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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
His (Salmonella typhimurium)
Trp (E. coli)
Species / strain
Species / strain:
other: Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone - induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment (SPT): 0; 22; 110; 550; 2750 and 5500 μg/plate (TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA)
2nd Experiment (SPT): 0; 0.4; 2; 10; 50 and 100 μg/plate (TA 1535, TA 100, TA 1537); 0; 0.8; 4; 20; 100 and 200 μg/plate (TA 98). Reason: Strong bacteriotoxicity was observed in the 1st Experiment.
3rd Experiment (PIT): 0; 0.2; 1; 5; 25 and 50 μg/plate (TA 1535, TA 100, TA 1537); 0; 0.4; 2; 10; 50 and 100 μg/plate (TA 98); 0; 22; 110; 550; 2750 and 5500 μg/plate (E. coli)
Vehicle:
Dimethylsulfoxide (DMSO)
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and conditions:
POSITIVE CONTROLS:
With S9 mix:
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix:
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 – 72 hours at 37 °C

NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY: 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
Evaluation 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 ≥ 10 e8/mL.

The test item is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test item is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
depending on the strain and test conditions
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 10 μg/plate onward. No bacteriotoxicity was observed with E. coli WP2uvrA up to the highest applied dose of 5500 μg/plate in the absence and presence of metabolic activation. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 25 µg/plate onward using four salmonella tester strains. In the preincubation assay with E. coli WP2uvrA weak bacteriotoxicity was observed only in the presence of metabolic activation at 5500 μg/plate.

PRECIPITATION: Test item precipitation was found from 550 μg/plate onward with and without S9 mix.

MUTAGENICITY: The 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 three experiments carried out independently of each other (standard plate test and preincubation assay). (see table 1)

Any other information on results incl. tables

Table 1: Number of revertants in the control or after treatment with the test substance in the a.) standard plate test and b.) preincubation test

a.) Standard plate test (0.4 - 100 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 1535 no 20 0.9 no negative
  yes 17 0.9 no negative
TA 100 no 121 1.0 no negative
  yes 132 1.1 no negative
TA 1537 no 12 1.0 no negative
  yes 14 0.9 no negative
TA 98  no 26 1.1 no negative
   yes 29 1.0 no negative
b.) Standard plate test (22 - 5500 µg/plate)
E. coli WP2 uvrA no 47 1.1 no negative
  yes 50 1.1 no negative
Preincubation test (0.2 - 50 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 1535 no 14 1.0 no negative
  yes 16 0.9 no negative
TA 100 no 95 1.0 no negative
  yes 97 1.0 no negative
TA 1537 no 9 0.9 no negative
  yes 9 0.9 no negative
TA 98 no 32 1.1 no negative
  yes 36 1.1 no negative
E. coli WP2 uvrA no 40 1.1 no negative
  yes 38 1.2 no negative

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative