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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 cross-reading from testing with N-Dodecyl-1,3-diaminepropane , N-Coco-1,3-diaminopropane and N-Oleyl-1,3-diaminopropane, representing the studies of highest quality and relevance to N-C12,14-alkyl-1,3-diaminopropane available within the category of alkyl-diamines. Further information on the applicability of the read-across from various diamines to C12/14-diamine can be obtained from the document "Category polyamines - 20170518.pdf" added to IUCLID Ch. 13.

N-Dodecyl-1,3-diaminepropane was negative in both an Ames test and anin vitromammalian chromosomal aberration study. N-Coco-1,3-diaminopropane was found negative in an in vivo micronucleus test, and N-oleyl-1,3-diaminopropane was found not mutagenic in anin vitromammalian mutagenicity study.

All studies were performed under GLP according to current guidelines.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-Mar-2003 to 11-Apr-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
other: Japanese Substance Control Law (JSCL) Test Guideline III.1 Gene Mutation Test with bacteria
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa uvrB (TA98 and TA100 also R)
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
(0.16), 0.5, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
vehicle: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium-azide, 9-aminoacridine, 2-nitrofluorene, 4-nitroquinoline-N-oxide and 2-aminoanthracene
Details on test system and experimental conditions:
TEST GROUPS
first plate incorporation test (10 % S9 rat liver homogenate):
a: with metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
b: without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
second plate incorporation test (10 % S9 rat liver homogenate):
a: with metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate (TA 100, TA 1535, WP2uvrA)
0.16, 0.5, 1.6, 5, 16 and 50 µg/plate (TA 1537, TA 98)
b: without metabolic activation: 0.16, 0.5, 1.6, 5, 16, 50 µg/plate
third plate incorporation test (30 % S9 rat liver homogenate):
a: with metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate
b: without metabolic activation: 0.5, 1.6, 5, 16, 50 and 160 µg/plate

Control groups : negative controls:
a: untreated controls
b: solvent controls (0 µg/plate)
positive controls:
a: without metabolic activation: sodium-azide for strain TA 100 and TA 1535
9-aminoacridine for strain TA 1537
2-nitrofluorene for strain TA 98
4-nitroquinoline-N-oxide for strain WP2uvrA
b: with metabolic activation: 2-aminoanthracene for all strains

Formulation of test compound: dissolved in ethanol at appropriate concentrations immediately before use

Formulation of reference compounds: sodium-azide dissolved in deionized water final concentrations: 1.0 µg/plate for strain TA 1535
2.0 µg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0 µg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5 µg/plate for strain TA 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0 µg/plate (plate inc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations:
1.5 µg/plate for strains TA 98, TA 100, TA 1535 and TA 1537 (10 % S9-mix)
20.0 µg/plate (plate inc.) for strain WP2uvrA (10 % S9-mix)
1.5 µg/plate for strains TA 100, TA 98 (30 % S9-mix)
10.0 µg/plate for strains TA 1535 and TA 1537 (30 % S9-mix)
30.0 µg/plate for strain WP2uvrA (30 % S9-mix)
The frozen stock solutions of each compound were diluted progressively up to the final concentration on the day of treatment.

Source of bacteria: stock cultures in the bank of "Genetic Toxicology", Aventis Pharma Germany, ProTox; prepared from the original bacterial strains

TEST ORGANISM
Salmonella typhimurium strains: TA 98 hisD3052 rfa uvrB +R,
TA 100 hisG46 rfa uvrB +R,
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
Escherichia coli: WP2uvrA pKM101

Experimental conditions in vitro: approx. 37 °C in an incubator

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., 1.57 Industrial Park Dr. Boone, NC 28607, (828) 264-9099. 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. One volume of S9 fraction (batch no. Moltox 1455 for two plate incorporation tests, protein concentration 39.3 g/l) was mixed with 9 volumes of the S9 cofactor solution (10%, which was kept on ice until used), respectively three volumes of S9 fraction (batch no. Moltox 1455) was mixed with 7 volumes of the S9 cofactor solution (30%). This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
100 mM phosphate buffer pH 7.4

BACTERIA
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. colt' was obtained from the E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures, which had been stored in liquide nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled.

ASSAY PROCEDURE
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second respectively third experiment were based on findings, including toxicity, in the first respectively second experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn andlor reduction (at least halving) of the number of spontaneously occurring mutants compared to the corresponding solvent control value.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM).
The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in ethanol)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (wlv) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.
Evaluation criteria:
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 significant and within the laboratory's normal range

CRITERIA FOR A POSITIVE RESPONSE
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
SOLUBILITY AND TOXICITY
Genamin LAP 100 D was in ethanol and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160, 50, 16, 5, 1.6, 0.5, 0.16 µg/plate were used in the different experiments.
Visible precipitation of Genamin LAP 100 D on the plates was observed at 5000 µg/plate.
In the first plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation in a dose range of 50 to 5000 µg/plate with the test strains TA 1537 and TA 98. In the other test strains toxicity in bacteria was observed with metabolic activation in a dose range of 160 to 5000 µg/plate. In the absence of metabolic activation the test compound proved to be toxic to all bacterial strains at concentrations of 50 µg/plate and above. As less than 5 doses were evaluable with all strains due to toxicity in bacteria a second test with lower doses was performed.

In the second plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation at a dose level of 50 µg/plate with test strains TA 1535, TA 1537 and TA 98 and at a dose level of 160 µg/plate with TA 100 and WP2uvrA. In the absence of metabolic activation Genamin LAP 100 D proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 5 µg/plate and above. With tester strain TA 1535 toxicity was observed in a dose range of 16 to 50 µg/plate and with the strain WP2uvrA at a concentration of 50 µg/plate.

In the plate incorporation test with 30 % S9-mix with metabolic activation toxicity in bacteria was observed at a concentration of 160 µg/plate with all Salmonella strains. In the absence of metabolic activation the test compound proved to be toxic to bacterial strain TA 100 at a concentration of 16 µg/plate and above. With the other tester strains toxicity was observed in a dose range of 50 to 160 µg/plate.
Thinning of bacterial lawns and/or a reduction in the number of colonies (at least halving) was observed at these dose levels.

MUTAGENICITY
In the independent mutation tests Genamin LAP 100 D was tested for mutagenicity with the same concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.
Genamin LAP 100 D did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of S9-mix in each mutation test. No dose-dependent effect was obtained.
All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.
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 was not found to be mutagenic in the bacterial reverse mutation test.
Executive summary:

N-Dodecyl-1,3-diaminepropanewas tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 ofSalmonella typhimuriumand withEscherichia coliWP2uvrA.

Three independent mutagenicity studies were conducted (two plate incorporation tests with 10% S9-mix and one plate incorporation test with 30 % S9-mix), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For all studies, N-Dodecyl-1,3-diaminepropane was dissolved in ethanol, and each bacterial strain was exposed to 5 dose levels in the first plate incorporation test with 10% S9-mix, to 6 dose levels in the second plate incorporation test with 10% S9-mix and also to 6 dose levels in the plate incorporation test with 30 % S9-mix.

Other common vehicles (deionized water, DMSO) were not appropriate due to insolubility of the test compound. As ethanol had to be chosen as vehicle, preincubation was not possible because of vehicle toxicity and therefore, a modified plate incorporation test with 30% S9-mix was performed as confirmatory assay.

The concentrations used for the first plate incorporation test (10% S9-mix) were 50, 160, 500, 1600 and 5000 µg/plate. Because of toxicity in bacteria with the concentrations used in the first experiment, dose levels from 0.16 to 50 µg/plate were chosen in the absence of metabolic activation for all tester strains in the second test. In the presence of metabolic activation dose ranges were variable in the second test to account for varying susceptibilities to toxic effects of the bacterial strains: low dose levels ranged from 0.16 to 50 µg/plate (TA 1537, TA98), and high dose levels ranged from 0.5 to 160 µg/plate (TA 100, TA 1535 and WP2uvrA).

For the plate incorporation test with 30% S9-mix dose levels from 0.5 to 160 µg/plate with and without metabolic activation were chosen with all strains.

Visible precipitation of N-Dodecyl-1,3-diaminepropane on the plates was observed at 5000 µg/plate.

The number of revertant colonies of the solvent controls in individual strains were slightly out of the historical control data range with ethanol. However the existing control data pool with ethanol is limited as this solvent is used rarely. When compared to the historical control data pool with DMSO all values are in the normal range indicating that the validity of the study is not influenced.

 

Toxicity was assessed after microscopic thinning of the bacterial lawn and / or reduction of the number of mutants compared to the corresponding solvent control (at least halving).

In the first plate incorporation test with 10% S9-mix, toxicity in bacteria was observed with metabolic activation in a dose range of 50 to 5000 µg/plate with the test strains TA 1537 and TA 98. In the other test strains toxicity was observed with metabolic activation in a dose range of 160 to 5000 µg/plate. In the absence of metabolic activation the test compound proved to be toxic to all bacterial strains at concentrations of 50 µg/plate and above. As less than 5 doses were evaluable with all strains due to toxicity in bacteria a second test with lower doses was performed.

In the second plate incorporation test with 10 % S9-mix toxicity in bacteria was observed with metabolic activation at a dose level of 50 µg/plate with test strains TA 1535, TA 1537 and TA 98 and at a dose level of 160 µg/plate with TA 100 and WP2uvrA. In the absence of metabolic activation N-Dodecyl-1,3-diaminepropane proved to be toxic to bacterial strains TA 100, TA 1537 and TA 98 at concentrations of 5 µg/plate and above. With tester strain TA 1535 toxicity was observed in a dose range of 16 to 50 µg/plate and with the strain WP2uvrA at a concentration of 50 µg/plate.

In the plate incorporation test with 30% S9-mix with metabolic activation toxicity in bacteria was observed at a concentration of 160 µ/plate with allSalmonella strains.In the absence of metabolic activation the test compound proved to be toxic to bacterial strain TA 100 at a concentration of 16 µg/plate and above. With the other tester strains toxicity in bacteria was observed in a dose range of 50 to 160 µg/plate.

 

In the presence and in the absence of the metabolic activation systemN-Dodecyl-1,3-diaminepropanedid not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated thatN-Dodecyl-1,3-diaminepropane is not mutagenicin this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation system.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to
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, (828) 264-9099 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of 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
Justification for type of information:
Further information on the applicability of the read-across from various diamines to C12/14-diamine can be obtained from the document "Category polyamines - 20170314.pdf" added to IUCLID Ch. 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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/10
6

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.

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 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol.
Justification for type of information:
Further information on the applicability of the read-across from various diamines to C12/14-diamine can be obtained from the document "Category polyamines - 20170314.pdf" added to IUCLID Ch. 13.
Qualifier:
according to
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 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 andphospholipid 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

Applicability cross-reading:

On the substance N-C12,14-alkyl-1,3-diaminopropane (CAS 90640-43-0, also referred to as C12-14-diamine) itself no data is available.

Use is made of cross-reading, using studies from N-Dodecyl-1,3-diaminepropane (CAS 5538-95-4), also referred as C12-Diamine, N-Coco-1,3-diaminopropane (CAS 61791-63-7, recently redefined as Amines, N-C12-18-alkyltrimethylenedi-, with CAS 68155-37-3), also referred to as Coco-diamine, and N-Oleyl-1,3-diaminepropane ((Z)-N-9-octadecenyl-1,3-diaminopropane, CAS 7173-62-8), futher also referred to as Oleyl-diamine.

C12-14-diamine consists for about 70% of C12-diamine and 30% C14-diamine. The data from C12-diamine are therefore fully relevant for the evaluation of C12-14-diamine.

C12-14-diamine and Coco-diamine are also very similar, sharing the same structure of an alkyl-diamine, but with some difference in variation in alkyl-chain length variation. But the average chain-length is almost the same for both substances, 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:

N-Dodecyl-1,3-diaminepropane (C12-diamine) was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA in accordance to OECD guideline 471 and under GLP. Adequate toxicity was observed, and appropriate reference mutagens produced significant increases in the number of revertant colonies, demonstrating the sensitivity of the assay. In both the presence and in the absence of the metabolic activation system, C12-diamine did not result in relevant increases in the number of revertants in any of the bacterial strains.

 

This result was confirmed in a test with Coco-diamine, also in accordance to OECD guideline 471 and under GLP. Coco-diamine was tested in the Ames Salmonella/microsome plate test up to 33.3 µg/plate in 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 was therefore considered to be not mutagenic in this testsystem.

 

C12-diamine was further tested in an in vitro mammalian 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 lung in 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 S9-mix.

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 from in vitro mammalian 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 an in vivo micronucleus 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 three in vivo estimations 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 Systems In 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 Systems In 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-C12,14 alkyl-1,3-diaminopropane indicate that genotoxic properties are rather unlikely.