Amines, C36-alkylenedi-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data is available from C36-alkylenediamine itself and from data based on Octadecenylamine (Oleylamine). Cross-reading from this substance is acceptable on the basis of similarities of structure with same functional groups, properties leading to common biological activity, and common metabolic degradation. For full justification see document “Justification in support of cross-reading from primary fatty amines (PFA) to Dimerdiamine”. C36-alkylenediamine did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Negative results for mutagenicity of Octadecenylamine ((Z)-octadec-9-enylamine, CAS No. 112-90-3) were obtained in a valid L5178Y TK+/- mouse lymphoma assay in the presence and absence of metabolic activation. No increases in mutation frequency as compared to solvent controls were found. Additionally, (Octadecenylamine did also not induce chromosomal aberrations in a valid cytogenetic study in vitro in CHO cells in the presence and absence of aroclor 1254 induced rat liver S9-mix.

Link to relevant study records

Reference

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:

21-Jun-2012 to 09-Jul-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 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:

- 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 a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate
Experiment 2:
Without and with S9-mix: 3, 10, 33, 100, 333 and 1000 µg/plate

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:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

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

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat 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 independently repeated experiment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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
- Precipitation: Precipitation was observed at dose levels of 3330 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 1000 μg/plate and above in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 333 µg/plate and above and with S9: 333 µg/plate and above
TA1537: without S9: 100 µg/plate and above and with S9: 333 µg/plate and above
TA98: without S9: 333 µg/plate and above and with S9: 333 µg/plate and above
TA100: without S9: 333 µg/plate and above and with S9: 333 µg/plate and above
WP2uvrA: without S9: 1000 µg/plate and above and with S9: 1000 µg/plate and above

Conclusions:

Interpretation of results (migrated information):
negative

Amines, C36-alkylenedi- is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Amines, C36-alkylenedi- did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

C36-alkylenediamine has not been found mutagenic in the Ames test.

Cross-reading can be done to data obtained on alkyl amines; most specifically on Octadecenylamine (Oleylamine) as C36-alkylenediamine, which can best be described as two molecules of Octadecenylamine that are linked together in the middle of the alkyl chain. With respect to toxicological properties of the dimerdiamine, the most relevant functional groups are the two primary amines, and the remaining large alkyl part with some level of unsaturation. As such it is not basically different from Octadecenylamine itself. Cross-reading to Primary alkyl amines can therefore be considered on the basis of similarities of structure with same functional groups, leading to common biological activity, and common metabolic degradation. For full justification see document “Justification in support of cross-reading from primary fatty amines (PFA) to Dimerdiamine”.

Also on the basis of results on Octadecenylamine used for read-across, no genotoxcicity is to be expected from C36-alkylenediamine.

 

Based on structure and mechanism of cytotoxicity, genototoxicity is also not expected. The cationic surfactant structure of C36-alkylenediamine leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and to move to the nucleus to interact with DNA.

 

Selecting in QSAR Toolbox all primary amines (from OECD HPV profile) (total 1750 selected), and removing all compounds that are not discrete chemical and having other atoms besides carbon and nitrogen results to 306 relevant primary amines. From these there are 764 genotoxicity data points reported belonging to 68 of these subcategorized substances. Evaluation of all mutagenicity related data (608 data points of the 763), there was only one positive mutagenic result present, belonging to naphthylethylenediamine. This indicates a lack of mutagenic properties for the selected category of chemicals.

 

Also supporting the lack of genotoxic properties comes from the profiling ofC36-alkylenediamine (QSAR Toolbox v.3.0). There are no alerts are found for DNA interaction.

Information from QSARs also showed no indication for mutagenicity:

- VEGA (Mutagenicity modelsCAESAR version 2.1.10; SarPy model, version 1.0.5-BETA): Predicts non-mutagenic, both with moderate reliability, with the indication could be out of the Applicability Domain of the model.

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

- TOPKAT (Accelrys ADMET Toxicity Prediction (Extensible)) predicts non-mutagen, with a probability for mutagenic ofC36-alkylenediamineactivity of 0%.

 

Justification for selection of genetic toxicity endpoint
For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity one study is available on the basis of testing with C36-alkylenediamine itself or from cross-reading.

Justification for classification or non-classification

Available studies show no concern for possible genotoxicity. Also further property data for C36-alkylenediamine indicate that genotoxic properties are rather unlikely.