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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12-Dec-2011 to 31-May-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:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 3, 10, 33, 100 and 333 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 3, 10, 33, 100, 333 and 1000 µg/mL

First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr fixation: 10, 48, 51 and 54 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 10, 45 and 60 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 5, 15 and 25 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in DMSO and DMSO is 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: 0.1 µg/mL
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: Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 33 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation and at dose levels of 33 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr

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.

In the first cytogenetic assay in the presence of S9-mix the highest concentration used for scoring of micronuclei (60 µg/ml) showed a percentage cytostasis of 38%, whereas the protocol stated about 55±5%. A concentration of 60µg/ml determined by a second scorer showed 46% cytotoxicity (quality control). Moreover the next concentration, which was just above the selected concentration (75 µg/ml) showed 81% cytotoxicity and was therefore too toxic. Therefore this deviation has no effect on the results of the study.

 

In the second cytogenetic assay the highest concentration used for scoring of micronuclei (25 µg/ml) showed a percentage cytostasis of 61%, whereas the protocol stated about 55±5%.The previous concentration, which was just below the selected concentration (20 µg/ml) showed 36% cytotoxicity and was therefore less suitable. In addition the deviation from the range mentioned in the protocol was only 1%. Therefore this deviation has no effect on the results of the study.

Conclusions:
Interpretation of results (migrated information):
negative

3-(isodecyloxy)-1-propanamine 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 acceptability criteria. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

3-(isodecyloxy)-1-propanamine 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.

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:
07-Nov-2011 to 28-Dec-2011
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
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. certificate)
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: 10, 33, 100, 333 and 500 µg/mL
Without S9-mix, 24 hours treatment: 10, 33, 100, 333 and 500 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 15, 17.5, 20 and 22.5 µg/mL
With S9-mix, 3 hours treatment: 3, 10, 35, 40, 45, 50, 55 and 60 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 7 µg/mL
With S9-mix, 3 hours treatment: 3, 10, 35, 50, 52, 56, 58 and 65 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
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
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:
The global evaluation factor (GEF) has been defined by the IWGT 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.

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.
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: Precipitation in the exposure medium was observed at dose levels of 333 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 10 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 10 µg/mL in the absence of S9, 24 hours treatment

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:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 87 and 95% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 93 and 78% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:
other: strain/cell type: L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

3-(isodecyloxy)-1-propanamine is not mutagenic in the mouse lymphoma L5178Y test system
Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

 

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, 3-(isodecyloxy)-1-propanamine 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, 3-(isodecyloxy)-1-propanamine 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.

 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
07-Jun-2010 to 17-Jun-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Cross-reading is proposed to Etheramine C13i. This substance has identical chemical structure as Etheramine C10i, except that the alkyl chain is two carbon units longer. This only has limited effects on physical properties, whereas chemical behaviour, reactivity and metabolism are the same. (See for more information endpoint summary Ch.7.1 Toxicokinetics)
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 0.3, 1, 3, 10, 33 and 100 µg/plate
Experiment 2:
TA1535, TA1537, TA98 and TA100
Without S9-mix: 0.1, 0.3, 1, 3, 10 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 100 µg/plate
WP2uvrA
Without S9-mix: 0.3, 1, 3, 10, 33 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 333 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
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9 Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
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: Slight 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 33 and 100 μg/plate and above in the absence and presence of S9-mix, respectively. In tester strain WP2uvrA, toxicity was observed at dose levels of 100 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: 33 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 33 µg/plate and above and with S9: 33 µg/plate and above
TA98: without S9: 33 µg/plate and above and with S9: 33 µg/plate and above
TA100: without S9: 33 µg/plate and above and with S9: 100 µg/plate and above
WP2uvrA: without S9: 100 µg/plate and above and with S9: 100 µg/plate and above
Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that 3-(Isotridecyloxy)-1-propane amine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of 3-(lsotridecyloxy)-1-propane amine in theSalmonella typhimuriumreverse mutation assay and theEscherichia colireverse mutation assay (with independent repeat).

 

3-(lsotridecyloxy)-1-propane amine was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain ofEscherichia coli(WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital andβ-naphthoflavone).

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

 

Batch 5001287 of 3-(Isotridecyloxy)-1-propane amine was a clear colourless liquid with a purity of 95.8% m/m (NMR). The test substance was dissolved in ethanol.

 

In the dose range finding test, 3-(lsotridecyloxy)-1-propane amine was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA.

3-(Isotridecyloxy)-1-propane amine precipitated on the plates at dose levels of 3330 µg/plate and upwards. The bacterial background lawn was reduced or absent at concentrations of 33 µg/plate and upwards in tester strain TA100 and at concentrations of 100 µg/plate and upwards in tester strain WP2uvrA. Since there were too many toxic dose levels in tester strain TA100 this part of the study was repeated in the first mutation assay. Results of WP2uvrA in the dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, 3-(lsotridecyloxy)-1-propane amine was tested in the first mutation assay at a concentration range of 0.3 to 100 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100.

Cytotoxicity was observed in all tester strains. In an independent repeat of the assay with additional parameters, 3-(lsotridecyloxy)-1-propane amine was tested in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The table below gives an overview of the concentrations tested in the second mutation assay. Cytotoxicity was observed in all tester strains.

 

Tester strains

Metabolic activation

Concentration range (µg/plate)

TA1535, TA1537, TA98 and TA100

10% (v/v) S9

Up to 33 IJg/plate

Up to 100 IJg/plate

WP2uvrA

-

10% (v/v) S9

Up to 100 µg/plate

Up to 333 µg/plate

- no 59-mix added.

 

3-(lsotridecyloxy)-1-propane amine 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 59-metabolic activation. These results were confirmed in an independently repeated experiment.

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

Genetic toxicity in vivo

Description of key information

No data available from an in vivo study.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, genototoxicity by etheramines is not expected. In physiological circumstances, the etheramines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures.

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity a GLP compliant study is available.

 

For the evaluation of Etheramine C10i for bacterial mutagenicity, cross-reading is proposed to Etheramine C13i. This substance has identical chemical structure as Etheramine C10i, except that the alkyl chain is two carbon units longer.This only has limited effects on physical properties, whereas chemical behaviour, reactivity and metabolism are the same.

Etheramine C13i was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA in accordance to OECD guideline 471 and incompliance to GLP. Adequate toxicity was observed, and appropriate reference mutagens produced significant increases in the number of revertant colonies, demonstrating the sensitivity of the assay. In both the presence and in the absence of the metabolic activation systemEtheramine C13i did not result in relevant increases in the number of revertants in any of the bacterial strains.

 

Lack of bacterial mutagenicity has also been reported for an Etheramine with a somewhat shorter chain length than C10i. NTP also reports negative results for 3-(2-ethylhexyl)oxypropylamine (Etheramine C8(branched)) in the Ames test (Zeiger,E, Anderson,B, Haworth,S, Lawlor,T And Mortelmans,K; Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals; Environ. Mol. Mutagen. 11(Suppl.12):1-158, 1988).

 

 

Etheramine C10i itself has been evaluated for both mammalian mutagenicity and mammalian clastogenicity:

 

The mutagenic activity of Etheramine C10i was evaluated in an in vitro mammalian cell gene mutation test in accordance to OECD 476 and incompliance to GLP. In this study the induction of forward mutations were evaluated 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 spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals induced appropriate responses. In the absence and presence of S9-mix, Etheramine C10i 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 without of S9-mix, or with modifications in the concentration of the S9 with metabolic activation.

 

 

Etheramine C10i has further been tested in an in vitro micronucleus assay in accordance to OECD 487 and incompliance to GLP. This study evaluated the effect of Etheramine C10i on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of Etheramine C10i was tested in two independent experiments. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the acceptability criteria. The positive control chemicals produced an adequate increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Etheramine C10i 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

Etheramine C10i is not clastogenic or aneugenic in human lymphocytes and is not mutagenic in the mouse lymphoma L5178Y test system. Cross-reading from available data on other Etheramines with slightly different alkyl chain lengths compared to iso-C10 (iso-C13 and 2-ethyl-hexyl) also indicates that Etheramine can be considered non-mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

All data therefore indicate that Etheramine C10i is not genotoxic.