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Genetic toxicity: in vitro

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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
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Direct plate incorporation method.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
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
Constituent 2
Reference substance name:
L-Asparaginsäure
IUPAC Name:
L-Asparaginsäure
Constituent 3
Reference substance name:
S-(+) amino succinic acid
IUPAC Name:
S-(+) amino succinic acid
Test material form:
solid: crystalline
Details on test material:
Supplier: Sponsor.
Batch No.: PRCA 015.
CAS No. 56-84-8.
Appearance: White, crystalline.
Purity: Approx. 99 %.
pH: 2.5 – 3.5 (4 g/L at 20 °C).
Melting range: 265 – 271 °C.
Solubility in water: 5 g/L at 25°C.
Conditions of storage: In the refrigerator, in the dark, may be used under light.
Date of expiry: 31 December 2002.

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction of rat liver, induced with Aroclor 1254.
Test concentrations with justification for top dose:
62 to 5000 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: water
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
2-nitrofluorene
sodium azide
other: 2-aminoanthracene; 1,8-dihydroxy-anthraquinone; 4-nitro-o-phenylenediamine; t-butyl-hydroperoxide
Details on test system and experimental conditions:
Preliminary toxicity test: Different concentrations of test substance solutions were mixed with phosphate buffer, bacteria (TA100) and top-agar, and spread over a plate with minimal agar. The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment.
The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 108 cells per mL.

Evaluation criteria:
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.

Statistics:
Means and standard deviation were calculated for the number of mutants in every concentration group.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was not toxic up to 5000 µg/petri dish.
A precipitate was visible when the test substance was mixed with the agar at the 5000 µg/plate samples.
In the preliminary test and in the main test no toxicity was seen up to 5000 µg/plate.

There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.
For details see the attachment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

L-aspartic acid did not induce mutagenicity either with or without metabolic activation in any of the bacterial strains at any level evaluated with the plate incorporation method at dose levels up to 5000 µg/plate.
Executive summary:

L-Aspartic acid was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part 8.13/14.

The test substance, dissolved in water, was tested at concentrations ranging from 62 to 5000 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An  independent   repetition of the experiment was performed.

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system. No toxicity of the test substance to the bacteria was observed up to 5000 µg per plate.

At the 5000 µg/plate samples a precipitate was visible when the test substance was mixed with the agar.

L-aspartic acid did not induce mutagenicity either with or without metabolic activation in any of the bacterial strains at any level evaluated with the plate incorporation method at dose levels up to 5000 µg/plate.

 

 

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