Magnesium dihydrogen di-L-aspartate

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Mutagenesis by normal metabolites in Escherichia coli: phenylalanine mutagenesis is dependent on error-prone DNA repair

Author:

Sargentini, N. J.; Smith, K.C.

Year:

1986

Bibliographic source:

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 161, Issue 2, July 1986, Pages 113-118

Materials and methods

Principles of method if other than guideline:

Scientific investigation. In search of a model for the production of 'spontaneous' mutations induced by DNA damage produced during normal metabolism, 19 amino acids were tested for mutagenicity in E. coli bacteria.

GLP compliance:

no

Remarks:

study performed prior to implementation of GLP

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aspartic acid

EC Number:

200-291-6

EC Name:

Aspartic acid

Cas Number:

56-84-8

Molecular formula:

C4H7NO4

IUPAC Name:

aspartic acid

Method

Target gene:

uvrB, uvrB, umuC, lac operon

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli, other: SR250 (uvrB5)

Details on mammalian cell type (if applicable):

derived from E coli K-12 strain, SR248 (wild-type)

Additional strain / cell type characteristics:

other: DNA repair-deficient strain

Species / strain 2

Species / strain / cell type:

E. coli, other: SR1034 ( uvrB5 umuC 122::Tn5)

Details on mammalian cell type (if applicable):

derived from E coli K-12 strain, SR248 (wild-type)

Additional strain / cell type characteristics:

other: DNA repair-deficient strain

Metabolic activation:

without

Test concentrations with justification for top dose:

2mM L-aspartic acid

Controls

Untreated negative controls:

yes

Remarks:

Glu 0 plates

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

Plate assay:
Cells were pelleted by centrifugation, washed once, and resuspended in PB (phosphate buffer) at 1.5 E+008 CFU/mL. Cells (0.1 mL) were spread on Glu-600 plates (lactose plates containing a growth-limiting amount of glucose, i.e., 600 µg/ml) ± the test compound at 2 mM and Glu-0 plates. Plates were incubated for 3 days at 37°C before scoring Lac+ mutants.

Tube assay:
Cells were grown overnight in SMM1, diluted 10E-005 with homologous medium ± phenylalanine at 2 mM, then dispensed into 9 tubes each (2-ml portions), and incubated with aeration for 48 h at 37°C. Then, cultures were assayed for mutants and viability.
For mutants, the plating conditions were: Lac+, 0.2 ml per Glu-0 plate; Rif‘, 1 ml plus 2.5 ml M-top agar per rifampicin plate; T6‘, 0.1 ml of 10-fold diluted cells plus 1 ml bacteriophage T6 lysate containing 10E+010 plaque- forming units (hold mixture 15 min at room temperature) plus 2.5 ml of R-top agar per R-plate.
Viability was determined on YENB plates. All plates were incubated for 2 days at 37°C (R-plates and YENB plates, 1 day). Median values for mutants and CFU were used to determine the median mutant frequencies for each assay. The phenylalanine effect was the ratio (test/control) of the median mutant frequencies.

Evaluation criteria:

Median values for mutants and CFU (colony forming units) were used to determine the median mutant frequencies for each assay.

Statistics:

Significant effects on mutagenesls were defined by either of two criteria.
That is, the mean mutant frequency + 1 SD (range) for the amino-acid-supplemented plates did not overlap the range for the control plates, and the mean relative (test/control) range for the uvrB strain did not overlap the mean relative range for the uvrB umuC strain.

Results and discussion

Test results

Key result

Species / strain:

E. coli, other: Both SR250 and SR1034

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not applicable

Applicant's summary and conclusion

Conclusions:

L-aspartic acid does not exhibit mutagenic activity under the conditions of this bacterial reverse mutation assay.