Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-14 - 2010-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

1

Reference substance name:

(Z)-N-9-octadecenylpropane-1,3-diamine

EC Number:

230-528-9

EC Name:

(Z)-N-9-octadecenylpropane-1,3-diamine

Cas Number:

7173-62-8

Molecular formula:

C21H44N2

IUPAC Name:

N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diamine

Details on test material:

Chemical registery number : 7173-62-8
Chemical name : (Z)-N-9-octadecenylpropane-1,3-diamine

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Method

Target gene:

Hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix (indued with ß-naphthoflavone and phenobarbital)

Test concentrations with justification for top dose:

Experiment I:
-S9: 0.350, 0.425, 0.500, 0.575, 0.650, 0.725, 0.800, 0.875 µg/mL
+S9: 0.05, 0.10, 0.25, 0.5, 1.0, 3.0, 4.0, 5.0 µg/mL

Experiment II:
-S9: 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 µg/mL
+S9: 1.0, 2.0, 3.8, 4.2, 5.0, 5.5, 6.0, 7.0 µg/mL

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 300 µg/mL ethylmethanesulphonate (EMS); 1.0 µg/mL (Experiment I) and 1.5 µg/mL (Experiment II) 7,12-dimethylbenzanthracene(DMBA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 20 hours
- Expression time (cells in growth medium): 48 to 72 hours after treatment

SELECTION AGENT (mutation assays): thioguanine (TG)

NUMBER OF REPLICATIONS: two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
- negative and/or solvent controls fall within the performing laboratories historical control data range: 1 - 39 mutants/10E6 cells
- the absolute cloning efficiency: ([number of positive cultures x 100] / total number of seeded cultures) of the negative and/or solvent controls is > 50%
- the spontaneous mutant frequency in the negative and/or solvent controls is in the range of historical control data
- the positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.

Atest is considered negative if there is no biological relevant increase in the number of mutants. There are several criteria for determining a positive result:
- a reproducible 3-times higher mutation frequency than the solvent control for at least one of the concentrations
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a 3-fold increase of the mutant frequency is not observed.

Statistics:

No data

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 0.875 µg/mL (-S9) and at 5.0 µg/mL (+S9); Experiment II: at 0.9 µg/mL (-S9) and at 7.0 µg/mL (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was based on data from pre-experiment. Eight concentrations were tested: 0.0625 - 4.0 µg/mL (-S9) and 0.125 - 3.0 µg/mL (+S9).
In experiment I 0.875 µg/mL (-S9) and 5 µg/mL (+S9) were selected as the highest concentrations. In experiment II 0.9 µg/mL (-S9) and 7 µg/mL [+S9) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 20 h long-term exposure assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
All values of the negative controls and test item concentrations found were within the historical control data

Any other information on results incl. tables

Table 1: Experiment I - without metabolic activation

Dose Group	Concentration [µg/mL]	Relative Growth [%]	Factor* (survived cells / seeded cells)	Mutant colonies per 10E6 cells	Mutation factor
NC1	0 0	125.1	0.78	11.48
NC2		133.3	0.82	14.58
S1	0 0	100.0 100.0	0.78	14.08
S2			0.69	6.48
5	0.350	104.1	0.73	18.49	1.80
6	0.425	98.6	0.71	25.42	2.47
7	0.500	90.9	0.81	8.07	0.79
8	0.575	80.5	0.81	24.66	2.40
9	0.650	53.5	0.75	22.09	2.15
10	0.725	51.9	0.74	14.29	1.39
11	0.800	33.2	0.74	24.19	2.35
12	0.875	13.6	0.63	3.96	0.39
EMS	300	105.9	0.82	150.37	14.62

Table 2: Experiment I - with metabolic activation

Dose Group	Concentration [µg/mL]	Relative Growth [%]	Factor* (survived cells / seeded cells)	Mutant colonies per 10E6 cells	Mutation factor
NC1	0 0	103.0	0.97	10.80
NC2		114.6	0.69	28.34
S1	0 0	100.0 100.0	0.84	11.96
S2			0.87	7.51
1	0.05	97.8	0.83	21.79	2.24
2	0.10	82.0	0.72	13.81	1.42
3	0.25	83.9	0.84	19.69	2.02
4	0.5	79.8	0.83	7.85	0.81
5	1.0	81.3	0.71	21.86	2.25
6	3.0	68.5	0.73	17.08	1.75
7	4.0	47.2	0.72	22.85	2.35
8	5.0	10.1	0.57	14.13	1.45
DMBA	1.0	67.0	0.68	147.19	15.12

Table 3: Experiment II - without metabolic activation

Dose Group	Concentration [µg/mL]	Relative Growth [%]	Factor* (survived cells / seeded cells)	Mutant colonies per 10E6 cells	Mutation factor
NC1	0 0	115.5	0.87	5.74
NC2		105.7	0.90	11.67
S1	0 0	100.0 100.0	0.87	5.76
S2			0.75	10.65
5	0.1	80.6	0.56	8.06	0.98
6	0.2	80.6	0.56	11.71	1.43
7	0.4	67.9	0.76	1060	1.29
8	0.5	62.0	0.51	13.70	1.67
9	0.6	38.2	0.94	8.55	1.04
10	0.7	27.7	0.84	12.54	1.53
11	0.8	26.7	0.67	2.98	0.36
12	0.9	12.6	0.99	9.06	1.10
EMS	300	49.3	0.57	184.12	22.44

Table 4: Experiment II - with metabolic activation

Dose Group	Concentration [µg/mL]	Relative Growth [%]	Factor* (survived cells / seeded cells)	Mutant colonies per 10E6 cells	Mutation factor
NC1	0 0	118.5	0.75	16.02
NC2		96.0	0.68	8.77
S1	0 0	100.0 100.0	0.86	16.36
S2			0.84	1.20
2	1.0	95.0	0.70	9.34	1.06
3	2.0	95.0	0.68	4.39	0.50
5	3.8	85.0	0.72	6.23	0.71
6	4.2	80.0	0.74	6.11	0.70
7	5.0	70.0	0.78	3.19	0.36
8	5.5	65.0	0.55	2.75	0.31
9	6.0	40.0	0.75	8.70	0.99
10	7.0	16.5	0.79	5.06	0.58
DMBA	1.5	80.0	0.87	116.09	13.23

NC: negative control / medium control

SC: solvent control (ethanol)

*: cloning efficiency x cells seeded

EMS: Ethylmethansulfonate

DMBA: 7,12 -Dimethylbenz(a)anthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test item N-Oleyl-1,3-diaminopropane is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Executive summary:

The test item N-Oleyl-1,3-diaminopropane was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to the OECD guideline 476.
The main experiments were carried out without and with metabolic activation. The experiments with metabolic activation were performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation.
The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476.
In experiment I 0.875 µg/mL (without metabolic activation) and 5.0 µg/mL (with metabolic activation) were selected as the highest concentrations. In experiment II 0.9 µg/mL (without metabolic activation) and 7.0 µg/mL (with metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 20 h long-term exposure assay.
The pH-value detected with the test item was within the physiological range. The test item was investigated at the following concentrations:
Experiment I
without metabolic activation:
0.350, 0.425, 0.500, 0.575, 0.650, 0.725, 0.800 and 0.875 µg/mL
and with metabolic activation:
0.05, 0.10, 0.25,0.5,1.0, 3.0, 4.0 and 5.0 µg/mL
Experiment II
without metabolic activation:
0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 µg/mL
and with metabolic activation:
1.0, 2.0, 3.8, 4.2, 5.0, 5.5, 6.0 and 7.0 µg/mL
No precipitation oft he test item was noted in experiment I and experiment II.
Toxicity:
A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.
In experiment I without metabolic activation the relative growth was 13.6% for the highest concentration (0.875 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5.0 µg/mL with a relative growth of 10.1%.
In experiment II without metabolic activation the relative growth was 12.6% for the highest concentration (0.9 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 7.0 µg/mL with a relative growth of 16 .5%.

Mutagenicity:
In experiment I without metabolic activation mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 1 - 39 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.
Mutation frequencies with the negative control were found to be 11.48 and 14.58 mutants/10⁶ cells, 14.08 and 6.48 mutants/10⁶ cells for the solvent control and in the range of 3.96 to 25.42 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.47 was found at a concentration of 0.425 µg/mL with a relative growth of 98.6%.
With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 2 - 28 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.
Mutation frequencies of the negative control were found to be 10.80 and 28.34 mutants/10⁶ cells, 11.96 and 7.51 mutants/10⁶ cells for the solvent control and in the range of 7.85 to 22.85 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.35 was found at a concentration of 4.0 µg/mL with a relative growth of 47.2%.
In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 1 - 39 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.
Mutation frequencies with the negative control were found to be 5.47 and 11.67 mutants/10⁶ cells, 5.76 and 10.65 mutants/10⁶

cells for the solvent control and in the range of 2.98 to 13.70 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent controls values) of 1.67 was found at a concentration of 0.5 µg/mL with a relative growth of 62.0%.
In experiment II with metabolic activation most mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 2 - 28 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.
Mutation frequencies of the negative control were found to be 16.02 and 8.77 mutants/10⁶ cells, 16.36 and 1.20 mutants/10⁶ cells for the solvent control and in the range of 2.75 to 9.34 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.06 was found at a concentration of 1.0 µg/mL with a relative growth of 95%.
DMBA (1.0 and 1.5 µg/mL) and (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.