Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assays, in vitro micronucleus assay and in vitro mammalian cell gene mutation test are available with oleyl diamine dioleate. All showed negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Aug-2011 to 21-Apr-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test, (exp 1):
Without S9-mix, 3 exposure; 24 hr fixation: 0.05, 0.17, 0.51, 1.7 and 5.1 µg/mL
With S9-mix, 3 exposure; 24 hr fixation: 0.51, 1.7, 5.1, 17 and 51 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 0.02, 0.05, 0.17, 0.51 and 1.7 µg/mL
Dose range finding test, (exp 2):
Without and with S9-mix, 3 exposure; 24 hr fixation: 1, 3, 10, 20 and 30 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 0.3, 1, 3, 5 and 10 µg/mL
Dose range finding test, (exp 3):
Without S9-mix, 24 exposure; 24 hr fixation: 3, 10, 20, 25 and 30 µg/mL
Dose range finding test, (exp 4):
Without S9-mix, 24 exposure; 24 hr fixation: 1, 10, 30, 50 and 100 µg/mL
First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr fixation: 5, 15, 20, 22 and 24 µg/mL
With and with S9-mix, 3hr exposure; 27 hr fixation: 10, 20, 28 and 30 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 24 and 30 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:

Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period

Positive control substance:

other: colchicine: of 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure period.

Remarks:

without S9

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: No precipitation was observed up to the highest tested dose level of 100 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- in the absence and presence of S9, 3 hr treatment/24 hr fixation: Toxicity was observed at dose levels of 20 µg/ml and above ;
- in the absence of S9 for the continuous treatment of 24 hr: the cytokinesis-block proliferation index could not be determined since the cytoplasm of the cells could not be observed, it was clear that at the concentrations of 30, 50 and 100 µg/ml the cells were lysed and that these concentrations were therefore cytotoxic.


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

It is concluded that this test is valid and that Oleyl diamine, dioleate is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemical cyclophosphamide produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical mitomycin C produced a statistically significant increase in the number of binucleated cells with micronuclei in the second cytogenetic assay and in the first cytogenetic assay in one of the duplicate cultures. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Oleyl diamine, dioleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 08 - November 07, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 µg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 0.01, 0.1, 0.3, 1 and 3.3 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.01, 0.05, 0.1, 0.5, 1, 2, 3 and 4 µg/mL
With S9-mix, 3 hours treatment: 1, 10, 20, 25, 30, 35, 40 and 45 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.05, 0.1, 1, 1.5, 2, 2.5, 3 and 3.5 µg/mL
With S9-mix, 3 hours treatment: 0.1, 1, 10, 20, 30, 40, 45 and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is = 50 per 10^6 survivors and = 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation in the exposure medium was observed in the main experiments.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity:
3 hours treatment: In the absence of S9-mix, the relative suspension growth was 26% at the test substance concentration of 3 µg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 10 µg/ml and above. In the presence of S9-mix, the relative suspension growth was 51% at the test substance concentration of 10 µg/ml compared to the relative suspension growth of the solvent control. Hardly any to no cell survival was observed at test substance concentrations of 33 µg/ml and above.
24 hours treatment: In the absence of S9-mix, the relative suspension growth was 94% at the test substance concentration of 1 µg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at a test substance concentration of 3.3 µg/ml

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First experiment: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 82% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 81% compared to the total growth of the solvent controls.
Second experiment: Since the relative suspension growth (after 48 hours cell culture) was 19% at the test substance concentration of 3.5 µg/ml in the absence of S9-mix, this dose level was selected as highest dose level to be tested. High toxicity was observed resulting in a reduction of 97% in the relative total growth compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 83% compared to the total growth of the solvent controls.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Summary tables see the attached background material.

Evaluation of the mutagenicity:

No significant increase in the mutation frequency at the TK locus was observed after treatment with Oleyl diamine, dioleate either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Oleyl diamine, dioleate treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

Conclusions:

Oleyl diamine, dioleate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study.

Executive summary:

Evaluation of the mutagenic activity of Oleyl diamine, dioleate in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat).

 

This report describes the effects of Oleyl diamine, dioleate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch 30411 of Oleyl diamine, dioleate was a brown-yellow very viscoud liquid with a purity of 99.32% weight. The test substance was dissolved in ethanol. Oleyl diamine, dioleate concentrations were prepared directly prior to use.

 

A range finding study was performed to establish the solubility of Oleyl diamine, dioleate and to set concentrations for the subsequent mutation experiments. Oleyl diamine, dioleate precipitated in the exposure medium at a test substance concentration of 100 µg/ml. Oleyl diamine, dioleate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data. To obtain more information about the toxicity of Oleyl diamine, dioleate a second dose range finding study was performed for the 24 hours exposure time.

 

In the first experiment, Oleyl diamine, dioleate was tested up to concentrations of 4 and 45 µg/ml in the absence and presence of 8% (v/v) S9-mix, respectively. The incubation time was 3 hours. Appropriate toxicity was observed at these dose levels in the absence of S9-mix. Oleyl diamine, dioleate was tested up to cytotoxic levels of 18 and 19% in the absence and presence of S9-mix, respectively.

 

In the second experiment, Oleyl diamine, dioleate was tested up to concentrations of 3.5 and 50 µg/ml, in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Appropriate toxicity was observed at these dose levels in the absence of S9-mix. Oleyl diamine, dioleate was tested up to cytotoxic levels of 3 and 17% in the absence and presence of S9-mix, respectively.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 11- and 15-fold for MMS in the absence of S9-mix, and by 9.1- and 11-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Oleyl diamine, dioleate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Oleyl diamine, dioleate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

 

It is concluded that Oleyl diamine, dioleate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2003 - 5 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver microsomes

Test concentrations with justification for top dose:

Preliminary test: with and without S9: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
Precipitate seen at = 2500 µg/plate
Experiment 1 and 2: All tester strains in triplicate:
Range of dose levels up to toxicity involved:
3.91, 7.81, 15.6, 31.3, 62.5, 93.75, 125, 250, 375 and 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation) for preliminary test, both experiments without S9 mix and the first experiment with S9 mix
- preincubation method: Second experiment with S9-mix: 60 minutes at 37°C before plating

DURATION
- Exposure duration: 48 to 72 hours of incubation at 37°C,

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

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

Acceptance criteria:
. the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.
Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (ethanol) at 100 mg/mL.
Consequently, with a treatment volume of 50 µL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
A marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels = 2500 µg/plate, toward the three strains used.
A moderate to marked toxicity was noted at dose-levels = 100 µg/plate in the TA 98 and TA 102 strains without S9 mix and at dose-levels = 500 µg/plate in the TA 98 and TA 102 strains with S9 mix and in the TA 100 strain, with and without S9 mix.

MUTAGENICITY EXPERIMENTS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix, a moderate to marked toxicity was generally noted at dose-levels = 31.3 µg/plate in the TA 1537 strain, = 62.5 µg/plate in the TA 1535, TA 98 and TA 102 strains, and = 125 µg/plate in the TA 100 strain.
With S9 mix, a moderate to marked toxicity was generally observed at dose-levels = 125 µg/plate in the TA 1537 strain, = 250 µg/plate in the TA 100 and TA 102 strains, at 375 µg/plate in the TA 98 strain, and at 500 µg/plate in the TA 1535 strain.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Test substance Oleyl diamine, dioleate was found to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The objective of this study was to evaluate the potential of the test item INIPOL 002 (batch No. 11638106, purity: 97.40%) to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of INIPOL 002 to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item INIPOL 002 was dissolved in ethanol.

The dose-levels of the positive controls were as follows:

without S9 mix:

• 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

• 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

• 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

• 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

with S9 mix:

• 2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

• 10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix, the selected treatment-levels ranged from 3.91 to 500 µg/plate.

A moderate to marked toxicity was generally noted at dose-levels = 31.3 µg/plate in the TA 1537 strain, = 62.5 µg/plate in the TA 1535, TA 98 and TA 102 strains, and = 125 µg/plate in the TA 100 strain.

With S9 mix, the selected treatment-levels ranged from 15.6 to 500 µg/plate.

A moderate to marked toxicity was generally observed at dose-levels = 125 µg/plate in the TA 1537 strain, = 250 µg/plate in the TA 100 and TA 102 strains, at 375 µg/plate in the TA 98 strain, and at 500 µg/plate in the TA 1535 strain.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

Under our experimental conditions, the test item INIPOL 002 (batch No. 11638106, purity: 97.40%) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oleyl-diamine dioleate was tested in the bacterial reverse mutation test according to OECD 471 and in compliance to GLP. The test item was then tested in two independent experiments, with and without a metabolic activation system, from a liver microsomal fraction of Aroclor 1254 induced rats. Both experiments were performed according to the direct plate incorporation method except for the second test with S9-mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of Salmonella typhimurium, TA1535, TA1537, TA98, TA100 and TA 102 were exposed to five dose-levels of the test item in triplicate.

A moderate to marked toxicity was generally observed at dose-levels from 31.3 µg/plate to 125 µg/plate without S9 and from 125 µg/plate to 500 µg/plate in the presence of S9-mix. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

 

A second older study also evaluated Oleyl-diamine dioleate in the bacterial reverse mutation according to OECD 471. In this study the evaluation was performed on S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100, in two independent experiments, with and without a S9-mix from Aroclor 1254 induced rat liver microsomes. Both experiments were performed according to the direct plate incorporation method. Each strain was exposed to five dose-levels 5000, 1500, 500, 150, and 50 µg/plate of the test item, and a solvent control, with three plates/dose-level.

Oleyl diamine, dioleate was found to be slightly toxic towards the tester strains at the highest dose level. No substantial increases in revertant colony numbers of any of the five tester strains were observed, either in the presence or absence of metabolic activation (S-9 mix).

 

Oleyl-diamine dioleate was recently assessed for its potential to induce gene mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells according to OECD 476 guidelines and in compliance to GLP. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). Oleyl diamine, dioleate precipitated in the exposure medium at a test substance concentration of 100 µg/ml. Oleyl-diamine dioleate was tested beyond the limit of the solubility to obtain adequate cytotoxicity data.

In the first experiment, Oleyl-diamine dioleate was tested up to concentrations of 4 and 45 µg/ml for 3 hours with and without S9-mix. Oleyl diamine, dioleate was tested up to cytotoxic levels of 18 and 19% in the absence and presence of S9-mix, respectively.

In the second experiment, Oleyl-diamine dioleate was tested up to concentrations of 3.5 and 50 µg/ml, in the absence and presence of S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Oleyl-diamine dioleate was tested up to cytotoxic levels of 3 and 17% in the absence and presence of S9-mix, respectively.

The positive and negative controls showed acceptable response.

In both experiments, Oleyl-diamine dioleate did not induce a significant increase in the mutation frequency with or without S9-mix.

 

Oleyl-diamine dioleate was further evaluated for possible clastogenicity and aneugenicity in an in vitro micronucleus assay in cultured peripheral human lymphocytes (with independent repeat) in the presence and absence of a metabolic activation system. The study was performed according to OECD 487 and in compliance to GLP. The results of the positive and negative controls indicate that the conditions of the test were adequate.

Oleyl diamine, dioleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Justification for classification or non-classification

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available. None of the studies indicate a potential concern for genotoxicity for Oleyl-diamine dioleate. Consequently, Oleyl-diamine dioleate need not be classified for genotoxicity.