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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-14 - 2008-09-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): N-Oleyl-1,3-diaminopropane
- Physical state: liquid
- Analytical purity: N-Alkyl-1,3-diaminopropanes: 92.3%
N-Alkyl-amines: 7.7%
C-Chain-distribution (R = alkyl):
R = C16: 7%; C18: 91%; C20: 2%
- Lot/batch No.: S000902
- Production date of the test item: August 13, 2007
- Expiration date of the lot/batch: September 30, 2010
- Storage condition of test material: at room temperature
- Product (name): Duomeen OV
- Colour: Max 7 Gardner
- Melting Point: 9 - 20°C
- pH: basic

Method

Target gene:
histidine operon (his)
Species / strain
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
Controls
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

Results and discussion

Test results
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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.