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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A mutagenicity study has been conducted in in-vitro test systems where the Ames test on Salmonella typhimurium and Escherichia coli (OECD 471), turned out to be negative.

This indicates clearly a non mutagenic potential in in-vitro conditions.

It was judged that the test item is non mutagenic in vitro.

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:
Experimental starting date: 13 February 2008 and Experimental completion date: 26 February 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- his+ and trp- trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-Naphthoflavone induced rat liver S9 is used as the metabolic activation system.
Test concentrations with justification for top dose:
- In the pre-experiment the concentration range of the test item was 3 – 5000 microg/plate. The pre-experiment is reported as experiment I.
Since no toxic effects were observed 5000 microg/plate were chosen as maximal concentration.

- The concentration range included two logarithmic decades. The following concentrations were tested in experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 microg/plate
Vehicle / solvent:
On the day of the experiment, the test item was dissolved in DMSO (MERCK, D-64293 Darmstadt; purity > 99 %).
The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
The test item precipitated in the test tubes and on the agar plates from 333 - 5000 microg/plate.
The undissolved particles had no influence on the data recording.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in deionised water
Positive control substance:
sodium azide
Remarks:
Sodium azide is the positive control without metabolic activation for strains TA 1535 and TA 100. NaN3 is used at 10 microg/plate.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
other: 4-nitro-o-phenylene-diamine, (4-NOPD)
Remarks:
4-NOPD is the positive control without metabolic activation for strains TA 1537 and TA 98. 4-NOPD is used at 10 microg/plate for TA 98 and at 50 microg/plate for TA 1537.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in deionised water
Positive control substance:
methylmethanesulfonate
Remarks:
MMS is the positive control without metabolic activation for strains WP2 uvrA. MMS is used at 3 microg/plate for WP2 uvrA.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
2-AA is the positive control with metabolic activation for strains TA1535, TA100, TA1537, TA98 and WP2 uvrA. 2-AA is used at 2.5 microg/plate for TA1535, TA1537, TA98 and TA100 and used at 10 microg/plate for WP2 uvrA.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- For the experiment I: plate incorporation
- For the experiment II: pre-incubation test

Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures:From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 LL ampicillin (25 Lg/mL) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
- 8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
- 5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar: The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt.

NUMBER OF REPLICATIONS: Each concentration, including the controls was tested in triplicate

Evaluation criteria:
Evaluation of Results
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.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

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.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: S. typhimurium TA 1535, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effect, evident as a reduction in the number of revertant were observed in the experiment II with metabolic activation at 5000 microg/L
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No toxic effect observed neither in Experiment I and II (with and without metabolic activation).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxic effect, evident as a reduction in the number of revertant were observed in the experiment II with and without metabolic activation at 2500 and 5000 microg/L with S9 mix and at 500 microg/plate without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 microg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 microg/plate

The plates incubated with the test item showed normal background growth up to 5000 microg/plate with and without S9 mix in both experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations:

 Strain     Experiment I Experiment II    
   Without S9 mix With S9 mix  Without S9 mix  With S9 mix 
 TA 1535  /  /  /  5000
 TA 1537  /  /  5000 2500/5000 
 TA 98  /  /  /  5000
 TA 100  /  /  /  5000
 WP2 uvrA  /  /  /  /

/ no toxic effects observed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance 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.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In 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.

Conclusions:
In 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, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 microg/plate

- Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 microg/plate

The plates incubated with the test item showed normal background growth up to 5000 microg/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 in experiment I. In experiment II, toxic effects were observed at 5000 microg/plate in strain TA 1537 without metabolic activation and in

strains TA 1535, TA 1537, TA 98, and TA 100 with metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In 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, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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

Additional information

A reliable bacterial reverse mutation test is available and was performed according to OECD/EC guidelines.

Regarding this in vitro test, the Ames test turnes to be negative on Salmonella typhimurium and Escherichia coli

The test item was found to be be negative in the Ames test (OECD 471).

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).