Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Oleyl-diamine mono-oleate: bacterial mutagenicity (Ames) negative

Oleic acid, like other natural fatty acids is considered to be devoid of genotoxic properties.

Both Oleyl-diamine (CAS 7173-62-8) andOleyl-diamine dioleate (CAS 34140-91-5)were negative inin vitrogenotoxicity studies involving bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2018 - 06 Augustus 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Direct plate assay
Dose-range finding test (without and with S9 (5% (v/v)); tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without S9 and with S9 (5% (v/v)); tester strains TA1535, TA1537 and TA98): 3.1, 6.3, 12.5, 25, 50, 250 μg/plate
Additonal experiment (without S9; Tester strain TA98): 0.78, 1.6 μg/plate

Second experiment (without S9; tester strains TA100, TA1535, TA1537, TA 98): 1.6, 3.1, 6.3, 12.5, 25 and 100 μg/plate
Second experiment (with S9 (10% (v/v)); tester strains TA100, TA1535, TA1537, TA 98): 3.1, 6.3, 12.5, 25, 50 and 250 μg/plate
Second experiment (with and without S9(10% (v/v)); tester strain WP2uvrA): 6.3, 12.5, 25, 50, 250 and 600 μg/plate

Vehicle / solvent:

- Vehicle used: Ethanol
- Justification for choice of vehicle: a previously performed solubility test showed that the test item can be dissolved in ethanol.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; ICR-191

Remarks:

For details on positive control substances, see Table 1 and Table 2

Details on test system and experimental conditions:

Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The second experiment was performed to obtain more information about the possible mutagenicity of the test item in the absence and presence of 10% (v/v) S9-mix.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (1E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in ethanol and
either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, presence of microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 512 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 164 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
- Precipitation of the test item on the plates was not observed at the start of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 1600 and 5000 μg/plate in the first experiment.
- Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period of the second experiment.

CYTOXICITY
In the first and second experiment cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester
strains in the absence and presence of S9-mix.

- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges except the negative control response for TA98 in the absence of S9-mix in the second experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (44 revertant colonies) when compared against relevant historical control data (41 revertant colonies), the validity of the test was considered to be not affected.

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, N-(Oleyl alkyl)-1,3- propanediamine mono oleate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Armolube 211 was tested in the Salmonella typhimurium (S.typhimurium; TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA) reverse gene mutation assay by direct plate incorporation method followed by an independent repeat.

Armolube 211 was a yellow paste. Ethanol was used as vehicle.

The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Comparable cytotoxicity was observed in the absence and presence of S9-mix.

Armolube 211 did not induce a significant dose-related increase in the number of revertant colonies in each of the tester strains both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusion: Armolube 211 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

2008-05-14 - 2008-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substance is a salt of (Z)-N-9-octadecenylpropane-1,3-diamine (Oleyl-diamine, CAS 7173-62-8) and Oleic acid (CAS 112-80-1), which dissociates into its components in aqueous environment, where the Oleyl-diamine will be present as a cationic charged structure under physiological pH (pKa = 10.7) and Oleic acid as anionic structure (pKa = 4,8). In aqueous conditions, no distinction can be made between solution of the Oleyl-diamine mono-oleate or solution of a mixture of Oleyl-diamine and oleic acid at the same molar concentration. Consequently, the evaluation of the individual components is just as relevant for the evaluation of genotoxicity as testing with the actual salt.
Oleic acid, like other natural fatty acids is considered to be devoid of genotoxic properties. Focus is therefore on the read-across from available data on Oleyl-diamine.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source (Oleyl-diamine) and target (Oleyl-diamine mono-oleate) only differ with respect to the co-presence of oleic acid. As oleic acid is of relative low toxicity, the overall toxicity is mostly based to the amount of Oleyl-diamine present. Additionally, available information from the same studies available on Oleyl-diamine dioleate (CAS 34140-91-5) for which basically the same argumentation applies, have demonstrated that the presence of oleic acid does not affect the outcome of the studies.

3. ANALOGUE APPROACH JUSTIFICATION
Toxicity of the substances is mostly driven by the Oleyl-diamine contents which has a relative higher toxicity than Oleic acid. This also applies for the cytotoxicity observed when comparing the relative toxicity of Oleyl-diamine and Oleyl-diamine dioleate.

4. DATA MATRIX
All relevant available data is included in this dossier.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimum essential medium) supplemented with 10% FCS (foetal calf serum)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix (induced with β-naphthoflavone and phenobarbital)

Test concentrations with justification for top dose:

Experiment I:
+S9: 0.5, 1.0, 2.0, 4.0, 5.0, 6.5 and 8.0 µg/mL
-S9: 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL

Experiment II:
+S9: 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 4.5 µg/mL
-S9: 0.05, 0.1, 0.2, 0.4, 0.55, 0.7, 0.85, 1.0 and 1.2 µg/mL

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 400 and 900 µg/mL ethylmethanesulphonate (EMS) and 0.83 µg/mL cyclophosphamide (CPA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in 2 independent experiments

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.5% (+S9) resp. 0.0% - 4.0% (-S9)
- the positive control substances should produce biologically relevant increases in the number of cells with structural chromosome aberrations

Criteria for determinig a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells (+S9) resp. 4.0% aberrant cells (-S9))

Statistics:

no data

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 1.0 µg/mL and higher (-S9) and 4.0 µg/mL and higher (+S9); Experiment II: at 0.4 µg/mL and higher (-S9) and 4.0 µg/mL and higher (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The concentration range for the Preliminary Toxicity Test was 0.0078 to 5000 µg/mL without metabolic activation and 1.95 to 5000 µg/mL with metabolic activation. The test item could be dissolved at a concentration of 500 µg/mL in ethanol. After dilution with cell culture medium, precipitation of the test item appeared in a concentration of 15.6 µg/mL and higher.
The selection of the concentrations used in experiment I and II based on data from the solubility and the pre-experiment which were performed according to guidelines. In experiment I (-S9) 1.2 µg/mL and (+S9) 4.0 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations. And in experiment II (-S9) 0.4 µg/mL and (+S9) 4.5 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.

COMPARISON WITH HISTORICAL CONTROL DATA: within historical reference range

Remarks on result:

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

Remarks:

Migrated from field 'Test system'.

Summary of aberration rates in experiment I

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	Mean % aberrant cells
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	4 h	20 h	2.0	0.0
S	0	4 h	20 h	4.5	1.5
5	0.85	4 h	20 h	3.5	1.0
6	1.0	4 h	20 h	0.5	0.0
7	1.2	4 h	20 h	0.5	0.0
EMS	900	4 h	20 h	11.5	8.5
with metabolic activation
C	0	4 h	20 h	5.0	3.5
S	0	4 h	20 h	2.0	1.5
2	1.0	4 h	20 h	4.5	1.5
3	2.0	4 h	20 h	4.5	2.0
4	4.0	4 h	20 h	3.5	1.0
CPA	0.83	4 h	20 h	10.5	9.0

Summary of aberration rates in experiment II

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	Mean % aberrant cells
				incl. Gaps	excl. Gaps
without metabolic activation
C	0	20 h	20 h	3.0	1.5
S	0	20 h	20 h	3.5	2.0
5	0.1	20 h	20 h	4.0	2.5
6	0.2	20 h	20 h	2.0	0.5
7	0.4	20 h	20 h	3.0	1.0
EMS	400	20 h	20 h	12.0	8.0
with metabolic activation
C	0	4 h	20 h	4.0	1.5
S	0	4 h	20 h	4.5	2.5
2	2.5	4 h	20 h	3.5	1.5
3	4.0	4 h	20 h	3.0	1.5
4	4.5	4 h	20 h	2.5	1.0
CPA	0.83	4 h	20 h	11.5	8.5

200 cells evaluated for each concentration

C: Negative control (culture medium)

S: Solvent control (Ethanol)

EMS: Positive control (-S9: ethylmethansulfonate)

CPA: Positive control (+S9: cyclophosphamide)

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item N-Oleyl-1,3-diaminopropane did not induce structural chromosome aberrations in the V79 Chinese hamster cell line. Therefore, the test item N-Oleyl-1,3-diaminopropane is considered to be non-clastogenic.

Executive summary:

The test item N-Oleyl-1‚3-diaminopropane was investigated for a possible potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation with S9 homogenate.
The selection of the concentrations used in experiment I and II based on data from the solubility test and the pre-experiment which were performed according to the guidelines.
In experiment I without metabolic activation 1.2 µg/mL and with metabolic activation 4.0 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations. In experiment II without metabolic activation 0.4 µg/mL and with metabolic activation 4.5 µg/mL were selected as highest dose groups for the microscopic analysis of chromosomal aberrations.
The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosomal aberrations.
The following concentrations were evaluated for microscopic analysis:
Experiment I:
with metabolic activation: 1.0, 2.0 and 4.0 µg/mL
without metabolic activation: 0.85, 1.0 and 1.2 µg/mL
Experiment II:
with metabolic activation: 2.5, 4.0 and 4.5 µg/mL
without metabolic activation: 0.1, 0.2 and 0.4 µg/mL
Precipitation:
No precipitation of the test item was noted with and without metabolic activation after the incubation at the concentrations evaluated.
Toxicity:
In experiment I without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 1.0 µg/mL and higher (48% at 1.0 µg/mL, 40% at 1.2 µg/mL). The cell density was not decreased. With metabolic activation a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at a concentration of 4.0 µg/mL (47% at 4 µg/mL). The cell density was also decreased at this concentration (61%).
In experiment II without metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 0.4 µg/mL (49%). The cell density was also decreased (65%). With metabolic activation, a biologically relevant decrease of the relative mitose index (decrease below 70% rel. mitose index) was noted at 4.0 µg/mL and higher (46% at 4.0 µg/mL, 30% at 4.5 g/mL). No decrease of the cell density was noted up to the highest dose evaluated.
Clastogenicity:
In the experiment without metabolic activation the aberration rates of the negative control (0.0%) and the solvent control (1.5%) were within the historical control data of the negative control (0.0% - 4.0%). The aberration rates of all dose groups evaluated were within the range of the historical control data. Mean values of 1.0% (0.85 µg/mL), 0.0% (1 and 1.2 µg/mL) aberrant cells were found.
In the experiment with metabolic activation the aberration rates of the negative control (3.5%) and the solvent control (15%) were within the historical control data (0.0% - 4.5%). The aberration rate of all dose groups evaluated were within the range of the historical control data. Mean values of 1.5% (1 µg/mL), 2.0% (2 µg/mL) and 1.0% (4 µg/mL) aberrant cells were found.
In experiment II without metabolic activation the aberration rate of the negative control (1.5%), the solvent control (2.0%) and all dose groups treated with the test item (2,5% (0.1 µg/mL, 0.5% (0.2 µg/mL) and 1.0% (0.4 µg/mL)) were within the historical control data of the testing facility (0.0% - 4.0%). With metabolic activation the aberration rates of the negative control (1.5%), the solvent control (2.5%) and all dose groups treated with the test item (1.5% (2.5 µg/mL), 1.5% (4.0 µg/mL) and 1.0% (4.5 µg/mL)) were within the historical control data of the testing facility (0.0% - 4.5%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control, In addition, no dose-response relationship was observed.
Polyploid cells
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

Reference 3

Endpoint:

in vitro gene mutation study in 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:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substance is a salt of (Z)-N-9-octadecenylpropane-1,3-diamine (Oleyl-diamine, CAS 7173-62-8) and Oleic acid (CAS 112-80-1), which dissociates into its components in aqueous environment, where the Oleyl-diamine will be present as a cationic charged structure under physiological pH (pKa = 10.7) and Oleic acid as anionic structure (pKa = 4,8). In aqueous conditions, no distinction can be made between solution of the Oleyl-diamine mono-oleate or solution of a mixture of Oleyl-diamine and oleic acid at the same molar concentration. Consequently, the evaluation of the individual components is just as relevant for the evaluation of genotoxicity as testing with the actual salt.
Oleic acid, like other natural fatty acids is considered to be devoid of genotoxic properties. Focus is therefore on the read-across from available data on Oleyl-diamine.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source (Oleyl-diamine) and target (Oleyl-diamine mono-oleate) only differ with respect to the co-presence of oleic acid. As oleic acid is of relative low toxicity, the overall toxicity is mostly based to the amount of Oleyl-diamine present. Additionally, available information from the same studies available on Oleyl-diamine dioleate (CAS 34140-91-5) for which basically the same argumentation applies, have demonstrated that the presence of oleic acid does not affect the outcome of the studies.

3. ANALOGUE APPROACH JUSTIFICATION
Toxicity of the substances is mostly driven by the Oleyl-diamine contents which has a relative higher toxicity than Oleic acid. This also applies for the cytotoxicity observed when comparing the relative toxicity of Oleyl-diamine and Oleyl-diamine dioleate.

4. DATA MATRIX
All relevant available data is included in this dossier.

Reason / purpose for cross-reference:

read-across source

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No need to perform in vivo studies.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genotoxicity by oleyl-diamine or oleic acid is not expected. Oleic acid, like other natural fatty acids is considered to be devoid of genotoxic properties.

Oleyl-diamine has in physiological circumstances 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

N-(Oleyl alkyl)- 1,3-propanediamine oleates- (Oleyl-diamine mono-oleate) is a salt of (Z)-N-9-octadecenylpropane-1,3-diamine (Oleyl-diamine, CAS 7173-62-8) and Oleic acid (CAS 112-80-1). Under aqueous conditions it dissociates into its components, and no distinction can be made between a solution of the Oleyl-diamine mono-oleate or a solution of a mixture of Oleyl-diamine and oleic acid at the same molar concentration. Consequently, the evaluation of the individual components is just as relevant for the evaluation of genotoxicity as testing with the actual salt. Oleic acid, like other natural fatty acids is considered to be devoid of genotoxic properties. Focus is therefore on the read-across from available data on Oleyl-diamine.

 

Oleyl-diamine (N-Oleyl-1,3-diaminopropane, CAS 7173-62-8), was tested in three in-vitro genotoxicity studies covering bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, which were all performed to current protocol and carried out to GLP with well-defined test substance. All three tests were negative, indicating that Oleyl-diamine is not genotoxic.

 

Oleyl-diamine dioleate,which only differs to Oleyl-diamine mono-oleate in that it has a higher ratio of Oleic acid to Oleyl-diamine (2:1 instead 1:1), was also testedin three in-vitro genotoxicity studies covering bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity, to current protocol and carried out to GLP with well-defined test substance. Also these studies were negative, proofing the presence of oleic acid does not impact the outcome of the evaluation.

 

Bacterial mutagenicity

Oleyl-diamine mono-oleate itself has only been tested for bacterial mutagenicity in the Salmonella typhimurium (S.typhimurium; TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA) reverse gene mutation assay by direct plate incorporation method followed by an independent repeat (OECD 471). 

The test substance was a yellow paste. Ethanol was used as vehicle. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Comparable cytotoxicity was observed in the absence and presence of S9-mix.

Results: There was no significant dose-related increase in the number of revertant colonies in each of the tester strains both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. It was concluded that Oleyl-diamine mono-oleate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Oleyl-diamine mono-oleate was not evaluated for genotoxic properties in mammalian cells. For this read-across to studies on Oleyl-diamine are marked as key studies, as this can be considered the main hazardous component of the salt.

 

Genotoxicity in mammalian cells:

Oleyl-diamine was investigated for a possible potential to induce structural chromosomal aberrations in Chinese hamster V79 cellsin vitroin the absence and presence of metabolic activation with S9 homogenate. The was performed under GLP according to OECD 473 guideline.

The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

- Experiment I: without S9: 0.85, 1.0 and 1.2 µg/mL; with S9: 1.0, 2.0 and 4.0 µg/mL

- Experiment II: without S9: 0.1, 0.2 and 0.4 µg/mL; with S9: 2.5, 4.0 and 4.5 µg/mL

No precipitation was noted at these evaluated concentrations.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

Without S9, cytotoxic effects were noted at concentrations of 1.0 µg/mL and higher in experiment I, and 0.4 µg/mL in experiment II. With S9, cytotoxic effects were seen at 4.0 µg/mL (both experiments) or higher.

Results: No biologically relevant increase in aberrant cells or in frequencies if polyploid cells was noted in any of the experiments. Oleyl-diamine is therefore considered to be non-clastogenic.

 

Mutagenicity in mammalian cells:

Oleyl-diamine was investigated for its potential to induce mutations at the HPRT locus using Chinese Hamster V79 cells in vitroin the absence and presence of metabolic activation with S9 homogenate. The was performed under GLP according to OECD 476 guideline.

Dose levels were based on data from the pre-experiments. Exposure duration was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. The following concentrations were evaluated:

- Experiment I: without S9: 0.350, 0.425, 0.500, 0.575, 0.650, 0.725, 0.800 and 0.875 µg/mL and with S9: 0.05, 0.10, 0.25,0.5,1.0, 3.0, 4.0 and 5.0 µg/mL

- Experiment II without S9: 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 µg/mL and with S9: 1.0, 2.0, 3.8, 4.2, 5.0, 5.5, 6.0 and 7.0 µg/mL

No precipitation was noted at these evaluated concentrations.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Cytotoxicity: Biologically relevant growth inhibition was observed in all experiments. Without metabolic activation the relative growth at the highest evaluated concentration was 13.6% (0.875 µg/mL) and 12.6% (0.9 µg/mL) in experiment I and II respectively. With metabolic activation this was 10.1% (5.0 µg/mL) and 16.5% (7.0 µg/mL) in experiment I and II respectively.

Results: In both experiments, no biologically relevant increase of mutants was found after treatment with the test item, with and without metabolic activation.

Justification for classification or non-classification

Oleyl-diamine mono-oleate is not mutagenic in the Ames test. Additionally, the most relevant constituent Oleyl-diamine isnegative inin vitrogenotoxicity studies involving bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity. The other constituent Oleic acid is as natural fatty acid also expected to be non-genotoxic. Further,Oleyl-diamine dioleate, with a higher level of Oleic acid was alsonegative in the standard battery ofin vitrogenotoxicity studies.

Consequently, no classification for genotoxic effects is required.