Amines, N-C12-18-alkyltrimethylenedi-

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

l7-Mar-2003 to 14-May-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-dodecylpropane-1,3-diamine

EC Number:

226-902-6

EC Name:

N-dodecylpropane-1,3-diamine

Cas Number:

5538-95-4

Molecular formula:

C15H34N2

IUPAC Name:

N-dodecylpropane-1,3-diamine

Constituent 2

Reference substance name:

N-Dodecyl-1,3-diaminepropane

IUPAC Name:

N-Dodecyl-1,3-diaminepropane

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

Name or number of compound: Genamin LAP 100 D
Formula of compound: R-NH-CH2-CH2-CH2-NH2 (R = Lauryl)
Chemical name: Laurylpropylendiamin
CAS-Register number: 5538-95-4
Molecular weight: 246 - 260 g/mol
Batch number: DEGE908461
Purity: approx. 100 %
Appearance: white wax
Storage conditions: at approximately 20 °C in a fume cupboard
Solubility in water: < 1 g/L
Solubility: solution in ethanol
Stability in the solvent: confirmed over 4 hours in ethanol

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEM (minimal essential medium) with Earle's salts and L-glutamine

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver induced with Aroclor 1254

Test concentrations with justification for top dose:

0.05, 0.1, 0.16, 0.2, 0.3, 0.32, 0.4, 0.5, 0.6, 0.64, 0.96, 1.28, 2.60, 5.10, 10.2, 20.4 µg/ml

Vehicle / solvent:

vehicle: ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: EMS (ethyl methane sulfonate) and CPA (cyclophosphamide)

Details on test system and experimental conditions:

TEST GROUPS:
First Experiment treatment time 3 h
without S9-mix with S9-mix
Part III Part II
Solvent control: 0.0 µg/ml 0.0 µg/ml
Positive control: EMS 1500.0 µg/ml CPA 7.5 µg/ml
Test group 1: 0.16 µg/ml $ 0.16 µg/ml $
Test group 2: 0.32 µg/ml $ 0.32 µg/ml $
Test group 3: 0.64 µg/ml $ 0.64 µg/ml $
Test group 4: 0.96 µg/ml § 1.28 µg/ml $
Test group 5: 2.60 µg/ml §
Test group 6: 5.10 µg/ml §
Test group 7: 10.2 µg/ml §
Test group 8: 20.4 µg/ml §Φ
§ not evaluated because of cytotoxicity
$ concentrations at which metaphase analysis was conducted
Φ macroscopic precipitation

Second Experiment treatment time 3 h
with S9-mix
Part II
Solvent control: 0.0 µg/ml
Positive control: CPA 7.5 µg/ml
Test group 1: 0.16 µg/ml $
Test group 2: 0.32 µg/ml $
Test group 3: 0.64 µg/ml $
Test group 4: 1.28 µg/ml $
Test group 5: 2.60 µg/ml §
Test group 6: 5.10 µg/ml §
Test group 7: 10.2 µg/ml §
Test group 8: 20.4 µg/ml §Φ

Second Experiment treatment time 20 h,
without S9-mix
Part III
Solvent control: 0.0 µg/ml
Positive control: EMS 400.0 µg/ml
Test group 1: 0.05 µg/ml #
Test group 2: 0.1 µg/ml $
Test group 3: 0.2 µg/ml $
Test group 4: 0.3 µg/ml $
Test group 5: 0.4 µg/ml §
Test group 6: 0.5 µg/ml §
Test group 7: 0.6 µg/ml §

§ not evaluated because of cytotoxicity
# not used because higher concentrations were evaluated
$ concentrations at which metaphase analysis was conducted

CONTROL GROUPS
Solvent controls: cultures treated with the solvent
Positive controls: a: without metabolic activation: EMS (ethyl methane sulfonate)
b: with metabolic activation: CPA (cyclophosphamide) = Endoxan®
Formulation of test compound: dissolved in ethanol at appropriate concentrations immediately before use
Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment
final concentration: 1.5 mg/ml (3 h treatment)
final concentration: 0.4 mg/ml (20 h treatment)
CPA dissolved in cell culture medium on the day of treatment,
final concentration in cell culture medium: 7.5 µg/ml
Source of cells: cell bank of "Genetic Toxicology", Aventis Pharma Germany GmbH, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Earle's salts and L-glutamine
Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks

PREPARATION AND STORAGE OF A LIVER HOMOGENATE FRACTION (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, [phone] and stored at approx - 80°C. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.

PREPARATION OF S9-MIX
Sufficient S9 fraction was thawed at room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. Moltox 1455) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until
used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

CELL CULTURE
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CO2 in 175 cm2 plastic flasks. About 5 x 10E5 to 1 x 10E6 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine The cells were subcultured twice a week.

TOXICITY EXPERIMENTS AND DOSE RANGE FINDING
For the determination of cytotoxic effects cell cultures on slides were treated with the test item. Evaluation of cell number was performed in the first experiment with the 3/20h treatment/sampling time with and without S9-mix. In the second experiment cell evaluation was performed with the 3/20h treatment/sampling time with S9-mix and the 20/20h treatment/sampling time without S9-mix. Using a 500 fold microscopic magnification the cells were counted in 10 fields of the slides. The cell number of the treatment groups is given as % cells in relation to the control.
The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level was determined by the solubility of the test compound up to the maximum of 10 mM, or the international limit dose, 5000 µg/ml.
Treatments were performed both in the presence and absence of the S9-mix metabolic activation system using a duplicate cell culture at each test point.

RATIONALE FOR DOSE SELECTION
The evaluated concentrations for mutagenicity are based on the results of the cell counting.
For non-toxic, freely soluble test compounds, the top dose is either 10 mM or 5000 µg/ml according to international testing guidelines.
For relatively insoluble test compounds that are not toxic at concentrations lower than the insoluble concentration, the highest dose used is a concentration above the limit of solubility in the final culture medium after the end of the treatment period.
For toxic compounds, a highest dose level is selected which reduced survival and/or the mitotic index below 50 %, of the corresponding solvent control.

MUTAGENICITY TEST
Unless positive results were obtained in the first test, two independent experiments were conducted using duplicate cultures of cells seeded onto slides (i.e. 2 per dose level) and at least three dose levels.
S9-Mix - + + -
Experiment I I II II
Exposure period [h] 3 3 3 20
Recovery [h] 17 17 17 0
Preparation time
[after start of
treatment in h] 20 20 20 20
However, if clearly positive results were obtained in the first experiment, the second assay was not evaluated. If equivocal or negative results were obtained in the first experiment, the second assay was evaluated for chromosomal aberrations.
Colcemide was added to each culture 2 hours before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined.
Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCI. Any effects on the osmolality during the study were described in the study report.
Exponentially growing cultures which were more than 50 % confluent were trypsinated by an approx. 0.25 % (v/v) trypsin solution ready for use (mfr. Gibco).
A single cell suspension (culture) was prepared. The trypsin concentration was approx. 025 % (v/v) in Ca-Mg-free salt solution.
Two slides were placed in Quadriperm'' dishes which were then seeded with cells to yield 3-4 x 10E4 cells/slide. Thus for each dose level and treatment time, duplicate cultures slides were used. The Quadriperm® dishes contained 6 ml MEM with approx. 10 % (v/v) FCS and approx. 0.1 % (w/v) neomycinsulfate.
After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent and in the presence of metabolic activation additionally 2 % (v/v) S9-mix.
For the 3 hours treatment time, the medium was replaced by normal medium following two rinses. In the second experiment the cells were exposed to the treatment medium without S9-mix for 20 h.
18 h after the start of the treatment, Colcemide was added (approx. 0.05 4g/ml/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared as follows:
The cultures were made hypotonic by adding about 5 ml of approx. 0.075 M potassium chloride solution at around 37 °C. The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 2 ml fixative.
Then the liquid was replaced by 6 ml fixative (methanol: glacial acetic acid, 3 : 1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:
- staining for 10 minutes in approx. 2 % (w/v) orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetonelxylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan® or Corbit®

Evaluation criteria:

ANALYSIS OF METAPHASES
The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed and classified as shown in chapter 9.1. Only metaphases with 22 +/- 2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, acentric chromatids (chromosomes), chromatid deletion, chromosome deletion, chromatid exchanges, chromosome exchanges including intrachanges, dicentrics and ring formation, pulverization, and multiple aberrations.
Furthermore the incidence of polyploid metaphases was determined in 100 metaphase cells of each cell culture.
Additionally the mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is expressed as a percentage.
After the metaphases had been evaluated, the code was broken. For each experiment the results from the dose groups were compared with those of the control group and the positive control at each sampling time.
CRITERIA FOR A VALID ASSAY
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are statistically significant and within the laboratory's normal range
CRITERIA FOR CLASTOGENICITY
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data and/or
- no significant increase in the number of structural chromosome aberrations is observed.
A test substance is classified as clastogenic if:
-the number of induced structural chromosome aberrations is above the range of our historical control data and
eith

Statistics:

The Biometry of the results was performed with a one-sided Fisher's exact test.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY TESTING
Genamin LAP 100 D was dissolved in ethanol.
Evaluation of the solubility of that solution in MEM Earle's salts cell culture medium showed that 2600 µg/ml was a practicable concentration. This concentration corresponds to 10 mM, which is the limit dose according to international guidelines.
Macroscopic precipitation was noted at concentrations of 162.5 µg/ml and above and microscopic precipitation at concentrations of 20.4 µg/ml and above.
First, the test substance was tested with and without S9-mix using a concentration range of 20.4 to 2600 µg/ml (named part I). However a complete repeat was necessary because all concentrations produced cytotoxicity in order that evaluation was not possible.
The repeat experiment performed with concentrations between 0.16 and 20.4 µg/ml (named part II) with S9-mix was evaluated. Four concentration groups were evaluated in order to cover a range of no to distinct toxicity. The experiment without S9-mix (3 hours treatment) had to be repeated due to inappropriate cytotoxicity: The highest evaluable concentration showed less than 50 % toxicity in contrast to the guideline requirements. Due to cytotoxicity only 2 concentrations were evaluable without S9-mix after 20 hours treatment and this experiment had also to be repeated using lower concentrations.
In the second repeat of the experiments without S9-mix (named part III) a dose range of 0.16 to 0.96 µg/ml was used for the 3 hours treatment time and a dose range of 0.05 to 0.6 µg/ml for the 20 hours treatment time.
Summarizing, the following concentrations were evaluated and reported:
Experiment Part S9 Treatment Sampling Evaluated canc.
time [h] time [h] [µg/ml]
1 (3/20 h) III - 3 20 0.16, 0.32, 0.64
1 (3/20 h) II + 3 20 0.16, 0.32, 0.64, 1.28
2 (3/20 h) II + 3 20 0.16, 0.32, 0.64, 1.28
2 (20/20H) III - 20 20 0.1, 0.2, 0.3
Evaluation of cytotoxicity by cell counting showed that Genamin LAP 100 D was toxic in a dose - related manner to the V79 cells in the absence and in the presence of metabolic activation. Cell survival was reduced below 50 % in the highest evaluated concentrations. Higher dose levels were not evaluable because of an insufficient number of metaphases.
The highest evaluated concentration after 3 hours treatment without metabolic activation (0.64 µg/ml) showed also a reduction of the mitotic index (39.5 %). The other evaluated concentration groups showed no significant reduction of the mitotic index.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of the test compound solution did not have any significant effect on osmolality, only a very slight reduction was noted with 2600 µg/ml which is not considered as relevant. The pH value was adjusted to pH 7.4 with HCI if necessary.

MUTAGENICITY TEST
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
The test compound Genamin LAP 100 D was assessed for its clastogenic potential in vitro in the chromosome aberration test in two independent experiments.
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. All values correspond to the historical control data range
Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Genamin LAP 100 D is not clastogenic in this in vitro chromosome aberration assay with cells of the V79 Chinese hamster cell line under the conditions described in this report.

Executive summary:

In this study the potential of N-Dodecyl-1,3-diaminepropane to induce chromosome aberrations was investigated in V79 cells of the Chinese hamster lungin vitro.The study was performed according to OECD 473 and under GLP

For each experiment duplicate cultures were used for each concentration.

The test compound was dissolved in ethanol. Evaluation of the solubility of that solution in MEM Earle's salts cell culture medium showed that 2600 µg/ml was a practicable concentration. Higher concentrations were not applied because of the 10mM limitation(OECDguideline).

 

Due to cytotoxic properties of the test compound, several experiments had to be repeated. Repeats are termed part II and part III, respectively.

 

The following concentrations were used.

Part I:

Experiment with and without S9-mix:

20.4!,40.7!, 81.3!,162.5!, 325.0!, 650.0!, 1300.0! and 2600.0!*µg/ml

Due to cytotoxicity the experiments had to be repeated using lower concentrations.

Part II:

First experiment with 3h treatment time

with S9-mix: 0.16$, 0.32$, 0.64$, 1.28$, 2.6!, 5.1!, 10.2! and20.4!µg/ml.

without S9-mix: 0.16, 0.32, 0.64, 1.28!, 2.6!, 5.1!, 10.2! and 20.4!µg/ml

 

The experiment without S9-mix had to be repeated due to inappropriate cytotoxicity: The highest evaluable concentration showed less than 50 % toxicity in contrast to the guideline requirements.

 

Second experiment with 3 h treatment time with S9-mix and 20 h without S9-mix:

With S9.-mix: 0.16$, 0.32$, 0.64$, 1.28$, 2.6!, 5.1!, 10.2! and20.4!µg/ml.

without S9-mix: 0.16, 0.32, 0.64!, 1.28!, 2.6!, 5.1!, 10.2! and 20.4!µg/ml

 

Due to cytotoxicity only 2 concentrations were evaluable without S9-mix and the experiment had to be repeated using lower concentrations.

 

Part III:

First experiment with 3 h treatment time without S9-mix:

0.16$, 0.32$, 0.64$ and 0.96 µg/ml

Second experiment with 20 h treatment time without S9-mix:

0.05, 0.1$, 0.2$, 0.3$, 0.4!, 0.5! and 0.6! µg/ml

 

* = 10 mM

$ = concentrations at which metaphase analysis was conducted

! = toxic concentration (not evaluable due to lack of cells, respectively metaphases)

 

Macroscopic precipitation was noted at concentrations of 162.5 µg/ml and above and microscopic precipitation at concentrations of20.4 µg/ml and above.

Cell survival was reduced below 50 % in the highest evaluated concentrations. The highest evaluated concentration after 3 hours treatment without metabolic activation (0.64 µg/ml) showed also a reduction of the mitotic index (39.5%). The other evaluated concentration groups showed no significant reduction of the mitotic index.

 

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.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9mix.

 

In conclusion, N-Dodecyl-1,3-diaminepropane did not induce chromosome aberrations in V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system.

N-Dodecyl-1,3-diaminepropane is not clastogenic in this in vitro chromosome aberration assay with V79 Chinese hamster lung cells.