Registration Dossier

Diss Factsheets

Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 26, 2007 - April 18, 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed in accordance to Directive 2000/32/EC, L1362000, Annex 4D, OECD No. 471

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
Identity: AES
Batch No.: LSF7
Aggregate state
at RT: Solid
Colour: White
Purity: 80.3 % (w/w)
Stability in solvent: Stable for 30 days in water at room temperature, in
refrigerator, and in freezer
Storage: In the refrigerator at +2 - +8°C, desiccator
Expiration Date: February 15, 2008


Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
and the Escherichia coli strain WP2 uvrA.
Additional strain / cell type characteristics:
other: frame shift mutations and base-pair substitutions
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Dose Selection:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment
is reported as experiment I. Since minor toxic effects were observed 5000 µg/plate were chosen as maximal
concentration and eight concentrations were tested.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment I and II:
3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Vehicle / solvent:
The test item AES was dissolved in deionised water. The solvent was chosen because of its solubility properties. The pH value of the stock
solution was adjusted to a pH value of 7 with 1 N hydrochloric acid.
No precipitation of the test item occurred up to the highest investigated dose.
Details on test system and experimental conditions:
Concurrent untreated and solvent controls were performed.

Without metabolic activation:
Strains: TA 1535, TA 100
Name: sodium azide, NaN3
Purity: at least 99 %
Dissolved in: water deionised
Concentration: 10 µg/plate

Strains: TA 1537, TA 98
Name: 4-nitro-o-phenylene-diamine, 4-NOPD
Purity: > 99.9 %
Dissolved in: DMSO (purity > 99 %)
Concentration: 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Strain: WP2 uvrA
Name: methyl methane sulfonate, MMS
Purity: > 99.0 %
Dissolved in: water deionised
Concentration: 3 µL/plate

With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Name: 2-aminoanthracene, 2-AA
Purity: 97.5 %
Dissolved in: DMSO (purity > 99 %)
Concentration: 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),
10 µg/plate (WP2 uvrA)

The stability of the positive control substances in solution is unknown but a mutagenic
response in the expected range will be sufficient evidence of biological stability.

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as
spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames (6). In
this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice
(strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent
second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic
potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is
not considered biologically relevant.
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Additional information on results:
The assay was performed in two independent experiments both with and without liver microsomal activation.
Each concentration, including the controls, was tested in triplicate.

Any other information on results incl. tables

Only in experiment I in strain TA 1537 a reduction in the number of revertants (below a factor of 0.5) was observed from

2500 µg/plate up to 5000 µg/plate with and without metabolic activation.

Applicant's summary and conclusion

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, AES is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AES at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.