Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized

EC Number:

291-716-4

EC Name:

Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized

Cas Number:

90459-71-5

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

Octadecanoic acid, reaction products with triethylenetetramine, chloromethane-quaternized

Test material form:

solid

Method

Target gene:

histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment I:
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate (TA98 and TA100)
1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 μg/plate (TA1535, TA1537 and TA102)
Experiment II:
0.50, 1.58, 5.00, 15.8, 50.0, 158, 500 and 1580 μg/plate

The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
due to the results of this pre-experiment, 2500 was chosen as the top concentration for the experiment.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

Five strains of S. typhimurium with the following characteristics were used:
TA98:
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100:
his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535:
his G 46; rfa-; uvrB-: base-pair substitutions
TA1537:
his C 3076; rfa-; uvrB-: frame shift mutations
TA102:
his G 428 (pAQ1); rfa-; R-factor: base-pair substitutions

The tester strains TA98, TA100 and TA102 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

Pre-Experiment for Toxicity

The toxicity of the test item is determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations are tested for toxicity and mutation induction with each three plates. The experimental conditions in this pre-experiment are the same as described below for the main experiment I.
In this study the highest concentration tested will be 5 mg/plate.
Toxicity may be detected by a 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.

Exposure Concentrations

At least five concentrations with and without metabolic activation will be tested.
The concentrations to be applied in the main experiments are chosen according to the results of the pre-experiment.
In case the results of the pre-experiment are in accordance with the criteria described above, these are reported as a part of the main experiment I.
For soluble, non-toxic test compounds the recommended maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest dose tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Alternatively, if it is demonstrated that toxicity occurs only at higher than the lowest insoluble concentration and in addition, there are positive responses in other mutagenicity test systems then the highest dose is based on toxicity irrespective of solubility.
Testing above the concentrations indicated has to be considered in case of substantial or potential mutagenic impurities. At least five different concentrations of the test item are tested with approximately half log intervals between test points for an initial test. Narrower spacing between dose levels is appropriate when a dose response is investigated.

Experimental Performance

For the plate incorporation method the following materials are mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
- 2000 µL overlay agar.

For the pre-incubation method 100 µL of the test item-preparation is 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.
For each strain and dose level, including the controls, a minimum of three plates is used.
After solidification the plates are inverted and incubated at 37 °C for at least 48 h in the dark.
 
Experiment I:
Experiment I is carried out according to the plate incorporation method.

Experiment II:
There is no requirement for verification of a clear positive response. If negative or equivocal results are obtained, they should be confirmed using a modification of experimental conditions.

Data Recording

The colonies are counted using a ProtoCOL counter. If precipitation of the test item precludes automatic counting the revertant colonies are counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 are counted manually.

Evaluation of Cytotoxicity

Cytotoxicity can be detected by a 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.

Criteria of Validity

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100 and TA102)
- the negative control wells (A. dest.) with and without S9 mix are within the ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2016 -2018))
- corresponding background growth on solvent control, negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity

The Mutation Factor is calculated by dividing the mean value of the revertant counts by 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 TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
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.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 μg/plate

Test results 2

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 μg/plate

Test results 3

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 316 μg/plate

Test results 4

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 316 μg/plate

Test results 5

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 2500 μg/plate

Test results 6

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 316 μg/plate

Test results 7

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 μg/plate

Test results 8

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 50 μg/plate

Test results 9

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 50 μg/plate

Test results 10

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 50 μg/plate

Test results 11

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 50 μg/plate

Test results 12

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 μg/plate

Test results 13

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 μg/plate

Test results 14

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 μg/plate

Test results 15

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 μg/plate

Test results 16

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 μg/plate

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Octadecanoic acid, reaction product with triethylenetretamine, chloromethane-quaternized did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Octadecanoic acid, reaction product with triethylenetretamine, chloromethane-quaternized is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Octadecanoic acid, reaction product with triethylenetretamine, chloromethane-quaternized did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, it is considered to be non-mutagenic in this bacterial reverse mutation assay.