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Genetic toxicity in vitro

Description of key information

The test material was examined for the induction of gene mutations in 5 strains of bacteria (Ames in S. typhimurium)and mammalian cells (HGPRT, V79 cells) at concentrationa up to 5000 µg/plate. No relevant increase of mutant responses was detected.

In an in vitro chromosomal aberration assay in V79 cells also not increased genotoxic responses were detected.

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
15.8, 50, 158, 500, 1580 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
negative controls: A. dest., solvent controls: EtOH
Untreated negative controls:
yes
Remarks:
A. dest., BSL Lot No. 120612, 120711, 120723
Negative solvent / vehicle controls:
yes
Remarks:
EtOH, AppliChem Lot No. 1Q010365
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)

DURATION
- 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:
Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment I precipitation of the test item was found at a concentration of 2500 µg/plate and higher (with and without metabolic activation). In experiment II precipitation of the test item was found at a concentration of 500 µg/plate and higher (with and without metabolic activation).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Toxic effects of the test item were noted in four tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at concentrations of 1000 µg/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 1537 at a concentration of 1000 µg/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

In experiment II toxic effects of the test item were noted in tester strain TA 98 at a concentration of 5000 µg/plate (without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1580 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were observed at concentrations of 500 µg/plate and higher (without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 1580 µg/plate and higher (without metabolic activation) and at concentrations of 500 µg/plate and higher (with metabolic activation).

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Fatty acids, C16-18-, reaction products with diethanolamine did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Fatty acids, C16-18-, reaction products with diethanolamine 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 Fatty acids, C16-18-, reaction products with diethanolamine at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 15.8, 50, 158, 500, 1580 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I and II).

Fatty acids, C16-18-, reaction products with diethanolamine 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:
2012-07-11 to 2012-10-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
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
- 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 oder Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (with and without metabolic activation):
10, 25, 50, 75, 100, 250, 500, 750, 1000 and 2500 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
0.05, 0.5, 1.0, 5.0, 20, 50, 200, 500 µg/mL
Experiment I
with and without metabolic activation: 1.0, 2.5, 5.0, 10, 25, 50, 75, 100, 250 and 500 µg/mL
Experiment II
without metabolic activation: 2.5, 5.0, 10, 25, 50, 60, 70, 80, 90 and 110 µg/mL
and with metabolic activation: 3.5, 5.0, 12.5, 20, 35, 50, 125, 200, 350 and 500 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was suspended in cell culture medium and processed by ultrasound for 10 min.
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 300 µg/mL
Positive controls:
yes
Positive control substance:
other: 7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; concentrations: 1µg/mL and 1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth
Evaluation criteria:
A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I without S9: ≥ 250 μg/mL; experiment I with S9: 500 μg/mL; Experiment II without S9: ≥ 60 μg/mL; Experiment II with S9:≥ 350 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Fatty acids, C16-18-, reaction products with diethanolamine is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to Fatty acids, C16-18-, reaction products with diethanolamine suspended in (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 1.0, 2.5, 5.0, 10, 25, 50, 75, 100, 250 and 500 µg/mL (with and without metabolic activation, Experiment I)

- 2.5, 5.0, 10, 25, 50, 60, 70, 80, 90 and 110 µg/mL (without metabolic activation, Experiment II)

- 3.5, 5.0, 12.5, 20, 35, 50, 125, 200, 350 and 500 µg/mL (with metabolic activation, Experiment II).

Fatty acids, C16-18-, reaction products with diethanolamine was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 39.7% for the higest concentration evaluated (500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 500 µg/mL with a relative growth of 52.0%. In experiment II without metabolic activation the relative growth was 16.2% for the highest concentration (110 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 500 µg/mL with a relative growth of 44.4%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.88 was found at a concentration of 5.0 µg/mL with a relative growth of 119.8%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.54 was found at a concentration of 1.0 µg/mL with a relative growth of 102.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.18 was found at a concentration of 70 µg/mL with a relative growth of 58.1%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.61 was found at a concentration of 20 µg/mL with a relative growth of 97.5%.

The positive controlsdidinduce the appropriate response. 

There was no evidence of a concentration related positive responseof induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

 

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:
2012-06-21 to 2013-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 Wistar and Sprague Dawley phenobarbital and β-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-Experiment
7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500, 5000 µg/mL


Experiment I:
Without metabolic activation, 4 h treatment, 24 h preparation interval:
250, 1000 and 1500 µg/mL
With metabolic activation, 4 h treatment, 24 h preparation interval:
250, 1000 and 3000 µg/mL

Experiment II:
Without metabolic activation, 24 h treatment, 24 h preparation interval:
1000, 3000 and 5000 µg/mL
With metabolic activation, 4 h treatment, 24 h preparation interval:
1000, 3000 and 5000 µg/mL


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test item was suspended in cell culture medium (RPMI) and diluted prior to treatment.
- Justification for choice of solvent/vehicle: The test item was suspended in cell culture medium (RPMI + 0% FBS 4h treatment; RPMI + 15% FBS 24 h treatment), processed by ultrasound for 10 min and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 400 and 600 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
24 hours (Experiment I without metabolic activation)

FIXATION INTERVAL: 24 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independet experiments
NUMBER OF CELLS SCORED: 200 per concentration (100 cells per culture)
DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; Proliferation Index BrdU

Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.0% aberrant cells).
Statistics:
According to the 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:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results of chromosome analysis
without metabolic activation
 
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%)    Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other Proliferations Index incl. Gaps excl. Gaps
Experiment I                              
negative control 200 no 1 5 0 0 2 0 0 0 100 1.20 0 4.0 2.5
250 µg/mL 200 no 3 3 0 0 0 0 0 0 89 1.18 0 3.0 1.5
1000 µg/mL 200 no 8 4 0 1 1 0 0 0 79 1.05 0 6.0 2.5
1500 µg/mL 200 yes 9 6 1 0 0 0 0 1 64 1.09 0 7.5 3.5
EMS 600 µg/mL 200 9 10 14 1 0 0 0 4 69 n.d. 0 15.5 12.0
Experiment II                                  
negative control 200 no 3 4 0 0 0 0 0 0 100 1.73 0 3.0 2.0
1000 µg/mL 200 no 2 1 0 0 0 0 0 0 110 1.67 0 1.5 0.5
3000 µg/mL 200 no 6 1 0 1 0 0 0 0 102 1.62 0 4.0 1.0
5000 µg/mL 200 no 6 4 0 0 0 0 0 0 107 1.66 0 4.5 2.0
EMS 400 µg/mL 200 5 23 11 3 3 2 0 2 78 n.d. 2 21.0 18.0
Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%)    Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other Proliferation Index incl. Gaps excl. Gaps
Experiment I                              
negative control 200 no 5 3 0 0 0 0 0 0 100 1.18 0 4.0 1.5
250 µg/mL 200 no 1 4 0 1 1 0 0 1 111 1.24 0 3.0 2.5
1000 µg/mL 200 no 0 0 0 0 2 0 0 0 93 1.10 0 2.0 1.0
3000 µg/mL 200 no 1 5 0 1 0 0 0 0 73 1.04 0 3.0 2.5
CPA 5 µg/mL 200 6 33 3 0 1 1 0 5 112 n.d. 0 17.0 14.5
Experiment II                                  
negative control 200 no 5 2 0 0 0 0 0 2 100 1.28 0 4.0 2.0
1000 µg/mL 200 yes 2 2 0 0 0 0 0 0 43 1.29 0 2.0 1.0
3000 µg/mL 200 no 10 3 1 0 0 0 0 0 87 1.13 0 5.5 2.0
5000 µg/mL 200 yes 1 3 0 0 0 0 0 0 69 1.14 0 2.0 1.5
CPA 5 µg/mL 200 5 23 10 0 3 1 1 5 104 n.d. 0 15.5 14.5

n.d.       =       no data

Conclusions:
In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Fatty acids, C16-18-, reaction products with diethanolamine did not induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, Fatty acids, C16-18-, reaction products with diethanolamine is considered to be non-clastogenic in this chromosome aberration test.
Executive summary:

A chromosome aberration assay was carried out in order to investigate a possible potential of Fatty acids, C16-18-, reaction products with diethanolamine for its ability to induce structural chromosome aberrations in Human Lymphocytes.

The metaphases were prepared 24 h after start of treatment with the test item. The treatment intervals were 4 h without and with metabolic activation (experiment I) and 24 h without and 4 h with metabolic activation (experiment II). Two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The test item was suspended in cell culture medium.

The following concentrations were evaluated:

Experiment I:

Without metabolic activation, 4 h treatment, 24 h preparation interval:

250, 1000 and 1500 µg/mL

With metabolic activation, 4 h treatment, 24 h preparation interval:

250, 1000 and 3000 µg/mL

Experiment II:

Without metabolic activation, 24 h treatment, 24 h preparation interval:

1000, 3000 and 5000 µg/mL

With metabolic activation, 4 h treatment, 24 h preparation interval:

1000, 3000 and 5000 µg/mL

Precipitation of the test item was noted in experiment I without and with metabolic activation at 1000 µg/mL and higher. In experiment II without and with metabolic activation precipitation was observed at 1000 µg/mL and higher. The precipitation was partly concentrated the tube wall.

Haemolytic effects were observed in experiment I without and with metabolic activation before the addition of colcemide at all evaluated concentrations. Haemolytic effects were noted in experiment II with metabolic activation after the treatment interval of 4 h at concentrations of 3000 µg/mL and higher and before the addition of colcemide without and with metabolic activation at all evaluated concentrations. The haemolytic effect was dose dependent.

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted in the highest evaluated concentration (1500 µg/mL) and with metabolic activation no biologically relevant decrease of the relative mitotic index was noted.

In experiment II with metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was detected at dose group 4 and 9 (1000 µg/mL and 5000 µg/mL). Without metabolic activation no biologically relevant decrease of the relative mitotic index was noted.

In experiment I and II no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

EMS (400 and 600 µg/mL) and CPA (5 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data.

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

Additional information

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to the test item at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 15.8, 50, 158, 500, 1580 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I and II).

The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

In a mammalian cell gene mutation assay (HPRT locus],V79 cells cultured in vitro were exposed to Fatty acids, C16-18, reaction products with diethanolamine at concentrations of 1 to 500 µg/mL (with and without metabolic activation, depending on experiment)

Fatty acids, C16-18, reaction products with diethanolamine was tested up to cytotoxic concentrations.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related increase of induced mutant colonies over background.

A chromosome aberration assay was carried out in Human Lymphocytes.

The following concentrations were evaluated: 250, 1000, 1500, 3000 and/or 5000 µg/mL with and or without metabolic activation. Haemolytic effects were observed in both experiments without and with metabolic activation before the addition of colcemide at several evaluated concentrations. The haemolytic effect was dose dependent. A biologically relevant decrease of the relative mitotic index was noted in some concentrations with metabolic activation. Without metabolic activation no biologically relevant decrease of the relative mitotic index was noted.

No biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

Positive controls induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects. There was no evidence of a concentration related clastogenic response in Human lymphocytes.

In conclusion no indication of genotoxic activiuty of the test item were detected in in-vitro assays in bacteria or mammalian cells.

Justification for classification or non-classification

Not classified.

No gene mutations were detected in bacteria or mammalian cells in vitro. No clastogenic effects were found in a chromosomal aberration assay.