C16-18-(even numbered)-alkyl fatty acid, compound with C16-18-(even numbered)-alkylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the registered salt:

-         a negative ames test (bacterial reverse mutation assay) is available;

For the other tests, a read across was applied from other diamine compounds:

-         Z-octadec-9-enylamines (CAS: 112-90-3) was tested for genetic toxicity in three in vitro tests (bacterial reverse mutation assay, mammalian cell gene mutations (HPRT) & chromosomal aberration test). All were negative;

-         octadecylamine (CAS: 124-30-1) and coco alkylamine (CAS: 61788-46-3)exhibited[GI1] a negative ames test.

Link to relevant study records

Reference

Reference 1

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:

11 January 2013 - 04 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

31.3, 62.5, 125, 250 and 500 µg/plate.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice: vehicle providing an homogenous suspension at 10 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

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

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 was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for TA 1535 and TA 98 only at 500 µg/plate

Vehicle controls validity:

valid

Untreated 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

Remarks:

only at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial 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 pre-incubation 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 was suspended in ethanol.

 

Results

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Since the test item was poorly soluble in the vehicle and non-toxic in the preliminary test, the highest dose-level selected for the main experiments was the highest achievable one.

 

The selected treatment-levels were 31.3, 62.5, 125, 250 and 500 µg/plate for the five strains in both mutagenicity experiments, with and without S9 mix.

 

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 250 µg/plate in the TA 1535 and TA 1537 strains and at 500 µg/plate in the TA 98, TA 100 and TA 102 strains.

 

Without S9 mix, a moderate toxicity was noted at 500 µg/plate in the TA 1535, TA 98 and TA 102 strains. With S9 mix, a moderate toxicity was only noted at 500 µg/plate in the TA 98 strain, in the second experiment.

 

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

 

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence ofa rat liver metabolizing system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

09 Jan - 02 Apr 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to acceptable standards including GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate: from adult male Sprague-Dawley rats that had been injected with Aroclor 1254 at 500 mg/kg bw ; 5 days after injection, the livers were excised and prepared

Test concentrations with justification for top dose:

without metabolic activation: 0.05, 0.15, 0.5, 1.5, 5 nL/mL
with metabolic activation: 0.2, 0.6, 2, 6, 20 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: triethylenemelamine and cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period in growth medium: 18-24 h
- Exposure duration: without S9 mix: 16 h; with S9 mix: 2 h
- Expression time (cells in growth medium): 14 h (only for the activated cells)
- Fixation time (start of exposure up to harvest of cells): 18.5 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: all assays in duplicate

NUMBER OF CELLS EVALUATED: 100 cells (50 cells/duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxic effects of treatment are expressed relative to the solvent-treated control (relative cell survival).

Evaluation criteria:

Chi-square-analysis using 2x2 contingency table was used to ascertain significant differences between the number of cells with aberrations in the treatment and control groups.
The number of cells with chromosome aberrations in the positive control must be at least 25% of the cells scored.

Statistics:

chi-square-test

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation: at 5 nL/mL; with metabolic activation: at 20 nL/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material octadecenylamine was not mutagenic in an in vitro chromosomal aberration test in CHO cells either with or without exogenous metabolic activation.

Executive summary:

Octadecenylamine (ODA-FG-11-27-84) did not induce chromosomal aberrations in CHO cells in the absence and presence of aroclor 1254 induced Sprague dawley rat liver S-9 mix. Cells were treated with0.05to 5 nl/ml of the test substance in the absence of S-9 mix and with 0.2 to 20 nl/ml in the presence of S-9 mix. Cell survival was reduced to 24% with and without S9 mix at the highest concentrations tested. The positive and negative controls fullfilled the requirements for a valid test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

05 May - 26 Sep 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to acceptable standards including GLP

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9-mix

Test concentrations with justification for top dose:

initial tests:
without metabolic activation: 0.13-10 nL/mL
with metabolic activiation: 1.3-100 nL/mL

confirmatory test.
without metabolic activation: 0.2-2 nL/mL
with metabolic activation: 1.5-15 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

aceton

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate in DMSO (nonactivated), 7,12-dimethylbenzanthracene in DMSO (activated)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): trifluorthymidine (final concentration: 4 µg/mL)

NUMBER OF REPLICATIONS: two initial assays (using single cultures per dose) were performed to achieve the desired range of toxicity and one confirmatory assay (using duplicate cultures per dose) was carried out

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined by comparing the cell population growth at each dose level with that of the solvent controls.

Evaluation criteria:

Positive - if there is a positive dose response and one or more of the 3 highest doses in the 10% or greater Total Growth range exhibit a mutant frequency wich is two-fold greater tahn the background level. All data including that from cultures with less than 10% Total Growth will be used to establish the dose response relationship. The first assay and the confirmatory assay must both demonstrate a positive response to call a test article a positive mutagen.

The spontaneous mutant frequency of the solvent control must be between 0.2 and 1.0 per 10000 surviving animals.
The plating efficiency of the solvent controls must be greater than 50%.
Mutant frequency of the positive control at least twice that of the solvent control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100% toxicity at 100 nL/mL (with metabolic activation); 100% toxicity at 10 nL/mL (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Confirmatory assay:
None of the nonactivated cultures that was cloned exhibited a mutant frequency which was two times the mean mutant frequency of the solvent controls. The total growths of these cultures ranged from 6% to 109%. A dose dependent response was not noted in the treated cultures. None of the S9 activated cultures that was cloned showed a mutant frequency which was twice the mean mutant frequency of the solvent controls. The total growth of these cultures ranged from 2% to 115%. A dose dependent response was not noted in the treated cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the study results the test article oleylamine was negative in both the presence and absence of exogenous metabolic activation in this Mouse Lymphoma Mutagenesis Assay.

Executive summary:

The test article Oleylamine was tested in the L5178Y TK+/- Mouse Lymphoma Mutagenesis Assay in the presence and absence of Aroclor induced rat liver S-9. Two initial and one confirmatory assays were conducted.

The nonactivated cultures selected for cloning in the first initial assay were treated with doses of 0.32 to 0.13 nl/ml and exhibited total growths from 75% to 99%. The S-9 activated cultures selected for cloning in the first initial assay were treated with doses of 10 to 1.3 nl/ml which produced from 6% to 106% total growths. None of the nonactivated or S-9 activated cultures that was cloned exhibited a mutant frequency which was at least twice the mean mutant frequency of the solvent controls. A dose dependent response was not noted in the treated cultures.

The nonactivated cultures selected for cloning in the second initial assay were treated with doses of 1.8 to 0.13 nl/ml and exhibited total growths from 3% to 110%. The S-9 activated cultures selected for cloning in the second initial assay were treated with doses of 13 to 1.3 nl/ml which produced from 13% to 107% total growths. None of the nonactivated or S-9 activated cultures that was cloned exhibited a mutant frequency which was at least twice the mean mutant frequency of the solvent controls. A dose dependent response was not noted in the treated cultures.

The nonactivated cultures selected for cloning in the confirmatory assay (duplicate cultures) were treated with doses of 1.0 to 0.2 nl/ml and exhibited total growths from 6% to 109%. The S-9 activated cultures selected for cloning in the confirmatory assay were treated with doses of 11 to 1.5 nl/ml which produced from 2% to 115% total growths. None of the nonactivated or S-9 activated cultures that was cloned exhibited a mutant frequency which was at least twice the mean mutant frequency of the solvent controls. A dose dependent response was not noted in the treated cultures.

The results indicate that, under the conditions of these mutagenicity tests, the test article Oleylamine was negative in both the presence and absence of exogenous metabolic activation in the Mouse Lymphoma Mutagenicity Assay.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

17 Dec 1984 - 22 Jul 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to acceptable standards including GLP

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl-transferase locus

Species / strain / cell type:

other: Chinese hamster Ovary (CHO-K2-BH4 cells)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate: from adult male Sprague-Dawley rats that had been injected with Aroclor 1254 at 500 mg/kg bw ; 2 days after injection, the livers were excised and prepared

Test concentrations with justification for top dose:

first assay:
without metabolic activation: 0.1, 0.5, 1.0, 1.5, 2.0 nL/mL
with metabolic activation: 9.0, 8.0, 7.0, 6.0, 5.0 nL/mL

second assay:
without metabolic activation: 2.0, 1.5, 1.0 nL/mL
with metabolic activation: 10.0, 9.5, 9.0, 8.0, 7.0 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate and benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS:
preliminary toxicity test: single culture per treatment
mutation assay: duplicate cultures per treatment

NUMBER OF CELLS EVALUATED: cloning efficiency: 100 cells; mutation assay: 2000 cells or 10000 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The assay is considered positive in the event a dose-dependent increase in mutation frequency is observed with one or more of th four concentrations tested inducing a mutation frequency which is at least twice that of the solvent control. The assay is considered suspect if there is no dose response but one or more doses induce a mutation frequency which is at least twice that of the solvent control.
The positive control must induce a mutation frequency at least three time that of the solvent control.

Species / strain:

other: Chinese hamster Ovary (CHO-K2-BH4 cells)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

=2.25 nL/mL (without S9); =10 nL/mL (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material is considered to be negative in the hgpr test either with or without exogenous metabolic activation.

Executive summary:

Mutagenicity of octadecenylamine (ODA-FG-11 -27 -84) at the hprt locus was investigated in Chinese hamster ovary cells at concentrations of 0.1 to 2.0 nl/ml without S-9 mix and of 5.0 to 10 nl/ml with S-9 mix. No relevant cytotoxicity (decrease in cloning efficiency) was found for the analysed concentrations; without S-9 mix concentrations higher than 2.0 nl/ml led to strong cytotoxicity so that cloning was not successful. In general, there was no increase in mutation frequencies after treatment, with the exception of the highest concentrations of 2.0 nl/ml (without S-9 mix) and 9.0 nl/ml (with S-9 mix) in the first experiment. Since no genetic effects were seen at lower concentrations and the second experiments were clearly negative with and without S-9 mix, these increased mutation frequencies can be interpreted as outliers (due to the low statistical power of this test system). Altogether, the test result is negative.

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An Ames test is available with the registered part. Since the negative result is in line with those observed with the primary alkyl amine compounds, a cross-reading could be applied for the in vitro mammalian tests (i.e. Chromosomal aberration and cell gene mutation tests) which show negative results.

 The bacterial reverse mutation assay available with the registered substance was not mutagenic with or without S9 mix in each of the five tester strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102). These results were confirmed in an independently repeated experiment.

                             

For the other tests, a read across was applied from other diamine compounds.

 

Z-octadec-9-enylamines (CAS: 112-90-3) was tested for genetic toxicity in threein vitrotests. In a bacterial reverse mutation assay the substance was not mutagenic with or without S9 mix. It was also tested for mammalian cell gene mutations at the HPRT locus using V79 cells of the Chinese hamster or in the L5178Y TK+/- Mouse Lymphoma and was not mutagenic. In addition, this primary alkyl amines was tested in a chromosomal aberration test in Chinese hamster ovary cells and was found to be not clastogenic. All tests were to current OECD/EU protocols carried out to GLP. Based on these results it can be concluded that Z-octadec-9-enylamines (CAS: 112-90-3) is not to be expected to be a genotoxic hazard to human health.

Other primary alkyl amine compounds such as, tallow alkylamine (CAS: 61790-33-8), octadecylamine (CAS: 124-30-1) and coco alkylamine (CAS: 61788-46-3) did not induce a significant dose-related increase in the number of revertant (His+) colonies in different tester strains (TA1535, TA1537, TA98, TA1538 or TA100) and/or in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

Justification for classification or non-classification

Based on the available negative in-vitro tests for genotoxicity, the registered substance does not require classification as a mutagen according to the criteria laid down in EU regulation (EC) n°1272/2008 (CLP).