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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
Study period:
August 2010 - November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): 1-Ethyl-3-methylimidazolium ethylsulphate
- Physical state: liquid
- Analytical purity: Approx. 99 area-%
- Lot/batch No.: lot. 100003p040
- Stability under test conditions: guaranteed until 2012-02-28
- Storage condition of test material: Room temperature (dry storage)
- Other: appearance: yellowish, clear

Method

Target gene:
histidine (S. typhimurium) and tryptophane (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: 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)
- Properly maintained: yes
- Periodically checked for deep rough character (rfa); UV sensitivity (delta uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy via the spontaneous rate in each experiment.
Additional strain / cell type characteristics:
other: histidine auxothroph ( his-)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: 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)
- Properly maintained: yes
- Periodically checked for UV sensitivity. Tryptophan auxotrophy is checked in each experiment via the spontaneous rate.
Additional strain / cell type characteristics:
other: tryptophan auxotroph (trp-)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
- 1rst experiment (SPT = standard plate test: plate incorporation method according to Ames et al. 1975):
0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate; +/- S9 mix; 3 plates per dosis and control
- 2nd experiment (PIT = preincubation test described by Yahagi et al. 1977 + Matsushima et al. 1980):
0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate, +/- S9 mix, 3 plates per dosis and control
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility in water
Controls
Untreated negative controls:
other: sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: for +/- S9 mix, for S. typhimurium + E. coli; details see below
Details on test system and experimental conditions:
DETAILS ON POSITIVE CONTROLS:
1) with S9 mix:
2-aminoanthracene (2-AA): 2.5 μg/plate for S. typhimurium, 60 μg/plate for E. coli
2) without S9 mix:
- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG): 5 μg/plate for TA 1535, TA 100;
- 4-nitro-o-phenylenediamine (NOPD): 10 μg/plate for strain TA 98
- 9-aminoacridine (AAC): 100 μg/plate for strain TA 1537
- 4-nitroquinoline-N-oxide (4-NQO): 5 μg/plate for E. coli

METHOD OF APPLICATION: in agar (plate incorporation) + preincubation

DURATION
- Preincubation period: 20 min (Experiment 2)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 - 72 h

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
MUTAGENICITY:
- mean number of revertant colonies per plate and standard deviations for all groups
- in general: 5 doses of the test substance with max. of 5 mg/plate
- triplicates

TITER: determination only in experiments with s9 mix for negative control and the 2 highest doses

TOXICITY: detection for all groups via decrease of number of revertants, clearing of diminution of the background lawn, reduction in the titer

SOLUBILITY: recording precipitation; if not interfering with colony scoring, max. dose (5 mg/plate) is generally selected and analyzed


ACCEPTANCE:
valid, if
- number of revertant colonies in negative control within range of historical negative control data for each tester strain
- no indication of bacterial contamination in sterility control
- positive control substances +/- S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or above
- titer of viable bacteria was ≥ 10exp8/mL

ASSESSMENT:
- mutagenic in this test, if 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 - or + S9 mix
- non-mutagenic in this test, if number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two independent experiments

Results and discussion

Test results
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:
no cytotoxicity
Remarks:
up to the max. dose (5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
other: sterility control: no contamination
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:
Results of all groups were within the range of the historical negative control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test and in the preincubation assay up to the highest required concentration.
Remarks on result:
other: other: all strains + E. coli WP2 uvrA
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

HISTORICAL NEGATIVE CONTROL DATA: revertants/plate (min.-max.)

Exp. vehicle S9 mix TA1535 TA 100 TA 1537 TA 98 WP2 uvrA
SPT water - 10-48 76-144 4-16 17-53 24-60
    + 10-35 81-155 5-17 21-55 24-121
PIT water - 9-25 75-150 5-16 18-48 21-56
    + 9-25 80-151 5-16 17-49 23-70

RESULTS FOR TEST SUBSTANCE: mean of revertants/plate

  TA1535 TA 100 TA 1537 TA 98 WP2 uvrA
Exp.  Dosis (µg/plate)  -S9 mix  + S9 mix  -S9 mix  + S9 mix  -S9 mix  + S9 mix  -S9 mix  + S9 mix  -S9 mix  + S9 mix
SIT 0 23 28 89 90 8 10 25 29 47 49
33 23 25 85 87 7 7 22 26 45 45
100 20 24 93 90 8 10 25 27 49 48
333 21 27 85 92 7 10 22 27 49 48
1000 23 33 79 91 7 9 23 25 53 48
2500 22 26 100 90 7 8 21 30 38 55
  5000 23 22 86 83 9 10 21 26 53 55
PIT 0 30 30 103 114 9 10 34 40 49 46
33 31 28 104 114 13 10 31 41 50 43
100 36 36 100 120 9 13 37 40 48 50
333 37 33 103 124 8 8 38 39 49 49
1000 34 30 103 105 10 11 28 39 48 47
2500 33 30 104 102 10 12 34 43 43 46
5000 24 28 98 106 9 8 40 39 51 34

SPT: Standard Plate Test

PIT: Preincubation Test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions chosen, it is concluded that 1-Ethyl-3-methylimidazolium ethylsulphate is not a mutagenic test substance in the bacterial reverse mutation test (Ames test) in the absence and the presence of metabolic activation.
Executive summary:

In a GLP OECD471 study (BASF SE 2010, 40M0104/06M004), 1-Ethyl-3-methylimidazolium ethylsulphate was tested with S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA +/- S9 mix in 2 independent experiments (standard plate test and preincubation assay) with doses from 33 - 5000 µg/plate. No toxicity was observed in any of the groups. The results of the negative and the positive controls were valid. 1-Ethyl-3-methylimidazolium ethylsulphate did not lead to a relevant increase in the number of revertant colonies +/- S9 mix.

Thus, under the experimental conditions chosen, it is concluded that 1-Ethyl-3-methylimidazolium ethylsulphate is not a mutagenic test substance in the bacterial reverse mutation test (Ames test) in the absence and the presence of metabolic activation.