5-potassium hydrogen L-glutamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997, corrected 26th June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

5-potassium hydrogen L-glutamate

EC Number:

243-094-0

EC Name:

5-potassium hydrogen L-glutamate

Cas Number:

19473-49-5

Molecular formula:

C5H9NO4.K

IUPAC Name:

potassium 5-oxido-5-oxo-L-norvaline

Test material form:

solid

Specific details on test material used for the study:

Name and Data of Test Item

Name: L-Glutamic acid potassium salt monohydrate[1]
Batch number: VG29748063 / Vendor Lot: SLCJ1327
CAS number: 6382-01-0
Molecular formula: C5H8KNO4 x H2O
Molecular weight: 203.23 g/mol (anhydrous form: 185.22 g/mol)
Description: White powder
Expiry date: 01 December 2022
Purity: 100 %
Storage conditions: Room temperature (15-25 ºC)

[1] Anhydrous substance name: 5-potassium hydrogen L-glutamate, CAS: 19473-49-5

Test item information was based on written information given by the Sponsor. Certificate of Analysis is attached to this report (Appendix VI).

Method

Target gene:

In addition to a histidine or a tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Freshly prepared S9 mix.

Test concentrations with justification for top dose:

Justification of concentrations:
Choice of the concentrations is done on the basis of the solubility trial and available background information [12] about the test item, in accordance with OECD 471 Guideline [6] recommendations.

The L-Glutamic acid potassium salt monohydrate concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
In the main experiments the test item solutions were prepared in the testing laboratory using ultrapure water (ASTM Type I). The formulation details are summarized in Table 1.
At the preparation of the test item solutions a correction (multiplier) factor of 1.097 – regarding monohydrate correction − was taken into consideration.

Vehicle / solvent:

ultrapure water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD, 2-Aminoanthracene, 2AA

Details on test system and experimental conditions:

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)

The test item solutions were prepared from the test item with ultrapure water (ASTM Type I) to obtain the dosing solutions (see: Table 1).
For the selected concentration range, the noticed solubility (adequately soluble) and available background information were taken into consideration in accordance with the recommendations in OECD 471 guideline [6].
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
In the main experiments the test item solutions were prepared in the testing laboratory using ultrapure water (ASTM Type I).
At the preparation of the test item solutions a correction (multiplier) factor of 1.097 – regarding monohydrate correction − was taken into consideration.
The test solutions were freshly prepared at the beginning of the experiments.

Controls

The tests were performed with parallel running controls: untreated, vehicle and positive reference. Table 5 contains the content of the control treatment mixtures.

Procedure for the Initial Mutation Test

A standard plate incorporation procedure was performed as an initial mutation test. Bacteria (cultured in Nutrient Broth No.2. (Section: 5.4.2)) were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per controls or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).

The content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain* 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL
* Salmonella typhimurium TA98, TA100, TA1535, TA1537 or E. coli WP2 uvrA, respectively.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37°C for about 48 hours.

Procedure for the Confirmatory Mutation Test

A pre-incubation procedure [1], [2], [5] and [6] was performed, as a confirmatory mutation test. Before the overlaying of the test item, the bacterial culture (5.3.6) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
The content of the tubes during incubation:
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain1) 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL
* Salmonella typhimurium TA98, TA100, TA1535, TA1537 or E. coli WP2 uvrA, respectively.
After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method (6.3). The entire test consisted of non-activated and activated test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37 °C for about 48 hours.

Rationale for test conditions:

The study includes a preliminary solubility trial, an initial mutation test (plate incorporation test) and a confirmatory mutation test (pre-incubation test).
In the initial mutation test the plate incorporation method was used.
Remark: in the frame of this study an informatory toxicity test, based on the available background information [12] was considered not necessary.

The pre-experiment on solubility of the test item was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance in the final report, but the raw data of this test will be archived under the study code of present study.

Concentrations:

Justification of concentrations:
Choice of the concentrations is done on the basis of the solubility trial and available background information [12] about the test item, in accordance with OECD 471 Guideline [6] recommendations.

Solubility Test

In the solubility trial the test item solubility was checked in ultrapure water (ASTM Type I), at the concentration of 50 mg/mL (with monohydrate correction). The test item was adequately soluble at this concentration level, and a clear solution was obtained.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 (Section: 5.4.2) for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37oC Benchtop Incubator Shaker.

Evaluation criteria:

A result is considered positive if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated, vehicle control, positive control and the test plates were determined visually by manual counting and the mean values, standard deviations and the mutation rates were calculated.

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.3	0.81	25.0	0.77	103.0	1.10	106.7	1.01	10.3	1.07	11.7	0.81	6.7	0.95	10.7	1.39	38.0	0.70	46.3	1.03
DMSO Control	24.3	1.00	21.3	1.00	–	–	100.0	1.00	–	–	12.0	1.00	6.3	1.00	11.7	1.00	–	–	47.7	1.00
Ultrapure Water Control	24.0	1.00	32.3	1.00	93.3	1.00	105.7	1.00	9.7	1.00	14.3	1.00	7.0	1.00	7.7	1.00	54.3	1.00	45.0	1.00
5000	28.0	1.17	23.3	0.72	113.7	1.22	95.3	0.90	7.0	0.72	12.7	0.88	5.7	0.81	8.0	1.04	41.7	0.77	49.0	1.09
1600	23.7	0.99	30.3	0.94	113.7	1.22	101.3	0.96	9.0	0.93	10.3	0.72	7.3	1.05	9.0	1.17	39.7	0.73	52.0	1.16
500	23.3	0.97	22.7	0.70	97.3	1.04	96.0	0.91	14.0	1.45	11.0	0.77	7.3	1.05	8.7	1.13	47.3	0.87	52.3	1.16
160	19.3	0.81	20.3	0.63	105.7	1.13	77.7	0.74	10.3	1.07	13.0	0.91	7.7	1.10	7.7	1.00	36.3	0.67	47.7	1.06
50	23.7	0.99	26.3	0.81	96.0	1.03	76.0	0.72	11.3	1.17	11.7	0.81	6.0	0.86	6.7	0.87	45.3	0.83	45.0	1.00
16	27.7	1.15	21.0	0.65	100.0	1.07	84.7	0.80	9.7	1.00	13.0	0.91	6.3	0.90	8.7	1.13	40.3	0.74	44.7	0.99
NPD (4mg/plate)	472.0	19.40	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	650.7	6.97	–	–	1020.0	105.52	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	292.0	46.11	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	632.0	11.63	–	–
2AA (2mg/plate)	–	–	944.0	44.25	–	–	917.3	9.17	–	–	159.0	13.25	–	–	185.7	15.91	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	318.7	6.69

MR:Mutation Rate;DMSO: Dimethyl sulfoxide;NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;

2AA: 2-Aminoanthracene

Remarks:          Ultrapure waterwas applied as vehicle of the test item and the positive control substances SAZ and MMS. The DMSO was applied as vehicle of the positive control substances NPD, 9AA and 2AA. The mutation rate obtained at the test item, at the untreated control; furthermore, at SAZ and MMS refers to the ultrapure water. The mutation rate obtained at NPD, 9AA and 2AA refers to DMSO.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item L-Glutamic acid potassium salt monohydrate has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

Purpose of the study:

The test itemL-Glutamic acid potassium salt monohydratewas tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation:

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

Experimental phases:

The study included a preliminary solubility trial, an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Vehicle, test item concentrations, rationale for dose selection:

For the selected concentration range, the noticed solubility (adequately soluble) and available background information [12] were taken into consideration in accordance with therecommendations in OECD 471 guideline [6]:

±S9:  5000, 1600, 500, 160, 50 and 16 µg/plate.

At the preparation of the test item solutions a correction (multiplier) factor of 1.097 – regarding monohydrate correction − was taken into consideration.

Solubility, precipitation:

In the initial and confirmatory mutation tests(following the plate incorporation and pre-incubation procedures), no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Cytotoxicity results:

In the initial and confirmatory mutation tests inhibitory effects of the test item on bacterial growth were not observed in the examined strains. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained within the range of the biological variability of the applied test system and thebackground lawn development was not affected in any case.

Mutagenicity results:

The revertant colony numbers of vehicle control (ultrapure water (ASTM Type I)) plates with and without S9 Mix demonstrated the characteristic mean numbers of spontaneous revertantsthat were in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment withL-Glutamic acid potassium salt monohydrateat any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusion:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test itemdid not induce gene mutations in the genome of the strains ofSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 and ofEscherichia coliWP2uvrA.

In conclusion, the test itemL-Glutamic acid potassium salt monohydratehas no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.