Acid D,L-aspart

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)

Rationale for reliability incl. deficiencies:

other: Well documented study according to international accepted guidelines and GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

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:

Acid D,L-aspart

EC Number:

210-513-3

EC Name:

Acid D,L-aspart

Cas Number:

617-45-8

Molecular formula:

C4H7NO4

IUPAC Name:

aspartic acid

Test material form:

solid: crystalline

Details on test material:

Test item D,L-aspartic acid
Alternative name D,L-aszparaginsav cfn. 100% Hat.
Batch No. S27170N
Physical state Solid
Loss on drying 13.5%
Active ingredient in dry substance 98.3%
Chloride ion 200 μg / g
Manufacturing date 20 July 2012
Expiry date 20 January 2014
Storage 15-30°C
Dose correction factor 1.176

Method

Target gene:

In addition to histidine or tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of 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 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Frozen stock cultures of all strains were prepared from the disc cultures immediately. The identification codes and expiry dates of the actual applied
stock cultures were the following:
Salmonella typhimurium TA98; 111005 Expiry date: 05 October 2013
Salmonella typhimurium TA100; 111005 Expiry date: 05 October 2013
Salmonella typhimurium TA1535; 111005 Expiry date: 05 October 2013
Salmonella typhimurium TA1537; 111005 Expiry date: 05 October 2013

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Frozen stock cultures of all strains were prepared from the disc cultures immediately. The identification codes and expiry dates of the actual applied
stock culture were the following:
Escherichia coli WP2 uvrA; 111005 Expiry date: 05 October 2013

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Justification of concentrations: Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test). The D,L-aspartic acid concentrations planned for the main experiments: 5000, 1581, 500, 158 and 50 μg/plate.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide; Ultrapure water

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Based on the solubility test, the stock solution with a concentration of 5 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate of the test item. The revertant colony numbers of vehicle control plates with and without S9 Mix were in line the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains. In comparison with the revertant colony numbers of the vehicle control plates sporadic changes slightly lower or higher revertant colony counts were observed in both strains examined at the test item.

In comparison with the revertant colony numbers of the vehicle control plates sporadic changes, slightly higher revertant colony counts were observed in S. typhimurium TA98 in the concentration range of 5000-15.8 μg/plate, without metabolic activation (-S9 Mix), furthermore in TA100 at the concentration of 5000 μg/plate, with addition of metabolic activation (+S9 Mix). These obtained changes were considered as reflecting the biological variability of the applied test system. The observed revertant colony numbers remained in the vehicle control data range, however were below the corresponding historical control data range (without any biological significance) in S. typhimurium TA100, in the concentration range of 500-15.8 μg/plate, in absence (-S9 Mix) and at the concentration level of 5 μg/plate in presence of metabolic activation (+S9 Mix). The experimental results (revertant colony numbers per plate, mutation factors, standard deviations) are summarised in Table 8 (Appendix I) and in Tables 11-12 (Appendix II).

Applicant's summary and conclusion

Conclusions:

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

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. 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 D,L-aspartic acid has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.