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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across is made with hydrogenated tallow alkylamines inside the primary alkylamines category.

Mutagenicity data on hydrogenated tallow alkylamines are not available. However, primary alkylamines in general have been tested in bacterial and mammalian cell systems. Uniformly, only negative results from tests on gene and chromosome mutations in mammalian cells in vitro as well as on chromosomal aberrations and micronuclei in vivo were revealed for this chemical category.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
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
Reason / purpose for cross-reference:
reference to same study
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:
other: In Vitro Mammalian Cell Gene Mutation Test
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.

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:
09 Jan - 26 Mar 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to acceptable standards including GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
- strain TA102 or E.coli WP2 was not tested
Principles of method if other than guideline:
Study was performed before actual guideline was adopted (21 Jul 1997).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate: from male Sprague-Dawley rats that had been injected with Aroclor 1254 at 500 mg/kg (Aroclor was diluted in corn oil 200 mg/mL); 5 days after injection, the livers were excised and prepared
Test concentrations with justification for top dose:
test 1:
with metabolic activation: 2, 10, 50, 100, 200 µg/plate
without metabolic activation: 0.2, 1, 5, 10, 20 µg/plate

test 2:
with metabolic activation: 2, 10, 25, 50, 100 µg/plate
without metabolic activation: 0.2, 1, 5, 10, 15 µg/plate
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: with metabolic activation: 2-aminoanthracene; without metabolic activation: 2-nitrofluorene, sodium azide, 9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 hours
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

Evaluation criteria:
For a test article to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article.
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with metabolic activation: 200 µg/plate; without metabolic activation: 20 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The results of a preliminary toxicity study of the test article conducted in the presence and absence of S9 mix indicated that the appropriate maximum dose level to be tested in the mutagenicity assay would be 200 µg/plate (with metabolic activation) and 20 µg/plate (without metabolic activation). However, in the mutagenicity experiments, cytotoxicity was observed at lower concentrations (test 1). Therefore, the experiment was repeated in a less toxic dose range (test 2).
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 proved to be negative in this Ames-test
Executive summary:

Octadecenylamine was not mutagenic in bacterial tester strains Salmonella typhimurium TA 98, TA 100, Ta1535, TA1537 and TA1538 in doses up to 20 µg/plate without metabolic activation and up to 200 µg/plate with metabolic activation (Arochlor induced rat liver S-9 mix).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan - 02 Apr 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to acceptable standards including GLP
Reason / purpose for cross-reference:
reference to same study
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
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
Reason / purpose for cross-reference:
reference to same study
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:
other: In Vitro Mammalian Cell Gene Mutation Test
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.

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:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, equivalent to guideline
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (S. typhimurium)
Tryptophan (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4, 20, 100 500, 2500, 10000 µg/plate (first experiment)
4, 20, 100 500, 2500, 5000 µg/plate (second experiment)
Vehicle / solvent:
- Vehicle/solvent used: ethanol
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: Sodium azide (TA 100, TA 1535), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA98, TA 1538), N-Methyl-N-nitro-N-nitrosoguanidine (WP2 uvrA); +S9: Benzo(a)pyrene, 2-Aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)



Species / strain:
other: all strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the test results it can be stated that Genamin 18 R 100 D is not mutagenic in the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA
Executive summary:

Genamin 18 R 100 D was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 and Escherichia coli WP2uvrA. The mutagneicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in literatur and comparable to historical controls. All the positive control compounds gave the expected increase in the number of revertant colonies. In the cytotoxicity trials, the test compound proved to be toxic to most of the bacterial strains at 100 microgram/plate. 5000 microgram/plate was chosen as top dose level for the mutagneicity studies. These tests demonstrated that in the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with genamin 18 R 100 D did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that Genamin 18 R 100 D is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

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:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (S. typhimurium)
Tryptophan (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4, 20, 100, 500, 2500, 10000 µg/plate (first experiment)
0.16, 0.8, 4, 20, 100, 500 µg/plate (second experiment)
Vehicle / solvent:
- Vehicle/solvent used: acetone
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: Sodium azide (TA 100, TA 1535), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA98, TA 1538), N-Methyl-N-nitro-N-nitrosoguanidine (WP2 uvrA); +S9: Benzo(a)pyrene, 2-Aminoanthracene (all strains)
Species / strain:
other: all strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

The test item was tested for its mutagenic potential in 5 Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA 1537, TA 1538) and 1 Escherichia coli strain (WP2 uvr A). The test substance proved to be very toxic to the strains at 20 µg/plate and to form precipitations at 500 µg/plate, the highest concentration tested in the second experiment.

Either with or without metabolic activation, the substance was not mutagenic in the present test system.

Conclusions:
Based on the study results it is concluded that the test material is not mutagenic in the bacterial test systems investigated either in the absence or in the presence of an exogenous metabolizing system (S-9).
Executive summary:

An investigation on induction of gene mutations in bacteria (OECD 471) was performed with coco alkylamines ("Genamin CC 100 D"). The test compound (purity approx. 100%) was solved in acetone and tested in doses of 0.16µg/plate up to 10 mg/plate with and without liver S-9 mix from Arochlor induced male Sprague Dawley rats. Only doses up to 100 µg/plate could be evaluated due to strain specific strong cytotoxic effects at doses of 20 or 100 µg/plate and higher doses. In TA1537 the bacterial background lawn was reduced already at 4µg/plate in the second experiment. Precipitaions were seen at 500 µg/plate and higher doses. No increases in the number of revertants were induced in any of the tester strains, e.g. Salmonella typhimurium TA100, TA1535, TA1537, TA1538, TA98 and E. coli WP2uvrA. Summarizing it can be stated that the test article genamin CC 100 D is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

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:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (S. typhimurium)
Tryptophan biosynthesis / deficiency in uvrA system for DNA repair (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4, 20, 100, 500, 2500, 10000 µg/plate (first experiment)
0.16, 0.8, 4, 20, 100, 500 µg/plate (second experiment)
Vehicle / solvent:
- Vehicle/solvent used: ethanol
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: Sodium azide (TA 100, TA 1535), 9-Aminoacridine (TA 1537), 2-Nitrofluorene (TA98, TA 1538), N-Methyl-N-nitro-N-nitrosoguanidine (WP2 uvrA); +S9: Benzo(a)pyrene, 2-Aminoanthracene (all strains)
Species / strain:
other: all strains tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Genamin TA 100 D is not mutagenic in the Ames-test .
Executive summary:

Genamin TA 100 D was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 0.16 or 0.8 microgram/plate or 2500 microgram/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in literature and were within historical controls. All the positive control compounds gave the expected increase in the number of revertant colonies. In the toxicity tests, the test compound proved to be very toxic to the bacterial strains at 100 microgram/plate. 500 or 2500 microgram/plate was chosen as top dose level for the subsequent mutagenicity study. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with genamin TA 100 D did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that Genamin TA 100 D is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read across is made with hydrogenated tallow alkylamines inside the primary alkylamines category.

In vivo mutagenicity studies on hydrogenated tallow alkylamines are not available. However, based on read-across no indications of a related potential exist from analogous compounds. A GLP-compliant bone marrow micronucleus test with tallow alkylamines (CAS No. 61790-33-8) in 50 Sprague Dawley rats led to a negative result after a single oral dose of 2000 mg/kg body weight.
Negative results are also reported from an in vivo chromosomal aberration test in mice. Administration of single doses up to 5000 mg/kg body weight in corn oil via gavage revealed no significant increases in aberrant cells although clinical signs of toxicity indicated that the test material was systemically available after oral application.

Link to relevant study records

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Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to acceptable standards including GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
Study was performed before actual guideline was adopted (21 Jul 1997).
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick , MD, USA
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: males: 28-38 g; females: 22-28 g
- Assigned to test groups randomly: The animals were assigned to 13 experimental groups of 5 males and 5 females each based on a computer-generated random number table.
- Housing: The animals were housed in plastic autoclavable cages with filter tops during quarantine and the experimental period. Hardwood chips were used for bedding.
- Diet (e.g. ad libitum): certified laboratory rodent chow which had been analysed for environmental contaminants; ad libitum
- Water (e.g. ad libitum): tap water; ad libitium

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25 °C
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): Sept1389J (Giant Foods, Washington, D.C., USA)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was dissolved in a total volume of 10 mL/kg bw.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
6, 12, or 24 h
Remarks:
Doses / Concentrations:
500, 2500, 5000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine: dissolved in sterile distilled water at a concentration of 0.05 mg/mL
- Route of administration: injection
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the preliminary dose-finding study, the doses for the main study were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Colchicine, used to arrest dividing cells at metaphase, was administered ip at 1 mg/kg to all mice two to four hours prior to sacrifice. Animals were sacrificed by carbon dioxide asphyxiation. Immediately following sacrifice, the femur was exposed and the bone marrow was aspirated.

DETAILS OF SLIDE PREPARATION:
The cells were centrifuged and resuspended in a fixative (methanol:acetic acid, 3:1, v/v). The cell suspension was dropped onto a wet glass slide and dried. 3 slides were prepared from each animal. The dry slides were stained with 5% Giemsa stain.

METHOD OF ANALYSIS:
A minimum of 50 metaphase cells containing 40±2 centromeres were examined from each animals and scored for chromatid-type and chromosome-type aberrations. Chromatid and isochromatid gaps were recorded but not included in the analysis. The mitotic index was recorded as the percentage number of cells in mitosis based upon 500 cells counted per animals.
Evaluation criteria:
The test article was considered to induce a positive response when the number of aberrant cells was significantly increased in a dose responsive manner relative to the vehicle control. A significant increase at the high dose only with no dose-response was considered suspect.
The percentage of cells in the solvent control group demonstrating aberrations of any type, other than gaps, must not exceed 4%. The cells with aberrations in the positive control group must be statistically increased (p=0.05, Fisher´s Exact Test).
Statistics:
Fisher´s Exact Test; Cochran-Armitage trend test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
One female receiving 2500 mg/kg died prior to colchicine administration.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 763, 1221, 1953, 3125, 5000 mg in corn oil/kg bw
- Clinical signs of toxicity in test animals: diarrhea, ruffled fur, crusty eyes and hunched posture (one female receiving 5000 mg/kg, one female receiving 3125 mg/kg, one female receiving 1221 mg/kg were found dead)
- Other: A single administration of 5000 mg/kg was selected as the high dose for the cytogenetics study.

RESULTS OF DEFINITIVE STUDY
- The marginal increases in percentage of aberrant cells observed in the high dose group were not statistically significant (p>0.05, Fisher´s exact test). In fact, the percentage of damaged cells was not significantly increased in the Oleylamine-treated animals, regardless of sex, dose or sacrifice time (p>0.05, Fisher´s exact test).
One female receiving 2500 mg/kg died prior to colchicine administration. No significant reduction in the rate of body weight gain was observed. Clinical observations in treated animals included diarrhea, piloerection, lethargy, crusty eyes and irregular breathings.
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the assay, Oleylamine at dose levels of 500, 2500 and 5000 mg/kg body weight was negative in the in vivo cytogenetic assay using male and female ICR mice. The negative and positive controls fulfilled the requirements for determination of a valid test.
Executive summary:

A chromosomal aberration test in mice bone marrow cells with octadecenylamine ("Oleylamine") was performed. Groups of five male and five female mice were administered single doses of 500, 2500 or 5000 mg/kg bw in corn oil by oral gavage at a volume of 10 ml/kg bw (purity >90%). Bone marrow cells were collected 6, 12 and 24 hours after treatment. No increases in percentages of aberrant cells were observed in the test substance treated animals regardless of dose or harvest time. One female in the 2500 mg/kg bw group died prematurely. No significant reduction in the rate of body weight gain was observed. Clinical signs of toxicity were observed in test substance-treated mice indicating that the test substance was systemically available after oral application. Based on the test results, oleylamine was negative in this in vivo mutagenicity assay. The concurrent negative and positive controls fullfilled the requirements for a valid test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999/2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study according to GLP
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River S.p.A., Claco, Italy
- Age at study initiation: 9 weeks
- Weight at study initiation: 187 - 257 g
TEST ANIMALS
- Source: Charles River S.p.A., Italy
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 187 - 275 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 hours peridocally
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: sesame oil

Details on exposure:
- Administered volume: 10 mL/kg
Frequency of treatment:
single treatment
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
limit test
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 15 mg/kg
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
2 slides/animal

ANALYSIS:
2000 polychromatic erythrocytes were scored for micronucleated cells. The ration of polychromatic to normochromatic erythrocytes (PCE/NCE) was calculated on 1 slide per animal by counting a total of 1000 PCEs.
Evaluation criteria:
Erythrocytes were classified as mature (normochromatic erythrocytes - NCE) or immature (polychromatic erythrocytes - PCE). The majority of micronuclei is circular, inside a PCE with normal morphology, where they are planar with the cytoplasm. A minority of micronuclei might be oval or almond-shaped, and a few are ring-shaped. Bodies with uncertain morphology or staining characteristics were not scored.

The test substance is considered to induce micronuclei if a statistically significant increase in micronucleus incidence (at p<0.05) is observed in any treatment group, in the pooled data for both sexes, or in the data for male or female groups alone. Where increases in the incidence of micronucleated PCEs were statistically significant but fell within the range of vehicle control values within the testing laboratory, concurrent and historical control data were used to evaluate the biological relevance of the findings.
Statistics:
Comparison of the micronucleus frequencies of the control group with the frequencies observed for the reference mutagen and treated with the test article was done using a non parametric method (Mann-Whitney).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection, hunched posture, hypoactivity, shallow breathing, one animal died about 48 h after treatment
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
Based on the study results, Genamin TA 100 is not mutagenic in the micronucleus test.
Executive summary:

A bone marrow micronucleus test (OECD TG 474) with tallow alkylamines (Genamin TA 100) in 50 Sprague Dawley rats (25 male and 25 female) led to a negative result after a single oral dose of 2000 mg/kg bw (Instituto di Ricerche Biomediche (2000c). The test substance was applicated in sesame oil by intragastric gavage. Sampling times were 24 h and 48 h after treatment. The tested dose induced clinical signs of toxicity (piloerection, hunched posture, hypoactivity and shallow breathing) in all animals. One male rat of the 48 h sampling time group died. For the mutagenicity evaluation 2000 polychromatic erythrocytes per animal were counted and scored for micronucleated cells. On the basis of the results obtained there was no significant difference between the micronucleus frequency in the treated groups in comparison with the negative control group, at any sampling time. The group of animals treated with Cyclophosphamide showed statistically significant frequency of micronucleated cells in comparison to the control group. Furthermore the ratio of polychromatic to normochromatic erythrocytes in both male and female animals remained unaffected by the tretament indicating that the test article is not toxic to bone marrow cells. Thus it is concluded that under the conditions of the present study Genamin TA 100 is not mutagenic in the micronucleus test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Mutagenicity data on hydrogenated tallow alkylamines are not available. However, primary alkylamines in general have been tested in bacterial and mammalian cell systems. Uniformly, only negative results from tests on gene and chromosome mutations in mammalian cells in vitro as well as on chromosomal aberrations and micronuclei in vivo were revealed for this chemical category. Alltogether, the negative data are considered as sufficient to exclude a mutagenic potential of hydrogenated tallow alkylamines in vivo. This conclusion is in line with the existing EU risk assessment on primary alkylamines in general.


Short description of key information:
Mutagenicity studies with hydrogenated tallow alkylamines are not available. However, primary alyklamines in general have been tested in a series of GLP compliant bacterial gene mutation tests (Ames-test) in Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 1538, TA 98 and E. coli WP2uvrA according to OECD TG 471 with and without endogenous metabolic activation. Uniformly, no increases in the number of revertants were induced in any of the tester strains used.

Additionally also for mammalian cell mutagenicity, data from closely related primary alkylamines can be used based on read-across. Data from (Z)-octadec-9-enylamine (CAS No. 112-90-3) indicate absence of mutagenicity at the hprt locus in mammalian CHO cells up to 10 nl/ml with and without S9-mix.

Negative results for mutagenicity of (Z)-octadec-9-enylamine (CAS No. 112-90-3) were also 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, (Z)-octadec-9-enylamine (CAS No. 112-90-3) 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.

In vivo mutagenicity studies on hydrogenated tallow alkylamines are not available. However, based on read-across no indications of a related potential exist from analogous compounds. A GLP-compliant bone marrow micronucleus test with tallow alkylamines (CAS No. 61790-33-8) in 50 Sprague Dawley rats led to a negative result after a single oral dose of 2000 mg/kg body weight.

Negative results are also reported from an in vivo chromosomal aberration test in mice. Administration of single doses up to 5000 mg/kg body weight in corn oil via gavage revealed no significant increases in aberrant cells although clinical signs of toxicity indicated that the test material was systemically available after oral application.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available database confirming the absence of a mutagenic potential in vitro as well as in vivo, respective classification of hydrogenated alkylamines, as well as of the whole category of primary alkylamines, is not warranted.