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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in bacterial was conducted on Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates (Amines, N-tallow alkyltrimethylenedi-, diacetates). The test item contained 25-35% of 2-butoxyethanol (CAS 111-76-2), which was not considered as impacting on the study since this solvent is not associated to known genotoxic properties. Under these experimental conditions, the test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. This result was further supported by reliable testing conducted on analogue substances.

 

The in vitro cytogenicity and in vitro gene mutation in mammalian cells were investigated using reliable studies conducted on known analogues of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates (Amines, N-tallow alkyltrimethylenedi-, diacetates). Under the conditions of the studies, the test substances returned negative results.

The read-across approach is considered as robust based on structural similaritiies between the substances.

 

Based on these negative results obtained in vitro, it is not deemed relevant to further investigate the genotoxicity of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates (Amines, N-tallow alkyltrimethylenedi-, diacetates), nor to conduct animal testing.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-14 - 2008-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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 ß-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

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 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

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:

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-Oleyl-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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Assay performed on a mixture containing 30-40% of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, 25-35% of 2-butoxyethanol (CAS 111-76-2) and water until complement to 100%. As the existing data on 2-butoxyethanol indicate no genotoxic potential, the presence of this solvent does not compromise the interpretation of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

In addition, to increase their sensitivity to mutagenic substances, further mutations have been added:
. the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
. the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
. the addition of the plasmid pKM 101 (conferring ampicillin resistance) to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to sorne mutagens,
. in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.

Test concentrations with justification for top dose:

Experiments without S9 mix:
The selected treatment-levels were:
• 3.13, 6.25, 12.5, 25 and 50 µg/plate, for the TA 1537, TA 98 and TA 102 strains in both experiments,
• 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 1535 and TA 100 strains in both experiments.

Experiments with S9 mix:
The selected treatment-levels were:
• 12.5, 25, 50, 100 and 200 µg/plate, for all the tester strains in the first experiment,
• 12.5, 25, 50, 100 and 300 µg/plate, for all the tester strains in the second experiment.

Vehicle / solvent:

The vehicle was water for injections, batch No. EVE 19D (Fresenius Kabi, Sèvres, France).
The test item was dissolved in the vehicle at concentrations of:
· 50 mg/mL for the preliminary toxicity test,
· 20 mg/mL for the first experiment,
· 30 mg/mL for the second experiment.
The preparations were made immediately before use.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see table 1 below

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method and preincubation method.

DURATION
The experiments were performed according to:
. direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test substance solution (0.1 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
. preincubation method (second experiment with S9 mix): test substance solution (0.1 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

ln two independent experiments, five dose-levels of the test item (three plates/dose-level) were tested on each strain, with and without S9 mix.

Treatment of results:
ln each experiment, for each strain and for each experimental point, the number of revertants per plate was scored.
The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Evaluation criteria:

This study is considered valid if the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with our historical data,
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 50 µg/plate (without S9) and at 100 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item was freely soluble in the vehicle (water for injections) at 50 mg/mL.
Consequently, with a treatment volume of 100 µL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A moderate to marked toxicity was noted towards the three strains used, at dose-levels = 100 µg/plate without S9 mix and at dose-levels = 500 µg/plate with S9 mix.

ACCEPTANCE CRITERIA :
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

RESULTS OF THE MAIN STUDY :
Experiments without S9 mix:
The selected treatment-levels were:
• 3.13, 6.25, 12.5, 25 and 50 µg/plate, for the TA 1537, TA 98 and TA 102 strains in both experiments,
• 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 1535 and TA 100 strains in both experiments.

A moderate toxicity was generally noted at 50 µg/plate in the TA 1537, TA 98 and TA 102 strains. A marked toxicity was induced at 100 µg/plate in the TA 1535 and TA 100 strains.
The test item did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains.

Experiments with S9 mix:
The selected treatment-levels were:
• 12.5, 25, 50, 100 and 200 µg/plate, for all the tester strains in the first experiment,
• 12.5, 25, 50, 100 and 300 µg/plate, for all the tester strains in the second experiment.

A marked toxicity was noted in the TA 98, TA 100 and TA 102 strains at 300 µg/plate.
A moderate to marked toxicity was noted in the TA 1537 strain at dose-levels = 100 µg/plate.
The test item did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains.

Table 2: Results table / First experiment without S9

ns	Doses (µg/plate)	T	P	Revertants per plate			mean	SD	ratio
TA 1535	0	0	0	19	18	28	22	6
	6.25	0	0	24	19	29	24	5	1.1
	12.5	0	0	36	24	24	28	7	1.3
	25	0	0	28	18	22	23	5	1.0
	50	0	0	20	18	18	19	1	0.9
	100	3	0	5 MSc+St	11 MSc+St	14 MSc+St	10	5	0.5
	NaN3 (1)	-	-	605	710	745	687	73	31.7
TA 1537	0	0	0	6	7	11	8	3	-
	3.13	0	0	6	8	11	8	3	1.0
	6.25	0	0	6	8	10	8	2	1.0
	12.5	0	0	1	8	11	8	7	1.0
	25	0	0	7	4	6	6	2	0.7
	50	3	0	4Mt	10Mt	4Mt	6	3	0.8
	9AA (50)	-	-	545	740	647	644	98	80.5
TA 98	0	0	0	30	26	29	28	2	-
	3.13	0	0	12	36	17	18	7	0.6
	6.25			25	24	18	22	4	0.8
	12.5	0	0	16	26	19	20	5	0.7
	25	0	0	31	24	36	30	6	1.1
	50	3	0	26Mt	23Mt	19Mt	23	4	0.8
	2NF (0.5)	-	-	199	236	176	200	25	7.1
TA 100	0	0	0	114	127	114	118	8	-
	6.25	0	0	116	109	145	123	19	1.0
	12.5	0	0	126	135	116	126	10	1.1
	25	0	0	103	98	125	109	14	0.9
	50	0	0	109	102	114	108	6	0.9
	100	3	0	79St	72St	81St	77	5	0.7
	NaN3 (1)	-	-	778	775	705	753	41	6.4
TA 102	0	0	0	380	389	440	403	32	-
	3.13	0	0	358	387	361	369	16	0.9
	6.25	0	0	381	350	474	402	65	1.0
	12.5	0	0	407	335	332	358	42	0.9
	25	0	0	337	370	335	347	20	0.9
	50	3	0	254Mt	284 Mt	261 Mt	266	16	0.7
	MMC (0.5)	-	-	2314	2558	2349	2407	132	6.0

0 : vehicle control (water for injections)

St : strong toxicity

Mt : moderate toxicity

SD : standard deviation

Ratio = number of revertants with the test substance / number of revertants with the vehicle

T= toxicity, P = precipitation

Msc : manual scoring

Table 3: Results table / Second experiment without S9

strains	Doses (µg/plate)	T	P	Revertants per plate			mean	SD	ratio
TA 1535	0	0	0	7	8	12	9	3	-
	6.25	0	0	10	18	11	13	4	1.4
	12.5	0	0	14	6	16	12	5	1.3
	25	0	0	7	8	7	7	1	0.8
	50	0	0	14	18	13	15	3	1.7
	100	3	0	10St	11St	8St	10	2	1.1
	NaN3 (1)	-	-	582	454	558	521	58	57.9
TA 1537	0	0	0	11	2	8	7	5	-
	3.13	0	0	2	8	5	5	3	0.7
	6.25	0	0	5	0	5	3	3	0.5
	12.5	0	0	8	8	5	7	2	1.0
	25	0	0	4	4	7	5	2	0.7
	50	0	0	5	6	2	4	2	0.6
	9AA (50)	-	-	297	265	355	306	26	13.6
TA 98	0	0	0	22	8	22	17	9	-
	3.13	0	0	23	11	19	18	6	1.0
	6.25	0	0	13	22	10	15	6	0.9
	12.5	0	0	16	17	11	15	3	0.9
	25	0	0	24	8	22	18	9	1.1
	50	3	0	12Mt	7Mt	20Mt	13	7	0.8
	2NF (0.5)	-	-	194	236	184	206	28	12.0
TA 100	0	0	0	91	91	96	93	3	-
	6.25	0	0	85	89	75	83	7	0.9
	12.5	0	0	87	93	113	98	14	1.1
	25	0	0	87	101	96	95	7	1.0
	50	0	0	90	101	102	98	7	1.1
	100	3	0	80St	74St	68St	74	6	0.8
	NaN3 (1)	-	-	628	517	489	545	74	5.9
TA 102	0	0	0	307	280	263	263	22	-
	3.13	0	0	334	338	333	335	3	1.2
	6.25	0	0	308	363	303	325	33	1.1
	12.5	0	0	311	306	346	341	28	1.1
	25	0	0	346	321	317	328	16	1.2
	50	0	0	340	339	326	335	8	1.2
	MMC (0.5)	-	-	1940	1743	1528	1737	206	6.1

 

0: vehicle control (water for injections)

St : strong toxicity

Mt : moderate toxicity

SD: standard deviation

Ratio = number of revertants with the test substance / number of revertants with the vehicle

T= toxicity, P = precipitate

Conclusions:

Under these experimental conditions, the test substance Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

The objective of this study was to evaluate the potential of the test substance Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates (batch No.94051339) to induce reverse mutation in Salmonella typhimurium.

A preliminary toxicity test was performed to define the dose-levels of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test substance was dissolved in water for injection.

The dose-levels of the positive controls were as follows:

without S9 mix:

1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

with S9 mix:

2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were as follows:

• 3.13, 6.25, 12.5, 25 and 50µg/plate, for the TA 1537, TA 98 and TA 102 strains in both experiments,

• 6.25, 12.5, 25, 50 and 100µg/plate, for the TA 1535 and TA 100 strains in both experiments.

A moderate toxicity was generally noted at 50µg/plate in the TA 1537, TA 98 and TA 102 strains.

A marked toxicity was induced at 100µg/plate in the TA 1535 and TA 100 strains.

The test item did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains.

Experiments with S9 mix:

The selected treatment-levels were as follows:

•12.5, 25, 50, 100 and 200µg/plate, for all the tester strains in the first experiment,

• 12.5, 25, 50, 100 and 300µg/plate, for all the tester strains in the second experiment.

A marked toxicity was noted in the TA 98, TA 100 and TA 102 strains at 300µg/plate. A moderate to marked toxicity was noted in the TA 1537 strain at dose-levels = 100µg/plate. The test item did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the five strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Under these experimental conditions, the test substance Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

An Ames test is available with Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates. Since the negative result is in line with those observed with the diamine compounds, a cross-reading could be applied for the in vitro mammalian tests (i.e. Chromosomal aberration and cell gene mutation tests) which show negative results.

 

The bacterial reverse mutation assay available with Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetateat 30 to 40 % of purity in mixture with 25 to 35% of 2-butoxyethanol and water was not mutagenic with or without S9 mix in each of the five tester strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102). These results were confirmed in an independently repeated experiment. The assay substance contained a high proportion of 2-butoxyethanol. However, the existing data on 2-butoxyethanol showed no alert on genotoxicity. Thus it is expected no impact of the solvent in the result (negative) observed.

For the other tests, a read across was applied from other diamine compounds.

 

N-Oleyl-1,3-diaminopropane, was tested for genetic toxicity in threein vitrotests. In a bacterial reverse mutation assay the substance was not mutagenic with or without S9 mix. It was also tested for mammalian cell gene mutations at the HPRT locus using V79 cells of the Chinese hamster and was not mutagenic. In addition, the substance was tested in a chromosomal aberration test in human lymphocytes and was found to be not clastogenic. All tests were to current OECD/EU protocols carried out to GLP and with a clearly defined and described test substance. Based on these results it can be concluded that N-Oleyl-1,3-diaminopropane is not to be expected to be a genotoxic hazard to human health.

 

The test substance, N-(hydrogenated tallow alkyl) trimethylenediamine, 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 inS. typhimuriumstrains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation.

 

N-C12-18-alkyltrimethylenediamine (Coco-diamine) was tested in theSalmonella/microsome plate test and did not induce a dose-related increase in the number of revertant(His+)colonies in each of the four tester strains (TA1535; TA1537;TA98 and TA100). These results were confirmed in an independently repeated experiment.

 

The potential of N-Dodecyl-1,3-diaminepropane to induce chromosome aberrations was investigated in V79 cells of the Chinese hamster lungin vitroaccording to OECD 473 and under GLP. No relevant or reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.

 

In conclusion, based on structure and mechanism of cytotoxicity, genotoxicity is also 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. 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.

For possible genotoxic effects the diamine or metabolite is needed to become available intra-cellular. It cannot be expected that intra-cellular presence and effects of a diamine molecule is different when the diamine derives from absorption of diamine from a diamine product or from a diamine acetate product.

Justification for classification or non-classification

Based on the available negative in-vitro tests for genotoxicity, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates does not require classification as a mutagen according to the European Union CLP/GHS criteria.