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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance was not mutagenic in a guideline conform Salmonella typhimurium reverse mutation assay (Ames test) using five tester strains with and without metabolic activation (S9-mix from induced rat liver). The submission substance was also not mutagenic in a guideline compliant mammalian cell gene (HPRT) mutation assay in V79 Chinese hamster cells. In addition, testing according to OECD TG 486 revealed that the registration substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

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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:
2013-06-20 to 2013-07-08
Reliability:
1 (reliable without restriction)
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 130510
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, Applichem Lot No. 2K005471
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II, with one exception: in experiment I toxic effects of the test item were noted in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-(2-Ethylhexyl)isononan-1-amide at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N-(2-Ethylhexyl)isononan-1-amide did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, N-(2-Ethylhexyl)isononan-1-amide is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to N-(2-Ethylhexyl)isononan-1-amide at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

N-(2-Ethylhexyl)isononan-1-amide was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

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:
2013-06-27 to 2013-08-13
Reliability:
1 (reliable without restriction)
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment I with and without metabolic activation: 0.2, 0.5, 2.5, 5.0, 7.5 and 10.0 mM
Pre-experiment II without metabolic activation (24 h long-term exposure): 0.005, 0.05, 0.5, 2.0, 5.0 and 10.0 mM

Experiment I
with metabolic activation: 0.05, 0.1, 0.2, 1.0, 2.5, 5.0, 7.5, 10.0 mM
without metabolic activation: 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 mM

Experiment II
without metabolic activation: 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1 mM
and with metabolic activation: 0.3, 0.7, 1.5, 3.0, 4.0, 6.0, 8.0, 10.0 mM


Vehicle / solvent:
Ethanol was used as solvent.
From the highest test item stock solution separate stock solutions of the test item were prepared for each of the concentrations by serial dilution. From these ethanol stock solutions 0.5% v/v was added to the cells in RPMI + 5% HS for short-term exposure or RPMI + 7.5% HS for long-term exposure.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
0.5% v/v Ethanol
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: 200 µg/mL and 300 µg/mL
Positive control substance:
other: methymethanesulfonat 8 µg/mL and 10 µg/mL
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in Ethanol
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item N-(2-Ethylhexyl)isononan-1-amide is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 10.0 mM (without and with metabolic activation) was selected as the highest concentration. In experiment II 0.1 mM (without metabolic activation) and 10.0 mM (with metabolic activation) were selected as the highest concentrations. Experiment I without and with metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

0.05, 0.1, 0.2, 1.0, 2.5, 5.0, 7.5, 10.0 mM

and with metabolic activation:

0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 mM

Experiment II

without metabolic activation:

0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1 mM

and with metabolic activation:

0.3, 0.7, 1.5, 3.0, 4.0, 6.0, 8.0, 10.0 mM

No precipitation of the test item was noted in the experiments.

Growth inhibition was observed in experiment I and II without and with metabolic activation.

In experiment I without metabolic activation the relative total growth (RTG) was 13.5% for the highest concentration (10.0 mM) evaluated. The highest concentration evaluated with metabolic activation was 10.0 mM with a RTG of 19.7%. In experiment II without metabolic activation the relative total growth (RTG) was 14.4% for the highest concentration (0.1 mM) evaluated. The highest concentration evaluated with metabolic activation was 10.0 mM with a RTG of 33.4%.

In experiment II without metabolic activation (24 h long-term treatment) the solvent ethanol had an effect on cell growth in the solvent controls. Suspension growth and cloning efficiency of the solvent controls were decreased. However, values for both parameters were found within the acceptance range. Therefore the experiment is considered as valid and can be used for the evaluation of results.

In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisifes the requirements for Test Guidelines OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward mutation) data.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: clastogenicity/aneugenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-06-07 to 2013-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
Pre-Experiement
without metabolic activation: 0.05, 0.50, 1.00, 2.00, 5.00 and 10.00 mM
with metabolic activation: 5.00 and 10.00 mM
Experiment I with short exposure (4 h):
without metabolic activation: 0.05, 0.10, 0.50, 1.00 and 5.00 mM
with metabolic activation: 0.40, 0.80 and 2.00 mM
Experiment II with extended exposure (24 h):
without metabolic activation: 0.012, 0.025 and 0.050 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium due to oily droplets. After ultrasonication for 5 min at room temperature oily striations were noticed. By using DMSO as solvent a cloudy suspension was obtained. After dilution with cell culture medium to a final concentration of 1 % v/v and additional 5 min ultrasonication at room temperature still a cloudy suspension was noticed. Therefore the test item was dissolved in Ethanol and it was possible to prepare a clear solution. To reach a final concentration of 1% v/v Ethanol in the samples the test item solution was diluted in cell culture medium. After the dilution with cell culture medium oily droplets were noticed. Following ultrasonication for 5 min at room temperature a cloudy, homogenous suspension was obtained, which was used within 1 hour. The solvent was compatible with the survival of the cells and the S9 activity. Based on the results of the solubility test Ethanol was chosen as solvent.
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive control substance:
ethylmethanesulphonate
Remarks:
clastogenic control without metabolic activation (1200 µg/mL and 900 µg/mL)
Positive control substance:
other: colcemide
Remarks:
aneugenic control without metabolic activation (1.20 µg/mL and 0.16 µg/mL)
Positive control substance:
cyclophosphamide
Remarks:
clastogenic control with metabolic activation (2.50 µg/mL)
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
24 hours (Experiment II without metabolic activation)

FIXATION INTERVAL:
24 hours (Experiment I and II)

STAIN (for cytogenetic assays): acridin orange

NUMBER OF REPLICATIONS: paralell cultures

NUMBER OF CELLS EVALUATED: 2000 cells per concentration (1000 cells per culture). In case of significant difference between both slides (generally factor ≥2) additional 1000 binucleated cells of the same concentration were screened to verify this analysis.

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI) (cytostasis respectivley)

Evaluation criteria:
There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative control range.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.
Statistics:
Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item N-(2-Ethylhexyl)isononan-1-amide did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, N-(2-Ethylhexyl)isononan-1-amide is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
Executive summary:

In order to investigate N-(2-Ethylhexyl)isononan-1-amide for a possible potential to inducemicronuclei in Chinese hamster V79 cells anin vitromicronucleus assay was carried out.

The selection of the concentrations was based on data from the pre-experiment. In main experimentIwithoutmetabolic activation 5.00 mM andwithmetabolic activation 2.00 mM test item was selected as the highest concentration.

In main experiment IIwithoutmetabolic activation 0.050 mM was selected as the highest concentration.

Experiment Iwith short exposure (4 h):

The following concentrations were evaluated for micronucleated frequencies:

withoutmetabolic activation: 0.05, 0.10, 0.50, 1.00 and 5.00 mM

withmetabolic activation: 0.40, 0.80 and 2.00 mM

Experiment IIwith extended exposure (24 h):

The following concentrations were selected for micronucleus analysis with regard to cytotoxicity:

withoutmetabolic activation: 0.012, 0.025 and 0.050 mM

According the OECD Guideline 487 the maximum of cytotoxicity should not exceed the limit of 55% ± 5%.Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. According to laboratory experience a culture showing reduced cell viability (more than 30% rel. cytostasis) of the negative control displays cytotoxicity. Due to this the acceptable limit of cytotoxicity is ≤ 70%.This corresponds to≥ 30% of rel. cytostasis.

In experiment Iwithoutmetabolic activation cytotoxcity exceeding 30% was noted at concentrations of ≥ 0.30 mM.

In experiment Iwithmetabolic activation cytotoxcity exceeding 30% was noted at all concentrations of ≥ 0.80, except at a concentration of 1.00 mM (0% cytostasis).

In experiment IIwithoutmetabolic activation cytotoxcity exceeding 30% was noted at concentrations of ≥ 0.025 mM.

In experiment I and II no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametricc² Test was performed to verify the results in both experiments. No statistically significant increase (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated (Table 11-13).

Ethylmethanesulfonate (EMS, 900 and 1200 µg/mL) for conditionswithoutmetabolic activation and Cyclophosphamide (CPA, 2.50 µg/mL) for conditionswithmetabolic activation were used as clastogenic controls. Colcemide (0.16 and 1.20 µg/mL) was used as aneugenic control for conditionswithoutmetabolic activation. All induced statistically significant and biologically relevant increases of micronucleus frequency. This demonstrates the validity of the assay.

This study is classified as acceptable. This study satisfies the requirements for Test Guideline OECD 487 for in vitro mammalian cell micronucleus test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on all availabe data, the registered substance is not consiered to be mutagenic. Accordingly, classification & labelling does not apply.