Amines, N-C12-18-alkyltrimethylenedi-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For each endpoint, bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity (both in vivo and in vitro), a GLP compliant study is available on the basis of testing on N-C12-18-alkyltrimethylenediamine (Coco-diamine) itself, or from cross-reading with studies on N-Dodecyl-1,3-diaminepropane , or N-oleyl-1,3-diaminopropane. These studies represent the studies of highest quality and relevance to N-C12-18-alkyltrimethylenediamine that are available within the category of alkyl-diamines. All studies were performed under GLP according to current guidelines.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

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

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

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.

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

2008-05-14 - 2010-04-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - 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 β-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

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

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

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

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:

Interpretation of results (migrated information):
negative

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, 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 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. (considered of no impact for diamines). 2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

All these strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.

S. typhimurium TA 1535 his G46 rfa -Δ uvr B -is derived from the his G46 strain but is a deep rough * excision repair deficient ** strain which is highly sensitive to base-pair substitution mutagens.
S. typhimurium TA 1537 his C3076 rfa -Δ uvr B -is a strain derived from a his C3076 mutant and carries deep rough and excision repair deficiency mutations. This strain is highly sensitive to frameshift mutagens.
S. typhimurium TA 98 his D3052 rfa - Δ uvr B - R + is also derived from a his 03052 mutant, but in addition to the deep rough and excision repair deficiency mutations it also carries a resistance transfer factor. This additional factor makes TA 98 very sensitive to the weaker frame-shift mutagens.
S. typhimurium TA 100 his G46 rfa -Δ uvr B -R + derived from the his G46 strain and carries the deep rough and excision repair deficiency mutations and also a resistance transfer factor. This additional factor makes TA 100 particularly sensitive to basepair substitution mutagens.
*All the five strains described above carry a mutation known as deep rough which affects the cell membrane so that the normal lipopolysaccharide cell coat is defective. These strains therefore allow a greater permeability of test agents across this membrane and into the cell.
** In bacteria almost all the primary dramage caused to DNA bya mutagen is repaired by excision and recombinatim repair systems so that normalIy only a small percentage of the potential mutagenic damage is expressed. The five tester strains used here lack this excision repair thus making them more sensitive to mutagens.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Toxicity study: 5000, 3330, 1000, 333, 100, 33.3, 10.0, 3.3, 1.0 µg/plate
Mutagenicity assay 1 and 2:
with S9-mix 33.3, 10.0, 3.3, 1.0, 0.33 µg/plate
without S9-mix 100, 33.3, 10.0, 3.3, 1.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to
use.
- Justification for choice of solvent/vehicle: test substance is soluble in dmso and stbel for at least 4 hours.

Untreated negative controls:

yes

Remarks:

dmso (vehicle)

Negative solvent / vehicle controls:

yes

Remarks:

dmso

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation (-S9-mix): Strain Chemical Concentration/plate Solvent TA1535 sodium azide (SA) (Fluka) 1 µg Saline TA1537 9-aminoacri

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate and an independent repeat was performed

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent) have been counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting have been counted manually.

- Other:
Selection of dose levels:
Selection of an adequate range o f doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity . The highest concentration of test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates.

Evaluation criteria:

An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range
for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding test with strain TA100 or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing has been done.
A test substance was considered negative (not mutagenic) in the Ames test if :
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or
without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect t o the number induced by the solvent control in
any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9-mix at 33.3 µg/plate and with S9-mix at 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Preliminary toxicity determination of the test substance in TA100

	Viable counts/plate (duplicate plates)
Concentartion (µg/plate)	Without S9-mix	With S9-mix
Control (dmso)	343;385	362;371
1.0	362;379	400;351
3.3	370;338	395;364
10.0	343;335	390;370
33.3	60;110	270;330
100	0;0	0;0
333	0;0	0;0
1000	0;0	0;0
3330*	0;0	0;0
5000*	0;0	0;0

*the test substance precipitaed slightly

Mutagenicity assay: experiment 1

	Mean number of revertant (His+) colonies/3 replicate plates (± S.D.) with different strains of S. typhimurium
dose µg/plate	TA1535	TA1537	TA98	TA100
	Without S9 -mix
0.33	10 ± 4	6  ± 1	17 ± 5	88 ± 11
1.0	9 ± 2	6 ± 3	18 ± 5	97 ± 18
3.3	10 ± 3	4 ± 1	21 ± 6	86 ± 5
10.0	9 ± 1	5 ± 2	18 ± 1	102 ± 20
33.3	8 ± 3	2 ± 1	3 ± 2*	85 ± 28
Solvent control (0.1 ml dmso)	11 ± 4	4 ± 1	21 ± 1	103 ± 10
Positive control	324 ± 25	366 ± 38	522 ± 27	806 ± 162

	With S9 -mix
1.0	7 ± 2	6  ± 2	24 ± 4	92 ± 4
3.3	8 ± 0	3 ± 0	24 ± 4	91 ± 7
10.0	10 ± 5	6 ± 3	18 ± 5	118 ± 15
33.3	9 ± 6	3 ± 1	5 ± 2*	118 ± 8
100	5 ± 2*	6 ±1 *	0 ± 0**	57 ± 3*
Solvent control (0.1 ml dmso)	10 ± 5	7 ± 0	29 ± 1	95 ± 15
Positive control	339 ± 16	403 ± 68	678 ± 43	692 ± 23

Mutagenicity assay: experiment 2

	Mean number of revertant (His+) colonies/3 replicate plates (± S.D.) with different strains of S. typhimurium
dose µg/plate	TA1535	TA1537	TA98	TA100
	Without S9 -mix
0.33	12 ± 6	10 ± 1	32 ± 7	83 ± 10
1.0	12 ± 6	11 ± 3	42 ± 6	99 ± 2
3.3	11 ± 3	12 ± 3	28 ± 2	88 ± 5
10.0	11 ± 2	14 ± 1	36 ± 14	97 ± 12
33.3	10 ± 1	12 ± 3	30 ± 3	96 ± 14
Solvent control (0.1 ml dmso)	13 ± 2	8 ± 2	28 ± 5	77 ± 15
Positive control	363 ± 10	203 ± 15	462 ± 43	750 ± 23

	With S9 -mix
1.0	9 ± 2	8 ± 3	35 ± 4	83 ± 6
3.3	12 ± 1	11 ± 2	32 ± 4	93 ± 9
10.0	12  ± 3	10 ± 3	33 ± 12	72 ± 7
33.3	8 ± 5	8 ± 4	25 ± 5	111 ± 9
100	7 ± 2*	2 ± 1*	2 ± 1**	75 ± 6*
Solvent control (0.1 ml dmso)	7 ± 2	6 ± 2	36 ± 4	68 ± 9
Positive control	412 ± 16	572 ± 15	497 ± 41	499 ± 70

* Bacterial background lawn slightly reduced

** Bacterial background lawn extremely reduced

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test substance can be considered as not mutagenic in the Ames Salmonella assay with tester strains TA98, TA100, TA1535 and TA1537.

Executive summary:

N-C12-18-alkyltrimethylenediamine (Coco-diamine) was tested in the Ames Salmonella/microsome plate test up to 33.3µg/platein the absence of S9-mix and up to 100 µg/plate in the presence of S9-mix. The test substance did not induceadose-related increase in the number of revertant(His+)colonies in each of the four tester strains (TA1535; TA1537;TA98and TA100). These results were confirmed in an independently repeated experiment.

The test substance can, therefore, be considered as not mutagenic in this testsystem.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Lack of genotoxic properties of alkyl-diamines was further confirmed in an in vivo micronucleus study with N-C12-18-alkyltrimethylenediamine (Coco-diamine) conducted according to OECD 474 guideline and under GLP.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed under GLP and according to standard protocol.

Qualifier:

according to guideline

Guideline:

other: 40 CFR Part 158 US-EPA-FIFRA, Section 158.340, Guideline 84-3

Principles of method if other than guideline:

This method is similar to OECD474. The humidity was out of the recommended range of 50-60%. It was8-53% this deviation is not thought to have influenced the outcome of the study.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: Approximately 6 weeks (born on approximately November 21, 1990)
- Weight at study initiation: one or two mice from each cage were selected randomly to be weighed. The weights of 17 male mice ranged from 21.8 to 26.7 g and those of 15 female mice from 18.7 to 22.7 g at the time of receipt.
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Mice were housed no more than 10 to a cage dufing quarantine, 3 to a cage for the range-finding assay, and 5 to a cage during the definitive assay. Polycarbmate cages with hardwood-chip bedding were used throughout the study.
- Diet (e.g. ad libitum): ad libitum; Purina Certified Rodent Chow #5002 ad libitum. Purina Mills, Inc., St. muis, MO. Lot no. NOV05901C.
- Water (e.g. ad libitum): Deionized tap water ad libitum via an automatic watering system.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25.5
- Humidity (%): 8-53
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/l2 hours dark


IN-LIFE DATES: From: 1990-11-10 To: -

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: standard; well-known, non-toxic
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 10ml/kg
- Lot/batch no. (if required): M05X1
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed well in corn oil immediately before dosing.
Assays to verify concentration, stability, and homogeneity of the test substance in the carrier vehicle were not performed.

Duration of treatment / exposure:

Animals w ill be dosed once each day on two consecutive days.

Frequency of treatment:

Animals w ill be dosed once each day on two consecutive days.

Post exposure period:

Animals are sacrificed 24 or 48 hours after the second dose.

Remarks:

Doses / Concentrations:
range finding: 0, 150, 300, 600, 1200, 2500, or 5000 mg/kg day
Basis:
other: administered by gavage

Remarks:

Doses / Concentrations:
main study:0, 31.3, 62.5, or 125 mg/kg/day
Basis:
other: administered by gavage

No. of animals per sex per dose:

range finding: 3
main study: 10

Control animals:

yes, concurrent vehicle

Positive control(s):

benzene
- Justification for choice of positive control(s): known clastogen
- Route of administration: oral gavage in corn oil
- Doses / concentrations: 500 mg/kg day
administered to male mice only

Tissues and cell types examined:

Peripheral blood smears were analyzed for the polychromatic erythrocyte (PCE) to red blood cell (RBC) ratio in the range-finding assay. Bone marrow smears were analyzed for micronucleus in both the range-finding and definitive assays.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: range finding study
TREATMENT AND SAMPLING TIMES: no additional data
DETAILS OF SLIDE PREPARATION:
Peripheral blood smears were analyzed for the polychromatic erythrocyte (PCE) to red blood cell (RBC) ratio in the range-finding assay. Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 µl of blood into a capillary lube. The sample was transferred to three clean, prelabeled microscope slides per mouse, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Two of the three prepared slides were coded, Both coded slides from each test animal were visually examined, and the slide with the most uniform
preparation of smear was stained with acridine orange. Unstained slides were filed for future use should extras be needed.

Bone marrow smears were analyzed in both the range-finding and definitive assays. The right femur from each mouse was removed and
flushed gently with 0.2 ml of fetal bovine serum (FBS) into 0.5 ml of FBS in a 2-ml conical polycarbonate tube. Cells were concentrated by
centrifugation and then resuspended in an equal volume of supernate. The sample was transferred to three clean, prelabeled microscope
slides per mouse, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Two of the three prepared slides
were coded. Both coded slides from each test animal were visually examined, and the slide with the most uniform preparation of smear was
stained with acridine orange. Unstained slides were filed for future use should extras be needed.
METHOD OF ANALYSIS:
Peripheral blood smears and bone marrow smears were evaluated using epifluorescence microscopy.

Other: Criteria for a Valid Assay
The data from this assay were considered acceptable if
(1) the frequency of micronucleated cells in the vehicle control group was within the normal historical range,
(2) administration of the positive
control substance resulted in a statistically significant elevation of micronucleated cells
(3) there were at least three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.

Evaluation criteria:

In the range-finding assay, peripheral blood smears and bone marrow smears were analyzed for the number of RNA-positive polychromatic erythrocytes in at least 500 and 200 erythrocytes, respectively, per animal, In the definitive assay, two parameters were determined in the bone marrow smears: (1) the number of micronucleated RNA-positive erythrocytes in at least 1000 RNA-positive erythrocytes per animal, which provides an index of - chromosomal damage; and (2) the number of RNA-positive erythrocytes in at least 200 erythrocytes per animal, which provides an index of cytotoxicity to the nucleated erythrocyte precursors.
The criteria used for MN are those described by Schmid (1976), with the additional requirement that the MN exhibit fluorescence characteristic of the stain used (i.e., bright yellow in the case of acridine orange). The ratio of RNA-containing erythrocytes to mature erythrocytes (RBC) was based on the number of RNA-positive cells in approximately 200 erythrocytes. Data from a given slide were directly captured by an IBM PC computer data file during scoring. After analysis, the slides were decoded and data summarized using a decoding program on an IBM PC.

Statistics:

Data were analyzed according to sex. The ratio of micronucleated RNA-containing erythrocytes (i. e. , micronucleated PCE) to RNA-positive
erythrocytes and the RNA-positive erythrocytes as a percentage of total erythrocytes were calculated for each animal. The statistical
significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace
analysis of variance on ranks (calculated using the SAS software package on an IBM PC). In experiments where the frequencies of micronucleated cells are determined by scoring 1000 cells per animal, data are not expected to be distributed normally. Such data were analyzed using the Cochran-
Armitage test (using an SRI-developed software package on an IBM PC) for trends in binomial proportions (to determine a significant doseresponse
relationship) and the normal test for equality of binomial proportions (to determine if values for individual dose groups were'
statistically different from those for controls) (Kastenbaum and Bowman, 1970; an SRI-developed software package on an IBM PC was
used). These tests and the rationale for each are discussed in the ASTM Standard Guide for Conduct of Micronucleus Assays in mammalian
Bone Marrow Erythrocytes (ASTM Committee, 1988) and in Margolin et al; (1983).

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Salient clinical signs included rough fur and loose stools in all Duomeen C dosage groups. One test-substance-related death was observed in the 62.5 mg/kg/day dosage group. Cytotoxicity, as indicated by a slight decrease in the PCE/RBC ratio was observed

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

see attached tables

Conclusions:

Interpretation of results (migrated information): negative
It was concluded that Duomeen C at dosages up to and including the MTD of 125 mg/kg/day did not induce increased incidences of
micronuclei in the bone marrow erythrocytes of male and female Swiss- Webster mice. Therefore, Duomeen C was considered to be nongenotoxic
under these test conditions.

Executive summary:

The genotoxic potential of orally administered N-C12-18-alkyltrimethylenediamine (Coco-diamine) to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice.

The study was conducted according to OECD 474 guideline and under GLP.

 

In the range-finding assay, three mice per sex received orally administered Coco-diamine in corn oil at dosage levels of 150, 300, 600, 1200, 2500, or 5000 mg/kg body weight/day (mg/kg/day) to determine a maximum tolerated dosage (MTD) that would be used in setting dosages for the definitive study. A control group of three male and three female mice received corn oil only. All mice were observed and dosed for two consecutive days. Mice surviving the dosing regimen were euthanized 48 hours after the administration of the last dose and evaluated for specific signs of cytotoxicity reflected in hematopoietic indices.

All mice receiving dosages of 300 mg/kg/day or greater died on study, while in the 150 mg/kg/day dosage group only one male mouse died. Adverse clinical observations reported for the 150 mg/kg/day dosage group included decreased body weight in female mice. A set number of erythrocytes in both bone marrow and peripheral red blood cell (RBC) pools from mice surviving to euthanasia were examined and the number of RNA-positive (polychromatic) erythrocytes was counted to determine cellularity and the frequency of PCEs among erythrocytes. Suppression of PCE/RBC ratio to approximately 65% of that of the corn oil control group was observed in both pools from mice receiving doses of 150 mg/kg/day. From this suppression and the minimal mortality observed at 150 mg/kg/day, an MTD of approximately 125 mg/kg/day was determined for Coco-diamine.

In the definitive assay, at least 10 mice per sex per dosage group were orally administered Coco-diamine in corn oil dosage levels of 31.3, 62.5, or 125 mg/kg/day for two consecutive days. Five mice per sex per dosage group were euthanized 24 hours after the final dose and the same number 48 hours after the final dose; all were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil vehicle control group (10 mice per sex) and a benzene positive control group (10 male mice only) were treated similarly and evaluated concurrently with test groups. Salient clinical signs included rough fur and loose stools in all Coco-diamine dosage groups. One test-substance-related death was observed in the 62.5 mg/kg/day dosage group. Cytotoxicity, as indicated by a slight decrease in the PCE/RBC ratio was observed in both sexes in the top two dosage groups of Coco-diamine. However, all Coco-diamine-treated groups, when compared to that of the corn oil control group, had average micronucleus counts approximately equal to that of the control groups. Background micronucleus incidences in bone marrow' erythrocytes of male and female mice treated with corn oil alone averaged 0.18% and 0.22%, respectively. The benzene positive control induced micronucleus rates at least 5-fold greater than that of the background.

In summary, Coco-diamine at dosages up to and including the MTD of 125 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, Coco-diamine was considered to be non-genotoxic under these test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by alkyl-diamines is 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. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil.

 

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

Additional information from genetic toxicity in vivo:

Applicability cross-reading:

Not all the studies used for the evaluation of genotoxic potential of N-Coco-1,3-diaminopropane CAS 61791-63-7 (recently redefined as Amines, N-C12-18-alkyltrimethylenedi-, CAS 68155-37-3; also referred to as Coco-diamine), have been performed on Coco-diamine itself.

Use is additionally made of cross-reading, using studies from N-Dodecyl-1,3-diaminepropane (CAS 5538-95-4), also referred as C12-diamine, and N-Oleyl-1,3-diaminopropane ((Z)-N-9-octadecenyl-1,3-diaminopropane, CAS 7173-62-8), further referred to as Oleyl-diamine.

 

C12-diamine and Coco-diamine are very similar, sharing the same alkyl-diamine structure, with only a difference in the variation in alkyl-chain lengths. Coco-diamine is an alkyl-diamine for which the alkyl chain varies between C10 and C18, and the average of the chain lengths is somewhere between C12 and C14. The fraction of 12-diamine in Coco-diamine is about 50%. The average chain-length is comparable between Coco-diamine and C12-diamine, and therefore results obtained with the one product are also considered to be fully relevant for the other.

Additional data is available from Oleyl-diamine. Cross-reading from this substance is acceptable on the basis of identical alkyl-diamine structure, resulting to the same functional groups with similar properties leading to common biological activity, and common metabolic degradation. The higher level of unsaturation in oleyl-alkyl chains can be considered a worst case representation, although the higher average chain length could be regarded as a disadvantage in that respect.

 

Available data:

Coco-diamine was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 ofSalmonella typhimuriumin accordance to OECD guideline 471 and under GLP.

Coco-diamine was tested up to 33.3µg/platein the absence of S9-mix and up to 100 µg/plate in the presence of S9-mix. Adequate toxicity was observed, and appropriate reference mutagens produced significant increases in the number of revertant colonies, demonstrating the sensitivity of the assay. The test substance did not induceadose-related increase in the number of revertant(His+)colonies in each of the four tester strains. These results were confirmed in an independently repeated experiment. The test substance was therefore considered to be not mutagenic in this testsystem.

Because of the omission of E.coli WP2 or S. typhimurium TA102 strains that are now part of the standard protocol, this was not suitable to identify certain oxidising mutagens, cross-linking agents and hydrazines. However, this is not considered to be relevant for diamines.

 

C12-diamine was tested in anin vitromammalian chromosomal aberration study according toOECD 473 and under GLP.In this study the potential of C12-diamine to induce chromosome aberrations was investigated in V79 cells of the Chinese hamster lungin vitro.The concentration range evaluated was sufficiently high to result to a reduction of cell survival below 50% in the highest evaluated concentrations.Appropriate positive controls showed a significant increase in chromosome aberrations, thus confirming the sensitivity of the assay, and the efficacy of the S9mix.

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. There was no relevant increase in the number of polyploid cells as compared with the solvent controls.

 

Additional data is available fromin vitromammalian mutagenicity studies based on Oleyl-diamine. The study was performed according to OECD 476 guideline under GLP. Oleyl-diamine was in this study found to be non-mutagenic to the target gene (HPRT-locus) in Chinese hamster V79 cells, in the presence and absence of metabolic activation. No increases in mutation frequency as compared to solvent controls were found.

 

Lack of genotoxic properties of alkyl-diamines was further confirmed in anin vivomicronucleus study with Coco-diamine, conducted according to OECD 474 guideline and under GLP.

The genotoxic potential of orally administered Coco-diamine to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice. Following a range-finding assay, 10 mice per sex per dose group were orally administered Coco-diamine in corn oil dosage levels of 31.3, 62.5, or 125 mg/kg/day for two consecutive days. Evaluations for cytotoxicity and micronucleus formation in bone marrow erythrocytes were done in half of the animals 24 hours after the final dose and for the remaining animals 48 hours after the final dose. A vehicle control group (10 mice per sex) and a benzene positive control group (10 male mice only) were treated similarly and evaluated concurrently with test groups. Toxicity was indicated by observed clinical signs. Cytotoxicity was indicated by a slight decrease in the PCE/RBC ratio in the top two dose groups. All Coco-diamine-treated groups showed an average micronucleus counts approximately equal to that of the control groups, whereas the benzene positive control induced micronucleus rates at least 5-fold greater. It was concluded that Coco-diamine at dosages up to and including the MTD of 125 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice.

 

 

Based on structure and mechanism of cytotoxicity, genototoxicity by alkyl-diamines is 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 transfer to the nucleus to interact with DNA.

 

Further information supporting the lack of genotoxic properties comes from the profiling of alkyl-diamines with varying chain length from C10 to C18, and including C18-unsaturated (Oleyl-diamine). (QSAR Toolbox v.3.0). There are no alerts are found for DNA interaction, protein interactions and no DNA alerts for Ames, MN and CA (OASIS v1.1).

Selecting in QSAR Toolbox all primary amines (from OECD HPV profile - total 1750 selected), and removing all compounds that are not discrete chemical and having other atoms besides carbon and nitrogen results to 306 relevant primary amines. From these there are 764 genotoxicity data points reported belonging to 68 of these subcategorized substances. Evaluation of all mutagenicity related data (608 data points of the 764), there was only one positive mutagenic result present, belonging to naphthylethylenediamine. This indicates a lack of mutagenic properties for the primary amines category of chemicals, to which the diamines also belong.

 

Information from QSARs on alkyl-diamine structures also showed no indication for mutagenicity:

- VEGA (Mutagenicity models CAESAR version 2.1.10; SarPy model, version 1.0.5-BETA): Predicts non-mutagenic, both with high reliability, but with the indication that compound could be out of the Applicability Domain of the model.

- DEREK (Derek Nexus: 3.0.1, Nexus: 1.5.0): Nothing to report on mutagenicity

- TOPKAT (Accelrys ADMET Toxicity Prediction (Extensible)) predicts non-mutagen, with high validity for oleyl-diamine, C18-diamine and C10-diamine (probability for mutagenicy of 0, 0.007 and 0.047 resp.).

- QSAR Toolbox v.3.0 contains series of QSAR for nodes under Human Health Hazards Genetic Toxicity that are all from Danish EPA DB. All eightIn vitroestimations and threein vivoestimations predicted negative genotoxicity for alkyl-diamine structures (in vitro:Ames test (Salmonella); UDS; DNA react. (Ashby fragments); Chrom. abber. (CHO); Mouse, COMET assay; HGPRT; Syrian hamster embryo cells; SCE;In vivo:Rodent, Dominant lethal; Drosophila sex-linked recessive lethal; Mouse micronucleus.).

 

 

Also the combined dataset of performed studies evaluating the genotoxicity hazards of alkyl-diamines substances indicate that alkyl-diamines do not have genotoxic properties. All of the available studies showed negative responses.

 

Available data on alkyl-diamines:

Test System or Species, Strain, Age, Number, and Sex of Animals	Biological Endpoint	S9	Chemical Form and Purity, vehicle	Dose	Results/Comments	Reference
Prokaryotic Systems
OECD 471, GLP S. typhimurium strainsTA 1535, TA 1537, TA 98 and TA 100	Increase in revertants due to mutations	+/-	Duomeen C Coco-diamine, 98.7% in DMSO	+S9: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate -S9: 3.3, 10.0, 33.3, 100 µg/plate	No mutagenic effects observed. Highest dose 1000 µg/plate was based on toxicity pre-test, Highest dose is selected to show slight toxicity.	Proprietary RCC Notox, 1990 031444
OECD 471, GLP S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA	Increase in revertants due to mutations	+/-	Genamin LAP 100D C12-diamine purity 100% in ethanol;	(0.16), 0.5, 1.6, 5, 16, 50, 160 µg/plate with and without S9	Precipitation at 5000 µg/plate No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 160 μg/ with and from 50 µg/plate without activation.	Proprietary Aventis, 2003 PT03-0028
OECD 471, GLP S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA	Increase in revertants due to mutations	+/-	Duomeen HT HT-diamine Purity np In ethanol	Exp.1: 0.316, 0.1, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µL/plate Exp.2: 0.158, 0.50, 1.58, 5.0, 15.8, 50 , 158, 500 and 1580 µL/plate	Precipitation from 31.6 µg/plate without S9 and from 500 µg/plate with S9. No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 10 μg/plate without and from 100 µg/plate with activation.	Proprietary BSL, 2008 081561
OECD 471, GLP S. typhimurium strainsTA 1535, TA 1537, TA 1538, TA 98 and TA 100	Increase in revertants due to mutations	+/-	Dinoram SH HT-diamine 90% In ethanol	1, 5, 10, 25 and 50 mg /plate.	No mutagenic effects observed under the test conditions. Toxicity was observed from 50 µg/plate and higher	Proprietary CIT, 1986 2097 MMO
OECD 471, GLP S. typhimurium strainsTA98, TA100, TA1535, TA1537;Escherichia coliWP2 uvrA	Increase in revertants due to mutations	+/-	Duomeen OV Oleyl-diamine 92.3% In Ethanol	Exp.1: 0.00010, 0.000316, 0.00100, 0.00316, 0.0100, 0.0316 and 0.1 µL/plate Exp.2: 0.000050, 0.000158, 0.00050, 0.00158, 0.0050, 0.0158, 0.05 and 0.1 µL/plate	No precipitation of the test item was observed. No mutagenic effects observed under the test conditions. Toxic concentration observed for bacteria from 0.00316 µL/plate without and from 0.0316 µL/plate with activation.	Proprietary BSL, 2008 081576
Mammalian SystemsIn Vitro
OECD 473, GLP CHL (V79)	Chromosomal aberration	+/-	Genamin LAP 100D C12-diamine purity 100% in ethanol;	+S9: 0.16, 0.32, 0.64, 1.28 µg/ml -S9 3h: 0.16, 0.32, 0.64 µg/ml -S9 20h: 0.1, 0.2, 0.3 µg/ml	The test material was classified as “negative” for chromosomal aberrations, under the test conditions. Cell survival was reduced below 50 % in the highest evaluated concentrations. Higher dose levels were not evaluable because of an insufficient number of metaphases.	Proprietary Aventis, 2003 PT03-0029
OECD 473, GLP CHL (V79)	Chromosomal aberration	+/-	Duomeen OV Oleyl-diamine 92.3% In Ethanol	+S9: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.5 and 8.0 µg/mL -S9 4h: 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL -S9 20h: 0.05, 0.1, 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL	The test material was classified as “negative” for chromosomal aberrations, under the test conditions. The lowest concentration producing cell toxicity was 4.0 mg/mL with metabolic activation and 1.0 (4h) and 0.4 (20h) mg/mL without metabolic activation.	Proprietary BSL, 2008 081575
OECD 476, GLP CHL (V79)	forward mutations (HPRT locus)	+/-	Duomeen OV Oleyl-diamine 92.3% In Ethanol	+S9: 0.05, 0.10, 0.25, 0.5, 1.0, 2.0, 3.0, 3.8, 4.0, 4.2, 5.0, 5.5, 6.0, 7.0 µg/mL -S9 4h: 0.350, 0.425, 0.500, 0.575, 0.650, 0.725, 0.800, 0.875 µg/mL -S9 20h: 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 µg/mL	The test material was non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster. Cytotoxicity was observed from 5.0 mg/mL with metabolic activation and 0.875 (4h) and 0.9 (20h) mg/mL without metabolic activation.	Proprietary BSL, 2010 092013
Mammalian SystemsIn Vivo
OECD474, GLP MouseSwiss Webster, adult, M and F	MN induction	na	Duomeen C Coco-diamine, in corn oil	Oral main study: 0, 31.3, 62.5 or 125 mg/kg/day; 5 animals/dose/sex + 125 mg additional 9 animals/sex	Does not increase frequency in micronuclei in mouse bone marrow PCE. Toxicity: 65% suppression PCE/RBC ratio at 125 mg	Proprietary. SRI, 1991 1924-C01-90

np = not provided; na = not applicable

 

References:

- Aventis, 2003: Aventis Pharma, PT03-0028, 27-06-2003, GENAMIN LAP 100 D bacterial reverse mutation test.

- Aventis, 2003: Aventis Pharma, PT03-0029, 16-07-2003, GENAMIN LAP 100 D in vitro mammalian chromosome aberration test in V79 Chinese Hamster Cells.

- RCC Notox, 1990: RCC Notox, 031444, 09-05-1990, Evaluation of the mutagenic activity of Duomeen C in the Ames Salmonella/microsome test (with independent repeat).

- SRI, 1991: SRI International, 1924-C01-90, 28-03-1991, Measurement of micronuclei in bone marrow erythrocytes of Swiss-Webster mice treated with Duomeen C.

- BSL, 2008: BSL Bioservice, 081561, 22-09-2008, Reverse mutation assay using bacteria Salmonella typhimurium and Escherichia coli with N-(Hydrogenated tallow)-1,3-diaminopropane.

- CIT, 1986: CIT, CIT, 2097 MMO, 21-05-1986, Dinoram SH - Study on the mutagenic properties in vitro in the Ames test.

- BSL, 2008: BSL Bioservice, 081576, 23-09-2008, Reverse mutation assay using bacteria Salmonella typhimurium and Escherichia coli with N-Oleyl-1,3-diaminopropane

- BSL, 2008: BSL Bioservice, 081575, 08-10-2008, In vitro mammalian chromosome aberration test in Chinese hamster V79 cells with N-Oleyl.1,3.diaminopropane

- BSL, 2010: BSL Bioservice, 092013, 29-04-2010, In vitro mammalian cell gene mutation test (HPRT-locus) in Chinese hamster V79 cells with N-Oleyl.1,3-diaminopropane

Justification for classification or non-classification

Available studies show no concern for possible genotoxicity. Also further property data for N-C12-18-alkyltrimethylenediamine indicate that genotoxic properties are rather unlikely.