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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-07-02 to 2002-12-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Deviations from the OECD 471 (1997): titre demonstration was invalid for two strains (experiment 2: one strain invalid only); S9 in the S9 mix was only 4 % (guideline: 5 to 30 %); spontaneous revertants counts were not for all strains within historical controls given by the laboratory; historical controls for the spontaneous revertants were only given for cultures without S9 mix; determination of cytotoxicity was not plausible; cytotoxicity occurrs at concentration levels at which a separate cytotoxicity test does not lead to assume toxicity; second experiment used only three concentration instead of five concentration; variable results in the experiments were observed for the different strains, which lead to assumption that something is wrong with the strains; historical data for the positive control are missing
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies ("Remarks" field)
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2003-09-29
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: closed, light-protected plastic vessel
Target gene:
TA97a: his D 6610
TA98: his D3052
TA100 and TA1535: his G 46
TA102: his G 428
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium, other: TA97a
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 fraction was prepared from livers of male Wistar rats. Rats received 80 mg phenobarbital/kg bw via intraperitoneal injection thrice on one day and 80 mg β-naphthoflavone/kg bw via oral adminsitration on the following day.
- method of preparation of S9 mix:
water, dest., steril: 19.75 mL
phosphate buffer (pH: 7.4 ± 0.1): 25.0 mL
NADP solution: 2.0 mL
glucose-6-phosphate solution: 0.25 mL
KCl + MgCL2* 6H20 + water dest.: 1.0 mL
S9 fraction (4 %): 2.0 mL
Test concentrations with justification for top dose:
Experiment 1: 0.005, 0.015, 0.05, 0.15 and 0.5 mg/plate
Experiment 2: 0.125, 0.25 and 0.5 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
For each test procedure, a stock solution was prepared with the maximum soluble amount of the test article in DMSO. This was done by careful heating to about 50 °C. A dilution series was prepared with the cooled, sterile-filtered solution.

- Justification for choice of solvent/vehicle: since the test item is not soluble in water, a stock solution with the maximum solubility (5 g/L) in DMSO has been prepared. The solubility limit was observed at the concentration of 0.5 mg/plate based on precipitates observed in the final mixture.

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
cumene hydroperoxide
other: 4-Nitro-1,2-phenylendiamine (NPD) and 2-Aminoantracene (2-AA)
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation; experiment 1) and preincubation (experiment 2)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: quadruple
- Number of independent experiments : 2

TREATMENT:
1) Plate incorpotation method:
Four plates with and without S9 mix were used per strain and dose.
Top agar was melted and histidine / biotin solution was added. After mixing and cooling to 45° C, the mixture was kept in a water bath.
0.1 mL of the test article solution was placed in a sterile tube followed by adding 2 mL top agar, 0.1 mL of the overnight bacterial culture and 0.5 ml of phosphate buffer (testing without S9) or 0.5 mL S9 mix (testing with S9).
The tube was then swirled and the content was poured onto a plate containing minimal glucose. The content was spread evenly onto the plate. Then, the plate was sealed and covered with brown paper for protection agaist UV radiation. The plate was allowed to cool down until the agar solidifies and, then, the plate was turned over and placed in an incubator (37 ° C) for a duration of 48 hours.

2) Pre-incubation method:
Four plates with and without S9 mix were used per strain and dose.
Top agar was melted and histidin/biotin solution was added. After mixing and cooling to 45 °C, the mixture was kept in a water bath.
0.1 mL of the test item solution was placed in a sterile tube followed by adding 0.1 mL of the overnight bacterial culture and 0.5 mL phosphate buffer (testing without S9) or 0.5 mL S9 mix (testing with S9). These preparations were incubated for 20 minutes at 37° C. During the incubation, the preparations were gently shaken in order to ensure aeration of the preparations. Then, 2 mL top agar were added to the preparations and the content of the tube is poured onto a minimal glucose plate and the mixture was evenly spread. The plate was sealed and covered with brown paper for protection agaist UV radiation. The plate was allowed to cool down until the agar solidifies and, then, the plate was turned over and placed in an incubator (37 ° C) for a duration of 48 hours.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decline in the number of revertantes
Rationale for test conditions:
The solubility limit was observed at the concentration of 0.5 mg/plate based on precipitates observed in the final mixture.
Evaluation criteria:
A test item is considered to be a mutagen, if the test item induces an increase twice the number of mutants as seen in the negative control (induction rate I≥ 2) in at least one of the strains used, with or without S9-mix, or shows a concentration-response relationship.
Statistics:
Mean ± standard deviations
Key result
Species / strain:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
A) Experiment 1:
The controls used to validated the genotypes as well as to check for sterility met all the required criteria. The expected number of revertants was found for the spontaneous revertants and the positive controls, and they were within the historical control data of the laboratory. The values of the titre control were below the theoretical value for 2 strains (TA 97a and 98). Since all other criteria, however, were within the usual range, the test was nevertheless declared valid.

At the highest tested concentration of 0.5 mg/plate, cytotoxicity (criterion: decrease of revertant numbers) was not observed for any strain. At this concentration the additional toxicity control did not show any toxic effects. The other tested concentrations also showed no toxicity.

At the highest concentration, no significant increase in revertant numbers with and without metabolic activation was observed in any of the tested strains. A dose-response relationship could not be determined. In strain TA 97a with metabolic activation, a slight increase in numbers of revertants was observed, but the induction rate was below 2. Based on the findings, the test item is not considered to be a mutagen.

A) Experiment 2:
The controls used to validated the genotypes as well as to check for sterility met all the required criteria. The expected number of revertants was found for the spontaneous revertants and the positive controls, and they were within the historical control data of the laboratory. The values for the titre control were below the theoretical value for one strain (TA 97a). However, since all other criteria were within the usual range, the test was nevertheless declared as valid.

At the highest tested concentration of 0.5 mg/plate, cytotoxicity was not observed for any strain due to a decrease in revertant numbers. At this concentration the additional toxicity control did not show any toxic effects. The other tested concentrations also showed no toxicity.

At the highest concentration, no significant increase in revertant numbers with and without metabolic activation was observed in any of the tested strains. A dose-response relationship was not observed. Based on the findings, the test item is not considered to be a mutagen.

Please also refer to the field "Attached background material" below
Conclusions:
The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Based on a reliable study acceptable with restrictions, the substance was not observed to be mutagenic in a bacterial reverse mutation assay (OECD 471).

Justification for classification or non-classification

Genetic toxicity in vitro

The test substance should be considered void of genotoxicity based on a bacterial reverse mutation assay (OECD 471). Thus, the test substance has no mutagenic potenital and does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.