Tetradecan-1-ol, propoxylated, esters with propionic acid

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA OCSPP harmonised guideline

Deviations:

no

Test guideline 4

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

Test material information

Constituent 1

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

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

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 / strain

Species / strain 1

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 2

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.