Amides, tall-oil fatty, N,N-di-Me

Genetic toxicity: in vitro

Administrative data

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:

17 December 2012 to 05 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to relevant test guidelines, with no deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

DMATO

IUPAC Name:

DMATO

Constituent 2

Reference substance name:

Amides, tall-oil fatty, N,N-di-Me

EC Number:

269-665-4

EC Name:

Amides, tall-oil fatty, N,N-di-Me

Cas Number:

68308-74-7

Molecular formula:

not applicable for UVCB

IUPAC Name:

Amides, tall-oil fatty, N,N-di-Me

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): DMATO
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: 12J46399
- Expiration date of the lot/batch: October 2015
- Storage condition of test material: at room temperature

Method

Target gene:

Histidine (his) and tryptophan (trp)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

The vehicle was DMSO, chosen according to its solubility properties and its relative nontoxicity to the bacteria. The test material was formulated in DMSO on the day of the experiment.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

The strain cultures were stored as stock cultures in ampoules with nutrient broth +5% DMSO in liquid nitrogen. From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. The nutrient medium contained per litre: 8 g nutrient broth and 5 g NaCl. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37°C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E8-10E9 cells/mL).

A pre-experiment was conducted in all strains at 8 doses between 3 and 5000 µg/plate, according to the plate incorporation assay. The pre-experiment was reported as Experiment I since the following criteria were met: evaluable plates (>0 colonies) at five concentrations or more in all strains used. Minor toxic effects were noted so the second experiment was performed with 7 concentrations up to a maximum of 5000 µg/plate. Experiment II was performed as a pre-incubation assay. Test material concentrations and controls were tested in triplicate for each strain. The following materials were mixed in a test tube and poured onto selective agar plates: 100 µL test solution, solvent or positive control; 500 µL S9 mix or buffer; 100 µL bacterial suspension; 2000 µL overlay agar. In the pre-incubation assay 100 µL test solution, solvent or positive control, 500 µL S9 mix or buffer and 100 µL bacterial suspension were mixed in a test tubes and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was then poured onto minimal agar plates. After solidification the plates were incubated upside down for at 48 hours at 37°C in the dark.

Overlay agar for S. typhimurium strains contained 7 g Agar Agar, 6.0 g NaCl, 10.25 mg L-Histidine HCl H2O and 12.2 mg Biotin. Overlay agar for E. coli contained 7.0 g Agar Agar, 6.0 g NaCl and 10.2 mg Tryptophan. Sterilisations were performed at 121°C in an autoclave.

The colonies were counted using the Petri Viewer Mk2 with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test item, the revertant colonies were partly counted manually.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

The assay was considered acceptable if it meets the following criteria: regular background growth in the negative and solvent control; the spontaneous reversion rates in the negative and solvent control are in the range of historical data; the positive control substances should produce a significant increase in mutant colony frequencies; a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Statistics:

Not required.

Results and discussion

Test results

Test results 1

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

Remarks:

Minor toxic effects were observed only in the presence of metabolic activation in strains TA 1535 and TA 1537 in experiment I at 5000 µg/plate and in experiment II in strain TA 1535 at 1000 and 2500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 2500 and 5000 µg/plate without S9 mix in experiment I and from 1000 to 5000 µg/plates in experiment I with S9 mix and in experiment II with and without S9 mix. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Minor toxic effects were observed only in the presence of metabolic activation in strains TA 1535 and TA 1537 in experiment I at 5000 µg/plate and in experiment II in strain TA 1535 at 1000 and 2500 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with DMATO at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No further information.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test material did not induce gene mutations by base pair changes or frameshifts under the experimental conditions. DMATO is therefore considered to be non-mutagenic in the bacteria reverse mutation assay

Executive summary:

The potential for DMATO to induce gene mutation in the bacterial reverse mutation assay was evaluated in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without metabolic activation (S9 mix). The following concentrations were tested in Experiment I (plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. The following concentrations were tested in Experiment II (pre-incubation test): 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. Appropriate negative, solvent and positive controls were tested. All test solution concentrations and controls were tested in triplicate. The plates incubated with the test material showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Minor toxic effects were observed only in the presence of metabolic activation in strains TA 1535 and TA 1537 in Experiment I at 5000 µg/plate and in Experiment II in strain TA 1535 at 1000 and 2500 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, in the presence or absence of metabolic activation. There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The test material did not induce gene mutations by base pair changes or frameshifts under the experimental conditions. DMATO is therefore considered to be non-mutagenic in the bacteria reverse mutation assay