1537

447

528

112

 

TA 100

TA 1535

E. coli WP2 uvrA

TA 98

TA 1537

+

A. dest.

105

21

56

39

13

+

Vehicle control (EtOH)

81

22

53

37

9

+

0.000050µL

-

28

-

-

-

+

0.000158µL

108

29

67

37

11

+

0.0005 µL

89

25

64

42

6

+

0.00158 µL

97

25

61

45

8

+

0.005 µL

103

17

51

40

8

+

0.0158 µL

112

22

50

41

17

+

0.05 µL

24

0

66

0

0

+

0.1 µL

0

-

2

0

0

Positive

controls

+ S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

2.5 µg

2.5 µg

10 µg

2.5 µg

2.5 µg

Number of colonies/plate

1611

181

231

2440

328

NaN3= Sodium azide

MMS = Methyl methane sulfonate

4-NOPD = 4-Nitro-o-phenylene-diamine

2-AA = 2-Aminoanthracene

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N-Oleyl-1,3- diaminopropane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, N-Oleyl-1,3-diaminopropane is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

The test item N-Oleyl-1,3-diaminopropane was investigated for its potential to induce gene mutations according to the OECD guideline 471.

In two independent plate incorporation tests using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicates. The following concentrations of the test item were prepared and used in the experiments:

Experiment 1:

0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate

Experiment 2:

0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate (TA 98, TA 100, TA 1537, E. coli WP2 uvrA) and

0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158 and 0.05 µL/plate (TA 1535).

No precipitation of the test item was observed in any of the five tester strains used in experiment 1 and 2 with and without metabolic activation.

Toxic effects of the test item were noted in all tester strains evaluated in experiment 1 and 2.

In experiment 1 toxic effects of the test item were observed in tester strains TA 98 and TA 100 at doses of 0.00316 µL/plate and higher (without metabolic activation) and at doses of 0.0316 µL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at doses of 0.01 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were seen at doses of 0.01 µL/plate and higher (without metabolic activation) and at doses of 0.0316 µL/plate and higher (with metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were noted at doses of 0.0316 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation).

In experiment 2 toxic effects of the test item were noted in tester strains TA 98, TA 100 and TA 1537 at doses of 0.005 µL/plate and higher (without metabolic activation) and at doses of 0.05 µL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at doses of 0.005 µL/plate and higher (without metabolic activation) and at a dose of 0.05 µL/plate (with metabolic activation). In tester strain E. coli WP2 uvrA toxic effects of the test item were seen at doses of 0.0158 µL/plate and higher (without metabolic activation) and at a dose of 0.1 µL/plate (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-Oleyl-1,3-diaminopropane at any concentration level, neither in the presence nor absence of metabolic activation in experiment 1 and 2.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2008-05-14 - 2008-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (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:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix (induced with β-naphthoflavone and phenobarbital)
Test concentrations with justification for top dose:
Experiment I:
+S9: 0.5, 1.0, 2.0, 4.0, 5.0, 6.5 and 8.0 µg/mL
-S9: 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL

Experiment II:
+S9: 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 µg/mL
-S9: 0.05, 0.1, 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 400 and 900 µg/mL ethylmethanesulphonate (EMS) and 0.83 µg/mL cyclophosphamide (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in 2 independent experiments

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.5% (+S9) resp. 0.0% - 4.0% (-S9)
- the positive control substances should produce biologically relevant increases in the number of cells with structural chromosome aberrations

Criteria for determinig a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells (+S9) resp. 4.0% aberrant cells (-S9))
Statistics:
no data
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: at 1.0 µg/mL and higher (-S9) and 4.0 µg/mL and higher (+S9); Experiment II: at 0.4 µg/mL and higher (-S9) and 4.0 µg/mL and higher (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The concentration range for the Preliminary Toxicity Test was 0.0078 to 5000 µg/mL without metabolic activation and 1.95 to 5000 µg/mL with metabolic activation. The test item could be dissolved at a concentration of 500 µg/mL in ethanol. After dilution with cell culture medium, precipitation of the test item appeared in a concentration of 15.6 µg/mL and higher.
The selection of the concentrations used in experiment I and II based on data from the solubility and the pre-experiment which were performed according to guidelines. In experiment I (-S9) 1.2 µg/mL and (+S9) 4.0 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations. And in experiment II (-S9) 0.4 µg/mL and (+S9) 4.5 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: within historical reference range

Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Summary of aberration rates in experiment I

Dose Group

Concentration [µg/mL]

Treatment Time

Fixation Interval

Mean % aberrant cells

incl. Gaps

excl. Gaps

without metabolic activation

C

0

4 h

20 h

2.0

0.0

S

0

4 h

20 h

4.5

1.5

5

0.85

4 h

20 h

3.5

1.0

6

1.0

4 h

20 h

0.5

0.0

7

1.2

4 h

20 h

0.5

0.0

EMS

900

4 h

20 h

11.5

8.5

with metabolic activation

C

0

4 h

20 h

5.0

3.5

S

0

4 h

20 h

2.0

1.5

2

1.0

4 h

20 h

4.5

1.5

3

2.0

4 h

20 h

4.5

2.0

4

4.0

4 h

20 h

3.5

1.0

CPA

0.83

4 h

20 h

10.5

9.0

Summary of aberration rates in experiment II

Dose Group

Concentration [µg/mL]

Treatment Time

Fixation Interval

Mean % aberrant cells

incl. Gaps

excl. Gaps

without metabolic activation

C

0

20 h

20 h

3.0

1.5

S

0

20 h

20 h

3.5

2.0

5

0.1

20 h

20 h

4.0

2.5

6

0.2

20 h

20 h

2.0

0.5

7

0.4

20 h

20 h

3.0

1.0

EMS

400

20 h

20 h

12.0

8.0

with metabolic activation

C

0

4 h

20 h

4.0

1.5

S

0

4 h

20 h

4.5

2.5

2

2.5

4 h

20 h

3.5

1.5

3

4.0

4 h

20 h

3.0

1.5

4

4.5

4 h

20 h

2.5

1.0

CPA

0.83

4 h

20 h

11.5

8.5

200 cells evaluated for each concentration

C: Negative control (culture medium)

S: Solvent control (Ethanol)

EMS: Positive control (-S9: ethylmethansulfonate)

CPA: Positive control (+S9: cyclophosphamide)

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item N-Oleyl-1,3-diaminopropane did not induce structural chromosome aberrations in the V79 Chinese hamster cell line. Therefore, the test item N-Oleyl-1,3-diaminopropane is considered to be non-clastogenic.
Executive summary:

The test item N-Oieyl-1‚3-diaminopropane was investigated for a possible potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation with S9 homogenate.
The selection of the concentrations used in experiment I and II based on data from the solubility test and the pre-experiment which were performed according to the guidelines.
In experiment I without metabolic activation 1.2 µg/mL and with metabolic activation 4.0 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations. In experiment II without metabolic activation 0.4 µg/mL and with metabolic activation 4.5 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.
The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosomal aberrations.
The following concentrations were evaluated for microscopic analysis:
Experiment I:
with metabolic activation: 1.0, 2.0 and 4.0 µg/mL
without metabolic activation: 0.85, 1.0 and 1.2 µg/mL
Experiment II:
with metabolic activation: 2.5, 4.0 and 4.5 µg/mL
without metabolic activation: 0.1, 0.2 and 0.4 µg/mL
Precipitation:
No precipitation of the test item was noted with and without metabolic activation after the incubation at the concentrations evaluated.
Toxicity:
In experiment I without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 1.0 µg/mL and higher (48% at 1.0 µg/mL, 40% at 1.2 µg/mL). The cell density was not decreased. With metabolic activation a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at a concentration of 4.0 µg/mL (47% at 4 µg/mL). The cell density was also decreased at this concentration (61%).
In experiment II without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 0.4 µg/mL (49%). The cell density was also decreased (65%). With
metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 4.0 µg/mL and higher (46% at 4.0 µg/mL, 30% at 4.5 g/mL). No decrease of the cell density was noted up to the highest dose evaluated.
Clastogenicity:
In the experiment without metabolic activation the aberration rates of the negative control (0.0%) and the solvent control (1.5%) were within the historical control data of the negative control (0.0% - 4.0%). The aberration rates of all dose groups evaluated were within the range of the historical control data. Mean values of 1.0% (0.85 µg/mL), 0.0% (1 and 1.2 µg/mL) aberrant cells were found.
In the experiment with
metabolic activation the aberration rates of the negative control (3.5%) and the solvent control (15%) were within the historical control data (0.0% - 4.5%). The aberration rate of all dose groups evaluated were within the range of the historical control data. Mean values of 1.5% (1 µg/mL), 2.0% (2 µg/mL) and 1.0% (4 µg/mL) aberrant cells were found.
In experiment II without metabolic activation the aberration rate of the negative control (1.5%), the solvent control (2.0%) and all dose groups treated with the test item (2,5% (0.1 µg/mL, 0.5% (0.2 µg/mL) and 1.0% (0.4 µg/mL)) were within the historical control data of the testing facility (0.0% -
4.0%). With metabolic activation the aberration rates of the negative control (1.5%), the solvent control (2.5%) and all dose groups treated with the test item (1.5% (2.5 µg/mL), 1.5% (4.0 µg/mL) and 1.0% (4.5 µg/mL)) were within the historical control data of the testing facility (0.0% - 4.5%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control, In addition, no dose-response relationship was observed.
Polyploid cells
No biologically relevant increase in the frequencies of
polyploid cells was found after treatment with the test item.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

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

Genetic toxicity in vivo

Description of key information

Lack of genotoxic properties of alkyl-diamines was further confirmed in anin vivomicronucleus study with Coco-diamine, conducted according to OECD 474 guideline and under GLP.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol.
Justification for type of information:
Further information on the applicability of the read-across from various diamines to C12/14-diamine can be obtained from the document "Category polyamines - 20170314.pdf" added to IUCLID Ch. 13.
Qualifier:
according to guideline
Guideline:
other: 40 CFR Part 158 US-EPA-FIFRA, Section 158.340, Guideline 84-3
Principles of method if other than guideline:
This method is similar to OECD474. The humidity was out of the recommended range of 50-60%. It was8-53% this deviation is not thought to have influenced the outcome of the study.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: Approximately 6 weeks (born on approximately November 21, 1990)
- Weight at study initiation: one or two mice from each cage were selected randomly to be weighed. The weights of 17 male mice ranged from 21.8 to 26.7 g and those of 15 female mice from 18.7 to 22.7 g at the time of receipt.
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Mice were housed no more than 10 to a cage dufing quarantine, 3 to a cage for the range-finding assay, and 5 to a cage during the definitive assay. Polycarbmate cages with hardwood-chip bedding were used throughout the study.
- Diet (e.g. ad libitum): ad libitum; Purina Certified Rodent Chow #5002 ad libitum. Purina Mills, Inc., St. muis, MO. Lot no. NOV05901C.
- Water (e.g. ad libitum): Deionized tap water ad libitum via an automatic watering system.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25.5
- Humidity (%): 8-53
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/l2 hours dark


IN-LIFE DATES: From: 1990-11-10 To: -
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: standard; well-known, non-toxic
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 10ml/kg
- Lot/batch no. (if required): M05X1
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed well in corn oil immediately before dosing.
Assays to verify concentration, stability, and homogeneity of the test substance in the carrier vehicle were not performed.
Duration of treatment / exposure:
Animals w ill be dosed once each day on two consecutive days.
Frequency of treatment:
Animals w ill be dosed once each day on two consecutive days.
Post exposure period:
Animals are sacrificed 24 or 48 hours after the second dose.
Remarks:
Doses / Concentrations:
range finding: 0, 150, 300, 600, 1200, 2500, or 5000 mg/kg day
Basis:
other: administered by gavage
Remarks:
Doses / Concentrations:
main study:0, 31.3, 62.5, or 125 mg/kg/day
Basis:
other: administered by gavage
No. of animals per sex per dose:
range finding: 3
main study: 10
Control animals:
yes, concurrent vehicle
Positive control(s):
benzene
- Justification for choice of positive control(s): known clastogen
- Route of administration: oral gavage in corn oil
- Doses / concentrations: 500 mg/kg day
administered to male mice only
Tissues and cell types examined:
Peripheral blood smears were analyzed for the polychromatic erythrocyte (PCE) to red blood cell (RBC) ratio in the range-finding assay. Bone marrow smears were analyzed for micronucleus in both the range-finding and definitive assays.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: range finding study
TREATMENT AND SAMPLING TIMES: no additional data
DETAILS OF SLIDE PREPARATION:
Peripheral blood smears were analyzed for the polychromatic erythrocyte (PCE) to red blood cell (RBC) ratio in the range-finding assay. Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 µl of blood into a capillary lube. The sample was transferred to three clean, prelabeled microscope slides per mouse, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Two of the three prepared slides were coded, Both coded slides from each test animal were visually examined, and the slide with the most uniform
preparation of smear was stained with acridine orange. Unstained slides were filed for future use should extras be needed.

Bone marrow smears were analyzed in both the range-finding and definitive assays. The right femur from each mouse was removed and
flushed gently with 0.2 ml of fetal bovine serum (FBS) into 0.5 ml of FBS in a 2-ml conical polycarbonate tube. Cells were concentrated by
centrifugation and then resuspended in an equal volume of supernate. The sample was transferred to three clean, prelabeled microscope
slides per mouse, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Two of the three prepared slides
were coded. Both coded slides from each test animal were visually examined, and the slide with the most uniform preparation of smear was
stained with acridine orange. Unstained slides were filed for future use should extras be needed.
METHOD OF ANALYSIS:
Peripheral blood smears and bone marrow smears were evaluated using epifluorescence microscopy.

Other: Criteria for a Valid Assay
The data from this assay were considered acceptable if
(1) the frequency of micronucleated cells in the vehicle control group was within the normal historical range,
(2) administration of the positive
control substance resulted in a statistically significant elevation of micronucleated cells
(3) there were at least three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.
Evaluation criteria:
In the range-finding assay, peripheral blood smears and bone marrow smears were analyzed for the number of RNA-positive polychromatic erythrocytes in at least 500 and 200 erythrocytes, respectively, per animal, In the definitive assay, two parameters were determined in the bone marrow smears: (1) the number of micronucleated RNA-positive erythrocytes in at least 1000 RNA-positive erythrocytes per animal, which provides an index of - chromosomal damage; and (2) the number of RNA-positive erythrocytes in at least 200 erythrocytes per animal, which provides an index of cytotoxicity to the nucleated erythrocyte precursors.
The criteria used for MN are those described by Schmid (1976), with the additional requirement that the MN exhibit fluorescence characteristic of the stain used (i.e., bright yellow in the case of acridine orange). The ratio of RNA-containing erythrocytes to mature erythrocytes (RBC) was based on the number of RNA-positive cells in approximately 200 erythrocytes. Data from a given slide were directly captured by an IBM PC computer data file during scoring. After analysis, the slides were decoded and data summarized using a decoding program on an IBM PC.
Statistics:
Data were analyzed according to sex. The ratio of micronucleated RNA-containing erythrocytes (i. e. , micronucleated PCE) to RNA-positive
erythrocytes and the RNA-positive erythrocytes as a percentage of total erythrocytes were calculated for each animal. The statistical
significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace
analysis of variance on ranks (calculated using the SAS software package on an IBM PC). In experiments where the frequencies of micronucleated cells are determined by scoring 1000 cells per animal, data are not expected to be distributed normally. Such data were analyzed using the Cochran-
Armitage test (using an SRI-developed software package on an IBM PC) for trends in binomial proportions (to determine a significant doseresponse
relationship) and the normal test for equality of binomial proportions (to determine if values for individual dose groups were'
statistically different from those for controls) (Kastenbaum and Bowman, 1970; an SRI-developed software package on an IBM PC was
used). These tests and the rationale for each are discussed in the ASTM Standard Guide for Conduct of Micronucleus Assays in mammalian
Bone Marrow Erythrocytes (ASTM Committee, 1988) and in Margolin et al; (1983).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Salient clinical signs included rough fur and loose stools in all Duomeen C dosage groups. One test-substance-related death was observed in the 62.5 mg/kg/day dosage group. Cytotoxicity, as indicated by a slight decrease in the PCE/RBC ratio was observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
see attached tables
Conclusions:
Interpretation of results (migrated information): negative
It was concluded that Duomeen C at dosages up to and including the MTD of 125 mg/kg/day did not induce increased incidences of
micronuclei in the bone marrow erythrocytes of male and female Swiss- Webster mice. Therefore, Duomeen C was considered to be nongenotoxic
under these test conditions.
Executive summary:

The genotoxic potential of orally administered N-C12-18-alkyltrimethylenediamine (Coco-diamine) to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice.

The study was conducted according to OECD 474 guideline and under GLP.

 

In the range-finding assay, three mice per sex received orally administered Coco-diamine in corn oil at dosage levels of 150, 300, 600, 1200, 2500, or 5000 mg/kg body weight/day (mg/kg/day) to determine a maximum tolerated dosage (MTD) that would be used in setting dosages for the definitive study. A control group of three male and three female mice received corn oil only. All mice were observed and dosed for two consecutive days. Mice surviving the dosing regimen were euthanized 48 hours after the administration of the last dose and evaluated for specific signs of cytotoxicity reflected in hematopoietic indices.

All mice receiving dosages of 300 mg/kg/day or greater died on study, while in the 150 mg/kg/day dosage group only one male mouse died. Adverse clinical observations reported for the 150 mg/kg/day dosage group included decreased body weight in female mice. A set number of erythrocytes in both bone marrow and peripheral red blood cell (RBC) pools from mice surviving to euthanasia were examined and the number of RNA-positive (polychromatic) erythrocytes was counted to determine cellularity and the frequency of PCEs among erythrocytes. Suppression of PCE/RBC ratio to approximately 65% of that of the corn oil control group was observed in both pools from mice receiving doses of 150 mg/kg/day. From this suppression and the minimal mortality observed at 150 mg/kg/day, an MTD of approximately 125 mg/kg/day was determined for Coco-diamine.

In the definitive assay, at least 10 mice per sex per dosage group were orally administered Coco-diamine in corn oil dosage levels of 31.3, 62.5, or 125 mg/kg/day for two consecutive days. Five mice per sex per dosage group were euthanized 24 hours after the final dose and the same number 48 hours after the final dose; all were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil vehicle control group (10 mice per sex) and a benzene positive control group (10 male mice only) were treated similarly and evaluated concurrently with test groups. Salient clinical signs included rough fur and loose stools in all Coco-diamine dosage groups. One test-substance-related death was observed in the 62.5 mg/kg/day dosage group. Cytotoxicity, as indicated by a slight decrease in the PCE/RBC ratio was observed in both sexes in the top two dosage groups of Coco-diamine. However, all Coco-diamine-treated groups, when compared to that of the corn oil control group, had average micronucleus counts approximately equal to that of the control groups. Background micronucleus incidences in bone marrow' erythrocytes of male and female mice treated with corn oil alone averaged 0.18% and 0.22%, respectively. The benzene positive control induced micronucleus rates at least 5-fold greater than that of the background.

In summary, Coco-diamine at dosages up to and including the MTD of 125 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, Coco-diamine was considered to be non-genotoxic under these test conditions.

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

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by alkyl-diamines is not expected. In physiological circumstances, the diamines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil.

 

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

Applicability cross-reading:

Not all the studies used for the evaluation of genotoxic potential ofN-(hydrogenated tallow alkyl) trimethylenediamine, CAS 68603-64-5 (recently redefined as Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine, CAS 133779-11-0), also referred to as HT-diamine,, have been performed on HT-diamine itself.

Use is additionally made of cross-reading, using studies from N-Oleyl-1,3-diaminopropane ((Z)-N-9-octadecenyl-1,3-diaminopropane, CAS 7173-62-8), further referred to as Oleyl-diamine.

 

HT-diamine and Oleyl-diamine are very similar, sharing the same alkyl-diamine structure, resulting to the same functional groups with similar properties leading to common biological activity, and common metabolic degradation. There is only a slight difference in the variation in alkyl-chain lengths, and therefore results obtained with the one product are also considered to be fully relevant for the other. The higher level of unsaturation in oleyl-alkyl chains can be considered a worst case representation, although the higher average chain length could be regarded as a disadvantage in that respect. Further support for read-across can be obtained from the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.

 

Available data:

The test substance, N-(hydrogenated tallow alkyl) trimethylenediamine, CAS 68603-64-5 (recently redefined as Amines, N-C16-18-alkyl (evennumbered) propane-1,3-diamine, CAS 133779-11-0), also referred to as HT-diamine,did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Another bacterial mutagenicity performed independently also found no mutagenicity in S. typhimuriums trains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation.

 

Additional data is available from from in vitro mammalian chromosomal aberration and mutagenicity studies based on N-oleyl trimethylenediamine (Oleyl-diamine) 

Negative results for mutagenicity ofOleyl-diaminewere obtained in a validin vitromammalian cell gene mutation test (HPRT-locus) in Chinese hamster V79cells, in the presence and absence of metabolic activation. No increases in mutation frequency as compared to solvent controls were found.

Additionally, Oleyl-diaminedid also not induce chromosomal aberrations in a valid cytogenetic study in vitro in CHL cells in the presence and absence of metabolic activation.

 

Based on structure and mechanism of cytotoxicity, genototoxicity is also not expected. In physiological circumstances, the diamines have a cationic surfactant structure whichleads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and to move to the nucleus to interact with DNA.

 

Supporting the lack of genotoxic properties comes from the profiling of alkyl-diamines with varying chain length from C10 to C18, and including C18-unsaturated (Oleyl). (QSAR Toolbox v.4.1). There are no alerts are found for DNA interaction, protein interactions and no DNA alerts for Ames, MN and CA (OASIS v1.1).

Selecting in QSAR Toolbox all primary amines (from OECD HPV profile) (total 1750 selected), and removing all compounds that are not discrete chemical and having other atoms besides carbon and nitrogen results to 306 relevant primary amines. From these there are 764 genotoxicity data points reported belonging to 68 of these subcategorized substances. Evaluation of all mutagenicity related data (608 data points of the 763), there was only one positive mutagenic result present, belonging to naphthylethylenediamine. This indicates a lack of mutagenic properties for the primary amines category of chemicals.

  

Information from QSARs also showed no indication for mutagenicity:

- VEGA (Mutagenicity models CAESAR version 2.1.10; SarPy model, version 1.0.5-BETA): Predicts non-mutagenic, both with high reliability, but with the indication that compound could be out of the Applicability Domain of the model.

- DEREK (Derek Nexus: 3.0.1, Nexus: 1.5.0): Nothing to report on mutagenicity

- TOPKAT (Accelrys ADMET Toxicity Prediction (Extensible)) predicts non-mutagen, with high validity for oleyl-diamine, C18-diamine and C10-diamine ( probability for mutagenicy of 0, 0.007 and 0.047 resp.).

- QSAR Toolbox v.3.0 contains series of QSAR for nodes under Human Health Hazards Genetic Toxicity that are all from Danish EPA DB. AlleightIn vitroestimation and threein vivoestimatuions predicted negative genotoxicity for alkyl-diamine structures (in vitro:Ames test (Salmonella); UDS; DNA react. (Ashby fragments); Chrom. abber. (CHO); Mouse, COMET assay; HGPRT; Syrian hamster embryo cells; SCE;In vivo:Rodent, Dominant lethal; Drosophila sex-linked recessive lethal; Negative Mouse micronucleus.).

Also the dataset of performed studies indicate that alkyl-diamines do not have genotoxic properties. All of the available studies showed negative responses.

Available data on N-alkyl trimethylenediamines:

Test System or Species, Strain, Age, Number, and Sex of Animals

Biological Endpoint

S9

Chemical Form and Purity, vehicle

Dose

Results/Comments

Reference

Prokaryotic Systems

OECD 471, GLP

S. typhimurium strainsTA 1535, TA 1537, TA 98 and TA 100

Increase in revertants due to mutations

+/-

Duomeen C

Coco-diamine,

98.7%

in DMSO

+S9: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate

-S9: 3.3, 10.0, 33.3, 100 µg/plate

No mutagenic effects observed. Highest dose 1000 µg/plate was based on toxicity pre-test, Highest dose is selected to show slight toxicity.

Proprietary

RCC Notox, 1990

031444

OECD 471, GLP

S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA

Increase in revertants due to mutations

+/-

Genamin LAP 100D

C12-diamine

purity 100%

in ethanol;

(0.16), 0.5, 1.6, 5, 16, 50, 160 µg/plate with and without S9

Precipitation at 5000 µg/plate

No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 160 μg/ with and from 50 µg/plate without activation.

Proprietary

Aventis, 2003

PT03-0028

OECD 471, GLP

S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA

Increase in revertants due to mutations

+/-

Duomeen HT

HT-diamine

Purity np

In ethanol

Exp.1: 0.316, 0.1, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µL/plate

Exp.2: 0.158, 0.50, 1.58, 5.0, 15.8, 50 , 158, 500 and 1580 µL/plate

Precipitation from 31.6 µg/plate without S9 and from 500 µg/plate with S9.

No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 10 μg/plate without and from 100 µg/plate with activation.

Proprietary

BSL, 2008

081561

OECD 471, GLP

S. typhimurium strainsTA 1535, TA 1537, TA 1538, TA 98 and TA 100

Increase in revertants due to mutations

+/-

Dinoram SH

HT-diamine

90%

In ethanol

1, 5, 10, 25 and 50 mg /plate.

 

No mutagenic effects observed under the test conditions. Toxicity was observed from 50 µg/plate and higher

Proprietary

CIT, 1986

2097 MMO

OECD 471, GLP

S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA

Increase in revertants due to mutations

+/-

Duomeen OV

Oleyl-diamine

92.3%

In Ethanol

Exp.1: 0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate

Exp.2: 0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate

No precipitation of the test item was observed.

No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 0.00316 µL/plate without and from 0.0316 µL/plate with activation.

Proprietary

BSL, 2008

081576

Mammalian SystemsIn Vitro

OECD 473, GLP

CHL (V79)

Chromosomal aberration

+/-

Genamin LAP 100D

C12-diamine

purity 100%

in ethanol;

+S9: 0.16, 0.32, 0.64, 1.28 µg/ml

-S9 3h: 0.16, 0.32, 0.64 µg/ml

-S9 20h: 0.1, 0.2, 0.3 µg/ml

The test material was classified as “negative” for chromosomal aberrations, under the test conditions. Cell survival was reduced below 50 % in the highest evaluated concentrations. Higher dose levels were not evaluable because of an insufficient number of metaphases.

Proprietary

Aventis, 2003

PT03-0029

OECD 473, GLP

CHL (V79)

Chromosomal aberration

+/-

Duomeen OV

Oleyl-diamine

92.3%

In Ethanol

+S9: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.5 and 8.0 µg/mL

-S9 4h: 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL

-S9 20h: 0.05, 0.1, 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL

The test material was classified as “negative” for chromosomal aberrations, under the test conditions. The lowest concentration producing cell toxicity was 4.0 mg/mL with metabolic activation and 1.0 (4h) and 0.4 (20h) mg/mL without metabolic activation.

Proprietary

BSL, 2008

081575

OECD 476, GLP

CHL (V79)

forward mutations (HPRT locus)

+/-

Duomeen OV

Oleyl-diamine

92.3%

In Ethanol

+S9: 0.05, 0.10, 0.25, 0.5, 1.0, 2.0, 3.0, 3.8, 4.0, 4.2, 5.0, 5.5, 6.0, 7.0 µg/mL

-S9 4h: 0.350, 0.425, 0.500, 0.575, 0.650, 0.725, 0.800, 0.875 µg/mL

-S9 20h: 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 µg/mL

The test material was non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Cytotoxicity was observed from 5.0 mg/mL with metabolic activation and 0.875 (4h) and 0.9 (20h) mg/mL without metabolic activation.

Proprietary

BSL, 2010

092013

Mammalian SystemsIn Vivo

OECD474, GLP

MouseSwiss Webster, adult, M and F

MN

induction

na

Duomeen C

Coco-diamine,

in corn oil

Oral main study: 0, 31.3, 62.5 or 125 mg/kg/day;

5 animals/dose/sex + 125 mg additional 9 animals/sex

Does not increase frequency in micronuclei in mouse bone marrow PCE.

Toxicity: 65% suppression PCE/RBC ratio at 125 mg

Proprietary.

SRI, 1991

1924-C01-90

np = not provided; na = not applicable

 

References:

·        Aventis, 2003: Aventis Pharma, PT03-0028, 27-06-2003, GENAMIN LAP 100 D bacterial reverse mutation test.

·        Aventis, 2003: Aventis Pharma, PT03-0029, 16-07-2003, GENAMIN LAP 100 Din vitromammalian chromosome aberration test in V79 Chinese Hamster Cells.

·        RCC Notox, 1990: RCC Notox,031444,, Evaluation of the mutagenic activity of Duomeen C in the Ames Salmonella/microsome test (with independent repeat).

·        SRI, 1991: SRI International, 1924-C01-90, 28-03-1991, Measurement of micronuclei in bone marrow erythrocytes of Swiss-Webster mice treated with Duomeen C.

·        BSL, 2008: BSL Bioservice, 081561, 22-09-2008, Reverse mutation assay using bacteria Salmonella typhimurium and Escherichia coli with N-(Hydrogenated tallow)-1,3-diaminopropane.

·        CIT, 1986: CIT, CIT, 2097 MMO, 21-05-1986, Dinoram SH - Study on the mutagenic properties in vitro in the Ames test.

·        BSL, 2008: BSL Bioservice, 081576, 23-09-2008, Reverse mutation assay using bacteria Salmonella typhimurium and Escherichia coli with N-Oleyl-1,3-diaminopropane

·        BSL, 2008: BSL Bioservice, 081575,, In vitro mammalian chromosome aberration test in Chinese hamster V79 cells with N-Oleyl.1,3.diaminopropane

·        BSL, 2010: BSL Bioservice, 092013, 29-04-2010, In vitro mammalian cell gene mutation test (HPRT-locus) in Chinese hamster V79 cells with N-Oleyl.1,3-diaminopropane

Justification for classification or non-classification

Available studies show no concern for possible genotoxicity. Also further property data for N-(hydrogenated tallow alkyl) trimethylenediamine indicate no genotoxic properties.