Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 3,5,5-trimethylhexanoic, n-heptanoic, n-octanoic and n-decanoic acids

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:

2013-05-23 to 2013-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9510 TSCA bacterial reverse mutation test and the USA, EPA (TSCA) OCSPP harmonized guidelines.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Cas Number:

65870-94-2

Molecular formula:

C33H62O6

IUPAC Name:

Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Test material form:

liquid

Details on test material:

Description: clear colourless liquid
Expiry date: 29 June 2013
Storage conditions: room temperature, in the dark

Method

Target gene:

None

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):

Non-mammalian study

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9; S9 fraction was prepared from liver homogenates of male rats induced with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Mutation tests (plate incorporation method)
Experiment 1:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for all S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E. coli strain WP2uvrA, with and without S9-mix

Experiment 2:
50, 150, 500, 1500 and 5000 μg/plate for all S.typhimurium strains (TA1535, TA1537, TA98 and TA100) and E. coli strain WP2uvrA, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was 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. Acetone was therefore selected as the vehicle.
- Preparation of formulation: Test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent (Acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene - 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA; Benzo(a)pyrene - 5 µg/plate for TA98

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent (Acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitro-N-nitrosoguanidine - 2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535; 9-Aminoacridine - 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide - 0.2 µg/plate for TA98

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors).

DURATION
- Exposure duration: Treated plates were incubated at 37 °C ± 3 °C for approximately 48 h and scored for the presence of revertant colonies using an automated colony counting system.

NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

DETERMINATION OF CYTOTOXICITY
- Method: The treated plates were viewed microscopically for evidence of thinning (toxicity).

OTHER:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

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:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).

Results and discussion

Test results

Test results 1

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 2

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 3

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 4

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 5

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were comparable with historical negative, solvent and positive control data (2011 and 2012) of laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation in both experiments.

OTHERS:
- 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 used in each experiment was also checked and considered acceptable.

Any other information on results incl. tables

See the attached document for tables of results

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test material is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) with and without metabolic activation

Executive summary:

Test Guidance

OECD Guideline 471

Method and materials

Strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA)were exposed to the test item at the following concentrations using plate incorporation method.

 

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all strains

Experiment 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without S9 mix in all strains

 

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with phenobarbitone/β-naphthoflavone. Negative, vehicle and positive control groups were also included in mutagenicity tests.

Results

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in any of the experiment. A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 or 2. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

Conclusion

Under the test conditions, the substance is not considered as mutagenic in bacterial systems.

.