L-Glutamic acid, N-(1-oxododecyl)-, 1,5-dioctadecyl ester

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

807-033-7

EC Number:

807-033-7

IUPAC Name:

807-033-7

Test material form:

other: white solid

Details on test material:

- Name of test material (as cited in study report):Distearyl N-lauroyl- L- glutamate
- Physical state: white solid
- Analytical purity: >98%
- Lot/batch No.: Test01
- Expiration date of the lot/batch: 29 September 2016
- Impurities (identity and concentrations): Formulated concentrations were adjusted to allow for the stated water/impurity content (2%) of
the test item.
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% rat liver homogenate S9 in standard co-factors

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. Tetrahydrofuran was therefore selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity
content (2%) of the test item. The 200 mg/mL formulation was maintained at approximately 40 ºC to conserve solubility. As an aid to dosing, glass (instead of plastic) tubes were employed at the maximum concentration to prevent test item adherence. Tetrahydrofuran is toxic to the
bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. 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. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation: ENNG:2 µg/plate WP2uvrA;3 µg/plate TA100;5 µg/plate TA1535; 9-AA:80 µg/plate TA1537; 4NOQ:0.2 µg/plate TA98.

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

With metabolic activation: 2AA:1 µg/plate TA100; 2 µg/plate TA1535 & TA1537; 10 µg/plate WP2uvrA; BP :5 µg/plate for TA98.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiement 1: in agar (plate incorporation)
Experiement 2: preincubation.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hrs (both methods)

NUMBER OF REPLICATIONS: 3 (Experiments 1 & 2)

DETERMINATION OF CYTOTOXICITY
- Method: Visible reduction in the growth of the bacterial background lawn

Preparation of Microsomal Enzyme Fraction
Lot No. PB/βNF S9 12 October 2014 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) 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 -196C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

Preparation of S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Bacto agar (lot number 3252380 07/18) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.

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.

CARIELLO, N.F. and PIEGORSCH, W.W. (1996). The Ames Test: The two-fold rule revisited. Mutation Research., 369, pp. 23-31.

DE SERRES, F.J. and SHELBY, M.D. (1979). Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Environmental Mutagenesis., 1, pp. 87-92.

MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Statistics:

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

MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 (greasy and white in appearance) was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
Combined historical negative and solvent control ranges for 2012 and 2013 are presented in Appendix 2.
The historical ranges of the positive control reference items for 2012 and 2013 are presented in Appendix 2.

Remarks on result:

other: other: Mutation test - Experiment 1 (plate incorporation)

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Distearyl N-lauroyl- L- glutamate was considered to be non-mutagenic under the conditions of the bacterial reverse mutation (Ames) test.