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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 07, 2017 to July 02, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008 of 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA OCSPP harmonised guideline
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
Principles of method if other than guideline:
During test item preparation and analysis, all formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity, and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to Good Laboratory Practise (GLP) and has been reflected in the GLP compliance statement.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(1-tetradecoxypropan-2-yloxy)propan-2-yl propanoate (2 mol PO)
Molecular formula:
C23H46O4
IUPAC Name:
1-(1-tetradecoxypropan-2-yloxy)propan-2-yl propanoate (2 mol PO)
Constituent 2
Chemical structure
Reference substance name:
1-(1-tetradecoxypropan-2-yloxy)propan-2-yl propanoate (1 mol PO)
Molecular formula:
C23H46O4
IUPAC Name:
1-(1-tetradecoxypropan-2-yloxy)propan-2-yl propanoate (1 mol PO)
Constituent 3
Chemical structure
Reference substance name:
Tetradecyl propionate
EC Number:
228-300-9
EC Name:
Tetradecyl propionate
Cas Number:
6221-95-0
Molecular formula:
C17H34O2
IUPAC Name:
tetradecyl propanoate
Constituent 4
Reference substance name:
PG-2 Tetradecan-1-ol
IUPAC Name:
PG-2 Tetradecan-1-ol
Test material form:
liquid

Method

Target gene:
Histidine locus in the genome of Salmonella typhimurium, or tryptophan for Escherichia coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: Strain is tryptophan-dependent
Metabolic activation:
with and without
Metabolic activation system:
10% liver S9 in standard co-factors (S9-mix), prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation method) had a pre-determined dose range of 1.5 to 5000 µg/plate (1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate). 5000 µg/plate was the maximum recommended dose level. A repeat experiment was undertaken, Experiment 2 (pre-incubation method), with fresh bacteria and test item and an adapted dose range of 15 to 5000 µg/plate (15, 50, 150, 500, 1500, and 5000 µg/plate). Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
Vehicle / solvent:
- Vehicle used: Acetone, Fisher Scientific (Batch No. 1679355; purity: 99.98%; expiry: 2021-11)
- Justification for choice of vehicle: The test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house
Controls
Untreated negative controls:
yes
Remarks:
Untreated to assess spontaneous revertant colony rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 followed the plate-incorporation method with Vogel-Bonner agar. As this experiment was deemed to be negative, a second was performed (Experiment 2) in line with the pre-incubation method (described by Ames et al. 1975; Maron and Ames 1983; Mortelmans and Zeiger 2000)

EXPOSURE DURATION: In Experiment 1, all plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for revertant colonies. In Experiment 2, plates containing the bacterial strain culture; phosphate buffer (without activation) or S9-mix (with activation); and test item, solvent vehicle, or positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking). This was followed by the addition of molten, trace amino-acid supplemented media and plating onto Vogel-Bonner plates, which were then incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies

NUMBER OF REPLICATIONS: Assays were performed in triplicate for the test item and the positive, negative, and solvent controls
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A 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. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item 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, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
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
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first mutation test (plate incorporation method), a test item precipitate (fine and particulate in appearance) was noted under an inverted microscope from 1500 µg/plate and by eye at 5000 µg/plate. In the second mutation test (pre-incubation method), a test item precipitate (fine and particulate in appearance) was only noted under an inverted microscope at 5000 µg/plate. The precipitate observations did not prevent the scoring of revertant colonies.
No biologically relevant increase in the frequency of revertant colonies was recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the second mutation test at 5000 µg/plate (presence of S9-mix). This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at the statistically significant dose level was within the inhouse historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.2 times the concurrent vehicle control.

Applicant's summary and conclusion

Conclusions:
PPG-2 myristyl ether propionate was shown not to increase the frequency of revertant colonies to a biologically significant degree in Salmonella typhimurium and Escherichia coli bacterial strains with or without metabolic activation. The substance is subsequently considered to be non-mutagenic to bacteria under the conditions of this test.
Executive summary:

An in vitro gene mutation test in bacteria was performed with Salmonella typhimurium and Escherichia coli to determine the genetic toxicity of PPG-2 myristyl ether propionate. The following guidelines and regulations were adhered to without deviation: OECD Guideline 471 (Bacterial Reverse Mutation Assay); EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) of Commission Regulation (EC) No. 440/2008 of 30 May 2008; EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998); and Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries. The study was compliant with Good Laboratory Practise (GLP).

S. typhimurium strains TA1535, TA1537, TA98, and TA100 and E. coli strain WP2uvrA were treated with PPG-2 myristyl ether propionate using the plate incorporation method (Experiment 1) and pre-incubation method (Experiment 2) at up to eight dose levels, 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 Experiment 1 was predetermined: 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate. The experiment was repeated on a separate day using fresh bacterial cultures and fresh test item formulations. The pre-incubation method was followed in Experiment 2 as the results of Experiment 1 were found to be negative. The dose range was amended to 15, 50, 150, 500, 1500, and 5000 µg/plate. Six test item concentrations were selected in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

No biologically relevant increase in the frequency of revertant colonies was recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre-incubation method). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the second mutation test at 5000 µg/plate (presence of S9-mix). This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at the statistically significant dose level was within the inhouse historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.2 times the concurrent vehicle control.

Under the conditions of this in vitro test system, PPG-2 myristyl ether propionate was concluded to be non-mutagenic.

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