Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Genetic toxicity: in vitro

Administrative data

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:

The experimental phase of this study was performed between 31 August 2011 and 30 September 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 2

Qualifier:

according 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

Test material

Test material information

Constituent 1

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description:
Off-white powder
Purity: Mixture
Batch number: PS101103
Date received: 11 July 2011
Expiry date: 18 November 2012
Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-:
TA98 his D 3052; rfa-; uvrB-;R-factor frame shif mutations
TA1535 his G 46; rfa-; uvrB-:
TA100 his G 46; rfa-; uvrB-; R-factor base-pair mutations

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA- base-pair substitution

In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Five concentrations of the test item (50, 150, 500, 1500 and 5000 IJg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

In solubility checks performed in-house, the test item was was found to be insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/ml, acetone at 100 mg/ml and tetrahydrofuran at 200 mg/ml. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

For strains WP2uvrA, TA 100 and TA 1535 without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA 1537 without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

For strain TA 98 without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Positive control substance:

benzo(a)pyrene

Remarks:

For strain TA98 with S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2M)

Remarks:

For strain

Details on test system and experimental conditions:

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 25 minutes at 40°C on the day of each experiment. The 50 mg/ml formulation was kept at approximately 40°C throughout the dosing period to maintain the suspension. 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 GLP and has been reflected in the GLP compliance statement.

Microsomal Enzyme Fraction:
The S9 Microsomal fraction was prepared in-house (26 June 2011) from rats induced with Phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The 89 homogenate was produced by homogenising the liver in a 0.15M KCI solution (1 9 liver to 3 ml KCI) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/ml. Aliquots of the supernatant were frozen and stored at approximately -196°C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

Mutation test - experiment 1:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test item formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation test - experiment 2:
The second experiment was performed using fresh bacterial cultures, test item and control solutions. The test item dose range was amended slightly, following the change in test methodology, and was 15 to 5000 µg/plate.

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 µg/plate because of test item precipitation.

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).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test results

Test results 1

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

Remarks:

precipitate at and above 1500 mg/kg

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at and above 1500 mg/kg

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary test:
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutation Test Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

The test item caused no visible reduction in the growth of the bacterial background lawnat any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (cream-coloured and powdery in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The test item, PC-9S, was considered to be non-mutagenic under the conditions of this test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item, PC-9S, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the DECO Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item, PC-9S, using both the Ames plate incorporation and pre-incubation methods at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10°A> liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 (Jg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range of 15 to 5000 (Jg/plate, fresh cultures of the bacterial strains and fresh test item formulations. An additional dose level and an expanded dose range was selected in the second experiment in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item.

Results: The vehicle (dimethyl sulphoxide) 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 efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 (Jg/plate. A test item precipitate (cream-coloured and powdery in appearance) was noted at and above 1500 (Jg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. Conclusion. The test item, PC-9S, was considered to be non-mutagenic under the conditions of this test.