L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

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:

23 February 2000 to 06 March 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The mutagenic potential of the test material was assessed using a reverse mutation assay in which four S. typhimurium strains and one E. coli strain were tested with and without metabolic activation.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

EC Number:

269-084-6

EC Name:

L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

Cas Number:

68187-29-1

Molecular formula:

Not specified (UVCB substance)

IUPAC Name:

bis(tris(2-hydroxyethyl)azanium) (2S)-4-carboxy-2-dodecanamidobutanoate (2S)-4-carboxy-2-tetradecanamidobutanoate

Test material form:

liquid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The definite amount of test material was weighed at the time of use and water for injection of Japanese Pharmacopoeia was added to this to prepare the test material solution of high concentration. Purity conversion was performed at the time of preparation.
The test material solutions of low concentration were prepared by diluting the test material solution of high concentration with the same medium one after another.

Method

Target gene:

S. typhimurium: Histidine
E. coli: Tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The bacterial strains are widely used in mutagenicity studies for their capacity to induce reverse mutations because of high sensitivity to known mutagens.
Inherent genetic characteristics of the respective strains, amino acid requirement, membrane mutation rfa property, UV sensitivity, number of spontaneous revertants, drug resistance factor and sensitivity to know mutagens (relative value to solvent control, relative value to positive control and dose response) were confirmed.
- Methods for maintenance in cell culture if applicable: The microorganisms were dispersed in Nutrient broth No. 2 (Oxoid) overnight to make dispersions. As the seed bacteria, the bacterial dispersions were spread on Nutrient broth No. 2 agar plate and a single colony was dispersed again in Nutrient broth No. 2 and by the early time of the rest period it was incubated under shaking at 37 °C and 0.07 mL DMSO was added to each 0.8 mL of dispersion and stored at -80 °C until use.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient broth No. 2 was used for pre-incubation of test bacterial strains. The top agar prepared according to the standard defined in Ministry of Labour No. 77 was used (Bacto-Agar, Difco). Minimum glucose agar plate medium, which was prepared according to the same standard, was purchased and stored at room temperature until used.
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: For confirmation of genetic characteristics and sensitivity to known mutagens, bacterial strains stored by freezing at -80 °C were thawed and 100 µL of each thawed bacteria was inoculated in 30 mL of nutrient broth No. 2 and incubated at 37 °C under shaking for 10 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-Dose-finding test: 5, 20, 78, 313, 1 250 and 5 000 µg/plate (with and without S9 mix).
-Main study with S9 mix: 156, 313, 625, 1 250, 2 500 and 5 000 µg/plate.
-Main study without S9 mix: 39, 78, 156, 313, 625, 1 250, 2 500 and 5 000 µg/plate.
-Concentrations for the main study were determined as a result of the dose-finding test.

Vehicle / solvent:

- Vehicle: water for injection
- Justification for choice of solvent/vehicle: Water for injection of Japanese Pharmacopoeia was selected as the medium of this test substance because this substance is stable in water and the provided test material is an aqueous solution containing 30 % of the test material.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

PRE-INCUBATION OF TEST BACTERIUM
The test bacterium strain frozen and stored at -80 °C was thawed, 100 μL of it was inoculated in 30 mL of Nutrient broth No.2, and incubated at 37 °C under shaking for 10 hours. Remaining thawed bacterium strain was all discarded after inoculation. The inoculated Nutrient broth No.2 was stored in a refrigerator before the start of shaking incubation.
After the end of pre-incubation, the optical density (OD value) of bacterial suspension was measured with a photoelectric spectrophotometer, and confirmed that the viable count calculated from the OD value was almost constant (> 10^9 cells/mL).

PREPARATION OF S9 MIX
S9 was thawed. This thawed S9 was added to cofactor solution, which was prepared before use, to make 10 v/v% solution, and agitated gently to prepare the S9 Mix of the following composition. The prepared S9 Mix was stored in a refrigerator until it was used.
Composition in each mL of S9 Mix: S9 0.1 mL, MgCl2 8.0 µmol, KCl 33.0 µmol, Glucose 6 phosphate 5.0 µmol, NADPHA 4.0 µmol, NADH 4.0 µmol and Sodium phosphate buffer (pH 7.4)100.0 µmol.

TEST PROCEDURE
-A 0.1 mL volume of the test material solution was dispensed into a test tube, 0.5 mL of 0.1 M phosphate buffer solution (pH 7.4) if there was no metabolic activation or S9 Mix if there was metabolic activation was added, and then 0.1 mL of respective bacterial suspension was added, and pre-incubated under shaking at 37 °C for 20 minutes. To this, 2.0 mL of top agar was added, and after agitation, it was poured over minimum glucose agar plate medium. Similar operations were performed for the negative (solvent) control and positive control.
-Minimum glucose agar plate medium was prepared 2 plates each for each dose of all of negative (solvent) control, positive control and test substance. In addition, for S9 Mix, 0.1 M phosphate buffer, Nutrient broth, and test substance of high concentration, sterility test was performed, confirming existence or absence of contamination of microorganisms.
-The number of revertant colonies that appeared after incubation at 37 °C for 48 hours was counted, and existence or absence of antibacterial activity was observed by a stereoscopic microscope, and existence or absence of precipitation of test substance was observed visually. The counting of number of revertant colonies was performed by the equipment measurement method using counting loss correction of the equipment.
The results are shown by the actual number and mean value of number of revertant colonies on each plate. In addition, dose-response curve was added for each bacterium strain.

DOSE CONCENTRATIONS
-In order to set the dose levels of each test, a dose finding test was performed, and the number of revertant colonies, existence or absence of antibacterial activity and precipitation were observed.
-From the results of dose finding test, 5 000 μg/plate was set as the high dose and the common ratio was set to be 4, and a total of 6 doses were set.
-In the dose finding test the test material showed antibacterial activity at more than 1 250 μg/plate to Salmonella typhimurium TA100, TA1535, TA98, TA1537 without metabolic activation, and at dose of 5 000 μg/plate with metabolic activation. In both presence and absence of metabolic activation, no precipitation was observed at any dose step, and no increase of more than 2 times of number of revertant colonies relative to the negative (solvent) control was observed in any bacterium strains.
-From the above result, for Salmonella typhimurium TA100, TA1535, TA98, TA1537 in reference to the dose where antibacterial activity was observed, the dose steps of this test were set 1 250 μg/plate for the case of no metabolic activation and 5 000 μg/plate for the case of metabolic activation as the high dose, and setting the common ratio to be 2, dose steps were set 6 doses in total.
-Because no antibacterial activity was observed for Escherichia coli WP2 uvrA regardless of existence or absence of metabolic activation in the dose finding test, 5 000 μg/plate was set as the high dose and the common ratio was set to be 2, with a total of 5 doses set.

PREPARATION OF POSITIVE CONTROL SUBSTANCES
Among positive control substances, NaN3 was prepared to the following test concentration using water for injection of Japanese Pharmacopoeia and other positive controls were prepared to their respective test concentrations using DMSO. The prepared positive control substances were frozen at -20 °C and stored and were thawed at time of use. The frozen storage period was one year after the preparation of the positive control substance stock solution that was used after diluting to the test concentrations and the item diluted to the test concentration was 3 months after dispensing. It was confirmed that the frozen positive control substances are stable for more than 1 year.

Evaluation criteria:

When the number of revertant colonies of the treated group with the test material increased more than 2 times relative to the negative (solvent) control and the dose-response relationship and repeatability were evident, it was judged to be positive.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

-The test material did not increase the number of revertant colonies by more than 2 times of negative (solvent) control in any test with bacterium strain regardless of existence or absence of metabolic activation.
-It was concluded that the test material had no mutagenic potency under the conditions of the study.

CONTROL DATA
-There was no existence of intermingled bacteria in Nutrient broth, S9 Mix, 0.1 M phosphate buffer or test material of high concentration used in the test. This was confirmed by a sterility test.
-The number of revertant colonies of the negative (solvent) control and positive control were within the standard values based on background data. In addition, the positive control produced revertant colonies of more than 2 times of the negative (solvent) control in respective bacterium strains, demonstrating that the test was conducted appropriately and was therefore valid.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material did not produce precipitation at any dose step with or without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY
-The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action to Salmonella typhimurium TA98, TA100, TA1535, TA1537 and at more than 5 000 μg/plate with metabolic activation.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material is not considered to have mutagenic potency.

Executive summary:

The potential of the test material to cause mutagenic potency was investigated using five strains of bacteria: Salmonella typhimurium TA98, TA100, TA1535, and TA 1537 and Escherichia coli WP2 uvrA, both in the presence and absence of metabolic activation in the form of S9 mix.

A 0.1 mL volume of the test material solution was dispensed into a test tube, 0.5 mL of 0.1 M phosphate buffer solution (pH 7.4) if there was no metabolic activation or S9 Mix if there was metabolic activation was added, and then 0.1 mL of respective bacterial suspension was added, and pre-incubated under shaking at 37 °C for 20 minutes. To this, 2.0 mL of top agar was added, and after agitation, it was poured over minimum glucose agar plate medium. Similar operations were performed for the negative (solvent) control and positive control. Duplicate tests were performed for all test groups. The number of revertant colonies that appeared after incubation at 37 °C for 48 hours was counted.

The test material did not increase the number of revertant colonies by more than 2 times the negative (solvent) control in any test with bacterium strain regardless of existence or absence of metabolic activation.

There was no existence of intermingled bacteria in Nutrient broth, S9 Mix, 0.1 M phosphate buffer or test material of high concentration used in the test. This was confirmed by a sterility test. The number of revertant colonies of the negative (solvent) control and positive control were within the standard values based on background data. In addition, the positive control produced revertant colonies of more than 2 times of the negative (solvent) control in respective bacterium strains, demonstrating that the test was conducted appropriately and was therefore valid. The test material did not produce precipitation at any dose step with or without metabolic activation.

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action to Salmonella typhimurium TA98, TA100, TA1535, TA1537 and at more than 5 000 μg/plate with metabolic activation.

Under the conditions of this study the test material is not considered to have mutagenic potency.