Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 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

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:

not applicable

Species / strain

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

Controls

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

Results and discussion

Test results

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'.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.