Fatty acids, tall-oil, reaction products with triethylenetetramine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Link to relevant study records

Reference

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:

From March 04, 2015 to April 01, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

Target gene:

Strain Mutation name
TA97a hisD6610
TA98 hisD3052
TA100 hisG46
TA102 hisG428
TA1535 hisG46

Species / strain / cell type:

S. typhimurium, other: TA97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix, rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Nominal concentrations tested in the first experiment (1a and 1b): 500 μg/plate, 150 μg/plate, 50 μg/plate, 15 μg/plate and 5 μg/plate.

Nominal concentrations tested in the second experiment (2a and 2b): 500 μg/plate, 250 μg/plate, 125 μg/plate, 63 μg/plate, 31 μg/plate, 16 μg/plate and 8 μg/plate.

Vehicle / solvent:

- Vehicle/solvent used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

without metabolic activation for TA97a, TA98 and TA102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

with metabolic activation for TA97a, TA100, TA102 and TA1535.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation for TA98

Details on test system and experimental conditions:

PERFORMANCE OF THE STUDY

Culture of Bacteria
8 h before the start of each experiment, the nutrient broth was inoculated. For the incubation of strains TA97a, TA98, TA100, TA102; ampicilline was added to the nutrient broth (25 mg/L), for the incubation of strain TA102, tetracycline was added (2 mg/L), too. TA1535 was incubated without the addition of antibiotics. The flasks were incubated at 37±1°C for 8 h.

Conduct of Experiment

Preparations
In the days before each test, the media and solutions were prepared.

On the day of the test, the overnight cultures were checked for growth. The incubation chambers were heated to 37±1°C. The water bath was turned to 43±1°C. The table surface was disinfected.

The S9 mix was freshly prepared and stored at 0±1°C.

Experimental Parameters

Experiment 1a
Concentrations tested: 500, 150, 50, 15 and 5 μg/plate
Incubation time: 48 h
Incubation temperature: 37±1°C
Bacteria strains: TA97a, TA102
Method: Plate incorporation method

Experiment 1b
Concentrations tested: 500, 150, 50, 15 and 5 μg/plate
Incubation time: 48 h
Incubation temperature: 37±1°C
Bacteria strains: TA98, TA100, TA1535
Method: Plate incorporation method

Experiment 2a
Concentrations tested: 500, 250, 125, 63, 31, 16 and 8 μg/plate
Incubation time: 48 h
Incubation temperature: 37±1°C
Bacteria strains: TA97a, TA98, TA102, TA1535
Method: Pre-incubation method

Experiment 2b
Concentrations tested: 500, 250, 125, 63, 31, 16 and 8 μg/plate
Incubation time: 48 h
Incubation temperature: 37±1°C
Bacteria strains: TA100
Method: Pre-incubation method

Description of the Method

General preparation
Per strain and dose, 3 plates with and 3 plates without S9 mix were used. Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotinsolution 0.5 mM per 100 mL basis was added and the bottle was placed in the water bath at 43±1°C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
• 2,000 μL overlay agar (top agar)
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37±1°C for 20 minutes:
• 100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension
After pre-incubation, 2,000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate.
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37±1°C.

Evaluation criteria:

A test substance is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >=2) in at least one strain can be observed.

A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Species / strain:

S. typhimurium, other: TA97a

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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

Species / strain:

S. typhimurium TA 1535

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

Additional information on results:

FINDINGS

Confirmation of genotype is performed for each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities.

Experiment 1a and 1b

Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity
The test substance was dissolved in ethanol. A stock solution containing 5 g/L was prepared. In both experiment, the test substance showed no precipitates on the plates in all tested concentrations. Signs of toxicity towards the bacteria strain TA100 could be observed in the highest concentration (500 μg/plate) with and without metabolic activation. In the next lower concentration (150 μg/plate) the test substance showed toxicity towards the strain TA100 only in the treatment without metabolic activation. The background lawn was visible, but the number of revertant colonies was clearly reduced.

Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test substance is stated as not mutagenic under these test conditions.

Experiment 2a and 2b

Confirmation of the Criteria and Validity
The treatments for the sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity
The test substance was dissolved in ethanol. A stock solution containing 5 g/L was prepared. In both experiment, the test substance showed no precipitates on the plates in all tested concentrations. Signs of toxicity towards all bacteria strains could be observed in the 2 highest concentrations (500 and 250 μg/plate) in the treatment with and without metabolic activation.

In these concentrations the background lawn was not visible and the number of revertant colonies was clearly reduced.

Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

Therefore, the test substance is stated as not mutagenic under the test conditions.

RESULTS

Mutagenicity of Test substance

The test substance did not show mutagenic effects in all experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). In the experiment 1b cytotoxicity of the test substance was detected towards the strains TA100 in the highest concentration (500 μg/plate) in the treatment with and without metabolic activation. In the next lower concentration (150 μg/plate) the test substance showed toxicity towards the strain TA100 only in the treatment without metabolic activation.

In the experiment 2a and 2b signs of cytotoxicity towards all bacteria strains could be observed in the 2 highest concentrations (500 and 250 μg/plate) in the treatment with and without metabolic activation.

In these concentrations the bacterial background lawn was partly not visible and the number of revertants was clearly decreased.
On the base of these results, it can be stated, that under the test conditions, the test substance is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Acceptability of Study
All spontaneous revertants (except of 2 values in the first experiment) and all positive control values were within the range of the historical data. Difference of revertants lying outside the range is marginal. Therefore, the study is considered valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

The test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Two mutagenicity studies were conducted, one as the plate incorporation test (i.e., experiment 1a and 1b) and the other one as a preincubation test (i.e., experiment 2a and 2b). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. The test substance was tested for mutagenic effects without and with metabolic activation at concentrations in the range of 5 - 500 µg/plate in experiment 1a and 1b and 8 - 500 µg/plate in experiment 2a and 2b. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix). Cytotoxicity was observed in the strains TA100 in experiment 1b at the highest concentration (500 μg/plate) with and without metabolic activation and at the next lower concentration (150 μg/plate) without metabolic activation. In the preincubation tests no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix). Further, cytotoxicity was observed in all bacteria strains in both the experiments i.e., 2a and 2b at the two highest concentrations (500 and 250 μg/plate) with and without metabolic activation. In addition, spontaneous revertants were within the normal range in comparison with the historical data. The reference mutagens also showed the expected increase in induced revertant colonies. Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay (Andres I, 2015c).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Two mutagenicity studies were conducted, one as the plate incorporation test (i.e., experiment 1a and 1b) and the other one as a preincubation test (i.e., experiment 2a and 2b). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. The test substance was tested for mutagenic effects without and with metabolic activation at concentrations in the range of 5 - 500 µg/plate in experiment 1a and 1b and 8 - 500 µg/plate in experiment 2a and 2b. In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix). Cytotoxicity was observed in the strains TA100 in experiment 1b at the highest concentration (500 μg/plate) with and without metabolic activation and at the next lower concentration (150 μg/plate) without metabolic activation. In the preincubation tests no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix). Further, cytotoxicity was observed in all bacteria strains in both the experiments i.e., 2a and 2b at the two highest concentrations (500 and 250 μg/plate) with and without metabolic activation. In addition, spontaneous revertants were within the normal range in comparison with the historical data. The reference mutagens also showed the expected increase in induced revertant colonies. Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay (Andres I, 2015c).

Justification for selection of genetic toxicity endpoint
High quality study conducted according to internationally accepted Guidelines.

Justification for classification or non-classification

Based on the results of in vitro genotoxicity testing, the substance does not require classification for this endpoint according to the EU CLP criteria (EC 1272/2008).