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Genetic toxicity in vitro

Description of key information

Based on the results of the Ames test, the test substance is considered to be non-mutagenic, 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:
From May 26 , 2011 to July 14, 2011
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
GLP compliance:
yes (incl. certificate)
Remarks:
no analytical veryfication
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: 100%;
Appearance: off white solid
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: derived from strain LT2 through mutations in histidine locus
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (10% liver in standard co-factors)
Test concentrations with justification for top dose:
Preliminary toxicity test: 0; 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500; 5000 ug/plate;
Experiment 1:
All strains (absence of S9-mix): 5; 15; 50; 150; 500; 1500; 5000 ug/plate.
All strains (presence of S9-mix): 15; 50; 150; 500; 1500; 5000 ug/plate.
Experiment 2:
All Salmonell astrains (absence of S9-mix) and TA100 (presence of S9-mix): 50; 150; 500; 1500; 5000 ug/plate.
All strains (presence of S9-mix) except TA100 and WP2uvrA (absence of S9-mix): 5; 15; 50; 150; 500; 1500; 5000 ug/plate.
Vehicle / solvent:
Tetrahydrofuran
Negative solvent / vehicle controls:
yes
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA 1535
Negative solvent / vehicle controls:
yes
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80µg/plate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
without S9-mix
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2µg/plate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
with S9-mix
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
with S9-mix
Positive controls:
yes
Positive control substance:
other:
Remarks:
10µg/plate for WP2uvrA, 1µg/plate for TA100, 2µg/plate for TA 1535 and TA1537
Details on test system and experimental conditions:
Preparation of the test substance formulations:
The test substance was accurately weighted and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 40 minutes at room temperature on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 uL, therefore all of the formulations were prepared at the concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 uL aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium-alumino-silicate pellets with a normal pore diameter of 4*10-E4 microns.

Preliminary toxicity test:
The concentrations tested: 0; 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500; 5000 ug/plate. The test was perforemed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.025 mL of the test substance formulation and 0.5 mLof S9-mix or phosphate bussefand overlying onto sterile plates of Vogel-Bonner Minimal agar (30mL/plate).

Mutation Test - Experiment 1:
Up to secen concentrations of the test substance were assayed in triplicate against each tester strain, using the direct plate incorporation method. Dose levels were allocated as follows:
All strains (absence of S9-mix): 5; 15; 50; 150; 500; 1500; 5000 ug/plate.
All strains (presence of S9-mix): 15; 50; 150; 500; 1500; 5000 ug/plate.

Mutation Test – Experiment 2:
The second experiment was performed using fresh bacterial cultures, test substance and control solutions:
All Salmonell astrains (absence of S9-mix) and TA100 (presence of S9-mix): 50; 150; 500; 1500; 5000 ug/plate.
All strains (presence of S9-mix) except TA100 and WP2uvrA (absence of S9-mix): 5; 15; 50; 150; 500; 1500; 5000 ug/plate.

Rationale for test conditions:
The test substance was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. The test substance formed the best doseable suspension in tetrahydrofuran, therefore this solvent was selected as the vehicle.
Evaluation criteria:
There are several criteria for determining positive result. Any one or all of the following can be used to determine overall result of the study:
1. Dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statystical analysis of data as determined by UKEMS (Mahon et al. 1989).
5. Fold increase greater than two times the concurent solvent control for any tester strain (especially if acompanied by out-of-historical range response (Cariello and Piegorsch, 1996)).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.Although most experiments will give clear positive or negative results, is some instances the data generated will prohibit making a definite judgment about test substance activity. Results of this type will be reported as equivocal.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficiency of the S9-mix were validated.

In the first experiment (plate incorporation method) the test substance caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially at 500 ug/plate (TA1537). In the presence of S9-mix weakened lawns were noted to all of the Salmonella strains, initially from 1500 ug/plate (TA1537).

In the second experiment (pre-incubation method) a much weaker response was noted with weakened bacterial background lawns only observed for TA1537 (presence of S9-mix) at 5000 ug/plate. The sensitivity of the tester strains to the toxicity of the test substance varied between strain type, exposures with or without S9-mix and experimental methodology. The test substance was tested up to maximum recommended dose level of 500 ug/plate. A test substance precipitate (particulate in appearance) was noted at and above 500 ug/plate, this observation did not prevent the scoring of revertant colonies.
Conclusions:
Under the study conditions, the test substance was determined to be non-mutagenic, with and without metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', using bacterial reverse mutation assay (Ames test), according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA were treated with suspensions of the test substance using both the plate incorporation and pre-incubation methods at up to seven dose levels (5; 15; 50; 150; 500; 1500; 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay, where the test concentrations ranged between 5 and 5000 µg/plate, in the presence or absence of S9-mix. The experiment was repeated on separate day (pre-incubation methods) using a similar dose range to first experiment, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test substance. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Results for the positive and negative controls were also in line with the historical laboratory data. Thus, the sensitivity of the assay and the efficiency of the S9-mix were validated. In the first experiment (plate incorporation method) the test substance caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially at 500 µg/plate (TA1537). In the presence of S9-mix weakened lawns were noted in all of the Salmonella strains, initially from 1500 µg/plate (TA1537). In the second experiment (pre-incubation method) a much weaker response was noted with weakened bacterial background lawns only observed for TA1537 (presence of S9-mix) at 5000 µg/plate. The sensitivity of the tester strains to the toxicity of the test substance varied between strain type, exposures with or without S9-mix and experimental methodology. The test substance was tested up to maximum recommended dose level of 500 µg/plate. A test substance precipitate (particulate in appearance) was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies. Under the study conditions, the test substance was determined to be non-mutagenic, with and without metabolic activation (Harlan, 2012).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the mutagenic potential of the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate', using bacterial reverse mutation assay (Ames test), according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. Salmonella typhimurium (TA1535, TA1537, TA98 and TA 100) and Escherichia coli strain WP2uvrA were treated with suspensions of the test substance using both the plate incorporation and pre-incubation methods at up to seven dose levels (5; 15; 50; 150; 500; 1500; 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay, where the test concentrations ranged between 5 and 5000 µg/plate, in the presence or absence of S9-mix. The experiment was repeated on separate day (pre-incubation methods) using a similar dose range to first experiment, fresh cultures of the bacterial strains and fresh test substance formulations. Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test substance. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Results for the positive and negative controls were also in line with the historical laboratory data. Thus, the sensitivity of the assay and the efficiency of the S9-mix were validated.In the first experiment (plate incorporation method) the test substance caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially at 500 µg/plate (TA1537). In the presence of S9-mix weakened lawns were noted in all of the Salmonella strains, initially from 1500 µg/plate (TA1537). In the second experiment (pre-incubation method) a much weaker response was noted with weakened bacterial background lawns only observed for TA1537 (presence of S9-mix) at 5000 µg/plate. The sensitivity of the tester strains to the toxicity of the test substance varied between strain type, exposures with or without S9-mix and experimental methodology. The test substance was tested up to maximum recommended dose level of 500 µg/plate. A test substance precipitate (particulate in appearance) was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies. Under the study conditions, the test substance was determined to be non-mutagenic, with and without metabolic activation (Harlan, 2012).

Justification for classification or non-classification

Based on the results of the Ames test, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' does not warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).