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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Mutagenicity in bacteria: negative (OECD 471) Gene mutation in mammalian cells: negative (OECD 476) Chromosome aberration: negative (OECD 473)
Link to relevant study records
Reference
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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- and guideline compliant
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his; trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from liver of phenobarbitaland β-naphthoflavone rats
Test concentrations with justification for top dose:
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 0.2; 1; 5; 25 and 50 μg/plate

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 20.8; 104; 520; 2 600 and 5 200 μg/plate

3rd Experiment
Strain: TA 100
Doses: 0; 31.3; 62.5; 125; 250 and 500 μg/plate

4th Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 20.8; 104; 520; 2 600 and 5 200 μg/plate (TA 1535, TA 1537, TA 98 and E. coli)
0; 2; 10; 50; 250 and 500 μg/plate (TA 100)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: limited solubility of the test item in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD),9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

1. Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. ( 5), with the exception of the amino acid solution, which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

2. Preincubation period: 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.



Evaluation criteria:
1. Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test item are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
2. Toxicity
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
3. Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
• The sterility controls revealed no indication of bacterial contamination.
• The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
• The titer of viable bacteria was ≥ 108/mL.
Statistics:
none
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:
cytotoxicity
Remarks:
from about 25 μg/plate onward in the standard plate tests; from about 250 μg/plate onward in the preincubation assay
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+
revertants, reduction in the titer) was observed in the standard plate tests depending on the
strain and test conditions from about 25 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the
number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain
and test conditions from about 250 μg/plate onward.

SOLUBILITY
Test item precipitation was found at 5 200 μg/plate onward with and without S9 mix.

MUTAGENICITY
T
he test item did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay).
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that ASA is not a mutagenic test item in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The test item was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay according to OECD 471 and GLP. The substance was tested in the standard plate test and in the preincubation test both with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. As a result, no increase in the number of revertants was found, so that the substance was not found to be mutagenic under the condition of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay according to OECD 471 and GLP. The substance was tested in the standard plate test and in the preincubation test both with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. As a result, no increase in the number of revertants was found, so that the substance was not found to be mutagenic under the condition of the test.

The substance was assessed for its potential to induce gene mutations at the HPRT locus in a study performed according to OECD 476 and GLP using V79 cells of the Chinese hamster. In the main experiments of this study (with and without S9 mix) the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 10E6cells. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

The potential to induce structural chromosomal aberrations in human lymphocytes was assessed in a study in accordance with OECD 473 and GLP in two independent experiments, each with and without S9 mix. The highest applied concentration in the pre-test on toxicity (3500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. In the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations being far in the range of test item precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and precipitating concentrations.


Justification for selection of genetic toxicity endpoint
Guideline study according to GLP

Justification for classification or non-classification

The available data are conclusive but not sufficient data for classification with regard to genetic toxicity in accordance with Directive 67/548/EEC or the CLP Regulation (EC) No 1272/2008.