L-aspartic acid, sodium salt

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:

2022

Report date:

2022

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

Constituent 1

Reference substance name:

sodium 2-amino-3-carboxypropanoate

Cas Number:

323194-76-9

Molecular formula:

C4H7NO4.xNa

IUPAC Name:

sodium 2-amino-3-carboxypropanoate

Test material form:

solid: particulate/powder

Details on test material:

Name: L-aspartic acid, sodium salt monohydrate 1
Batch/Lot number: Z201125/ VG29848563
CAS number: 323194-76-9
Anhydrous substance name: L-aspartic acid, sodium salt, CAS: 17090-93-6
Appearance: Solid, white, crystalline powder
Purity: 98%
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed
container)

Specific details on test material used for the study:

5.1.1 Name and Data of Test Item

Test item: L-aspartic acid, sodium salt monohydrate
Lot No.: Z201125 / VG29848563
CAS No.: 323194-76-9 *
Purity: 99.9 %
Molecular formula: C4H6NO4Na • H2O
Molecular weight: 173.1 g/mol
Appearance: Solid, white crystalline powder
Expiry date: 24 November 2022
Storage conditions: At room temperature, protected from humidity (tightly closed container)
Safety precautions: According to the SDS

* The L-aspartic acid sodium salt (anhydrous) CAS Number is: 17090-93-6.

Test item information is based on written information provided by the Sponsor. The copy of Certificate of Analysis is attached (see: Appendix VII).

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 about the test item, in accordance with OECD 471 Guideline [6] recommendations.

The L-aspartic acid, sodium salt monohydrate concentrations investigated in the initial and confirmatory mutation tests:
±S9: 5000, 1600, 500, 160, 50 and 16 µg/plate.
As indicated in section 6.1.2, at the chosen top concentration precipitate was not expected on the minimal glucose agar plates.
The test solutions were freshly prepared at the beginning of the experiments as indicated in the Section 5.1.3 and Table 1.
At the preparation of the test item stock solution a correction (multiplier) factor of 1.116 (based on the monohydrate content of the test item) 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:

Controls

The tests were performed with parallel running controls: untreated, vehicle and positive reference. Table 6 contains the content of the control treatment mixtures.

Table 6: Summary of Controls
Summary of Controls:
Type of control Activation with S9 mix Vehicle
(Section 5.2.3.) Top agar Strain-specific positive chemical solutions
(Section 5.2.1.) Phosphate buffer
Untreated - - + - +
Untreated + - + - -
Vehicle - + + - +
Vehicle + + + - -
Positive control - - + + +
Positive control + - + + -

6.2 Experimental Method

This study followed the methods described by Ames et al. [1] and Maron and Ames [2], Kier et al. [3], Venitt and Parry [4], OECD Guideline No 471, 1997, 2020 [6], EPA Guideline, OPPTS 870.5100, 1998 [7] EC No 440/2008; B13/14, 2008 [8] and ICH Guidance: ICH; S2(R1), (2011)[9].

6.3 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. 2 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 and E. coli WP2 uvrA.

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.

6.4 Procedure for the Confirmatory Mutation Test

A pre-incubation procedure (in line with references [1], [2], [5] and [6]) was performed, as a confirmatory mutation test. Before the overlaying of the test item, the bacterial culture (Section: 5.3.6) and the S9 mix or phosphate buffer was added into appropriate tubes to allow 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. After the incubation period, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method (Section: 6.3). The entire test consisted of non-activated and activated test conditions (metabolic) 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:

Experimental phases:
The study included preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).


The pre-experiment on solubility of the test item and the concentration range finding test were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.


Concentrations:

Justification of concentrations:Selection of the concentration range was done on the basis of the preliminary solubility test and a concentration range finding test (informatory toxicity test) according to the OECD 471 guideline [6] recommendations.



Solubility Test

In the solubility tests, the test item behavior was investigated in the applied test system. In the solubility test, the test item was dissolved and further diluted in ultrapure water (ASTM Type I). The obtained solution with the solution of top agar (Section: 5.4.4) and phosphate buffer (Section: 5.5.3) were examined in a test tube without test bacterium suspension. The test item behavior in the vehicle and in the applied test system is summarized in Table 5.

Table 5: Behavior of the Test Item in Ultrapure Water
Concentration of test item
in water
(mg/mL) Solubility
in water Solubility in the top solution
(test item solution 100 µL
+ phosphate buffer 500 µL
+ top agar 2 mL) Test item concentration in the test tube
g/tube
50 Clear solution Clear solution 5000

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

Table 8: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimurium tester strains																Escherichia coli
	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	20.3	1.33	25.7	1.33	77.3	1.01	121.3	1.08	13.0	0.98	14.0	1.35	4.0	1.00	8.0	1.85	46.0	0.80	48.0	1.04
DMSO Control	18.3	1.00	16.7	1.00	–	–	116.7	1.00	–	–	11.7	1.00	5.0	1.00	6.3	1.00	–	–	44.3	1.00
Ultrapure water Control	15.3	1.00	19.3	1.00	76.7	1.00	112.3	1.00	13.3	1.00	10.3	1.00	4.0	1.00	4.3	1.00	57.7	1.00	46.0	1.00
5000	22.7	1.48	24.0	1.24	91.7	1.20	106.0	0.94	11.3	0.85	8.3	0.81	7.7	1.92	7.0	1.62	54.0	0.94	44.0	0.96
1600	19.7	1.28	14.3	0.74	99.0	1.29	101.3	0.90	11.7	0.88	10.0	0.97	6.3	1.58	5.0	1.15	53.0	0.92	45.0	0.98
500	15.0	0.98	20.3	1.05	94.3	1.23	103.0	0.92	11.3	0.85	10.7	1.03	7.0	1.75	4.0	0.92	37.0	0.64	42.3	0.92
160	16.3	1.07	21.7	1.12	76.0	0.99	108.3	0.96	12.0	0.90	11.0	1.06	4.0	1.00	4.7	1.08	53.7	0.93	52.0	1.13
50	17.3	1.13	19.0	0.98	82.0	1.07	99.7	0.89	10.0	0.75	11.7	1.13	6.0	1.50	6.0	1.38	36.7	0.64	49.0	1.07
16	14.0	0.91	17.7	0.91	81.7	1.07	109.0	0.97	12.7	0.95	11.0	1.06	6.7	1.67	5.7	1.31	33.0	0.57	46.7	1.01
NPD (4 mg/plate)	357.0	19.47	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 mg/plate)	–	–	–	–	514.7	6.71	–	–	796.7	59.75	–	–	–	–	–	–	–	–	–	–
9AA (50 mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	255.3	51.07	–	–	–	–	–	–
MMS (2 mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	676.7	11.73	–	–
2AA (2 mg/plate)	–	–	1294.7	77.68	–	–	1701.3	14.58	–	–	99.7	8.54	–	–	90.3	14.26	–	–	–	–
2AA (50 mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	201.7	4.55

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 water was 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-aspartic acid, sodium 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 item L-aspartic acid, sodium salt monohydrate was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation:

Salmonella typhimurium TA98, target mutation: hisD3052; mutation type: frameshift;

Salmonella typhimurium TA100, target mutation: hisG46; mutation type: base pair substitution;

Salmonella typhimurium TA1535, target mutation: hisG46; mutation type: base pair substitution;

Salmonella typhimurium TA1537, target mutation: hisC3076; mutation type: frameshift;

Escherichia coli WP2 uvrA, target mutation: trpE; mutation type: base pair substitution.

Exogenous metabolic activation:

The experiments were carried out in the presence and absence of a cofactor supplemented post-mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The S9 mix contained 10 % (v/v) S9.

Experimental phases:

The study included preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Vehicle, test item concentrations, rationale for dose selection:

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations of the test item were prepared and investigated in the main experiments:

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

For the selected concentration range, the noticed cytotoxicity (absent, noticed in the informatory toxicity test), the solubility (adequately soluble in the applied test system), and the laboratory’s experience with used strains (for concentration choice at TA1535, TA1537 and Escherichia coli WP2 uvrA strains) were taken into consideration based on the recommendations in OECD 471 guideline [6].

At the preparation of the test item solutions a correction (multiplier) factor of 1.116 (based on the monohydrate content of the test item) was taken into consideration.

Validity of the Study:

In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.

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 revertants that 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 colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases.

 

Solubility, precipitation:

Based on the solubility investigations, the test item was adequately soluble in ultrapure water (ASTM Type I); therefore, in the experiments it was dissolved (stock solution: 50 mg/mL) and further diluted in ultrapure water, accordingly.

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 the background lawn development was not affected in any case.

 

Mutagenicity results:

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with L-aspartic acid, sodium salt monohydrate at 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 item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

In conclusion, the test item L-aspartic acid, sodium salt monohydrate has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.