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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
negative in OECD 471 study (Ames test, in vitro, with and without metabolic activation)
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:
June - July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD method without significant deviations
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The following five strains were used:
Base-pair substitution type: Salmonella typhimurlum TA 100, TA 1535, Escherichia coli WP2 uvrA
Frame-shift type: Salmonella typhimurium TA98, TA 1537
These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9mix from ORIENTAL YEAST, CO LTD. Lot.No. 14021412
Test concentrations with justification for top dose:
The dose range for the main test was determined from the preliminary test using the following seven dose levels: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate.
In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was observed at 313 µg/plate and more without metabolic activation, and at 1250 µg/plate and more with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 313 µg/plate dose was selected for all strains without metabolic activation, and the 1250 µg/plate dose was selected for all strains with metabolic activation, and these highest doses were diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.
Vehicle / solvent:
Based on preliminary tests THF appeared to be the best suited solvent for this test.
Name: Tetrahydrofuran (TH F)
Manufacturer: Dojindo Laboratories
Purity: Spectrophotometric grade, more than 99%
Lot No.: DB002
Based on the information from the sponsor that the test substance was insoluble in water, the solubility test was performed with DMSO, acetone, DMF, 1,4-dioxane and THF. The test substance was insoluble at 50 mg/mL in DMSO, acetone, DMF and 1,4-dioxane, and dissolved at 100 mg/mL in THF and neither exothermic reaction nor generation of gas was observed. Therefore, THF was used as the solvent for the test substance. In consideration of growth inhibition of test strains by THF, twice the normal concentration was made and 0.05 mL of the solution in each test tube was used and the test was performed in the plate method.
Untreated negative controls:
yes
Remarks:
treated with only THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Positive control used for TA98, TA100, and WP2uvrA without S9
Untreated negative controls:
yes
Remarks:
treated with only THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control used for TA1535 without S9
Untreated negative controls:
yes
Remarks:
treated with only THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Methoxy-6-chIoro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCI
Remarks:
Positive control used for TA1537 without S9
Untreated negative controls:
yes
Remarks:
treated with only THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control used for TA98, TA100, and TA1537 with S9
Untreated negative controls:
yes
Remarks:
treated with only THF
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Positive control used for TA1535, and WP2uvrA with S9
Details on test system and experimental conditions:
Test procedures (the plate method)
For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.05 mL of the test solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer. And then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. For the sterility test, 0.05 mL of the test solution of the highest dose and 0.5 mL of the S9 mix were put into each tube, 2.0 mL of top agar were then added to the tube, and the contents of each tube were poured over the surface of the minimal glucose agar plate. These operations were conducted under lamps with ultraviolet absorbent filter.
All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies was counted. Afierwards, growth inhibition of the test strains was checked using a stereoscopic microscope.
As top agar, a soft agar solution (0.6% Agar and 0.5% NaCl) with 1/10 by volume of the solution of 0.5 mM biotin-0.5 mM L-histidine and the same ratio of 0.5 mM L-tryptophan were used for the S. typhimurium TA strains and the E. coli strain, respectively. One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main tests which were performed at the same doses.
Counting procedure
The number of revertant colonies was counted visually due to the precipitate of the test substance on the plates. But the revertant colonies of positive controls were counted with a colony counter.
Measuring instrument
Model :Colony Analyzer CA-11, Manufacturer System Science Co., Ltd.
Correction: Count loss correction
Coefficient 1 - 100 colonies * 1.11, 101 - 400 colonies * 1.16, >400 - colonies * 1.25
Evaluation criteria:
If in the two main tests, the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ±3SD) in historical data, indicating that this study was performed correctly.
From these results, mutagenicity of the test substance was judged negative.
The growth inhibition of the test strains by the test substance was not observed. And the precipitate of the test substance on the plates was observed at 156 µg/plate and more without metabolic activation, and at 625 µg/plate andmore with metabolic activation.
In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

During the study period, there was no environmental factor that was thought to have affected the reliability of the study, and there was no deviation from the protocol. A graphical plot of the results from the two main tests is attached.

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

It is concluded that Hexadecanoic acid, 1,1'-[1,3-phenyIenebis(oxy-2,1-ethanediyl)] ester is not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.
Executive summary:

The mutagenicity potential of Hexadecanoic acid, 1,1'-[1,3-phenylenebis(oxy-2,1-ethanediyl)] ester was assessed with Salrnonela typhimurium TA 100, TA 1535, TA98, TA 1537 and Escheria coli WP2 uvrA.

In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. From the above, it is judged that hexadecanoic acid, 1,1'-[1,3-phenylenebis(oxy-2,1-ethanediyl)] ester has no mutagenicity forward to bacteria under the described study conditions.

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

Additional information

Additional information from genetic toxicity in vitro:

The mutagenicity potential of hexadecanoic acid, 1,1'-[1,3-phenylenebis(oxy-2,1-ethanediyl)] ester was assessed with Salmonella typhimurium TA 100, TA 1535, TA98, TA 1537 and Escheria coli WP2 uvrA.

In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. From the above, it is judged that hexadecanoic acid, 1,1'-[1,3-phenylenebis(oxy-2,1-ethanediyl)] ester has no mutagenicity forward to bacteria under the described study conditions.

Justification for classification or non-classification

Based on currently available mutagenicity data (Ames test, negative) the substance is not subject to classification for genetic toxicity according to CLP (Regulation EC No. 1272/2008).