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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
Study period:
from 30 March 2007 to 22 May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 471 compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
02 sept 2006
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver cells
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: usually recommended by the guideline.
Controls
Untreated negative controls:
yes
Remarks:
solvent served as negative controls
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; 4-nitro-o-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation method.

DURATION
- Preincubation period: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.
- Exposure duration: 48 hours

NUMBER OF REPLICATES: 3

NUMBER OF CELLS EVALUATED: The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

POSITIVE CONTROLS:
Without metabolic activation
Strains: TA 1535, TA 100
Name: sodium azide, NaN3
Concentration: 10 µg/plate

Strains: TA 1537, TA 98
Name: 4-nitro-o-phenylene-diamine, 4-NOPD
Concentration: 1 0 IJg/plate in TA 98, 50 IJg/plate in TA 1537

Strain: WP2 uvrA
Name: methyl methane sultanate, MMS
Concentration: 3 µL/plate

With metabolic activation
Strains: TA1535, TA 1537, TA 98, TA 100, WP2 uvrA
Name: 2-aminoanthracene, 2-AA
Concentration: 2.5 µg/plate {TA 1535, TA 1537, TA 98, TA 100), 1 0 µg/plate (WP2 uvrA)
Evaluation criteria:
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 (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
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.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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'.

Any other information on results incl. tables

Results for plate incorporation (mean of 3 replicates):

  TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA
Conc. without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix
µg/plate
0 36,3 42,7 105,3 111 18,3 19 14 17,3 32,3 46
3 41,7 52 118 101,3 15 19 12,7 15,7 33,7 42,7
10 36,7 43,7 109 96,7 13,7 18 10,3 15 43 44
33 33,3 38 108 101,3 15 16,7 12,3 16,7 35,3 38,7
100 38 43,3 116,7 108 22,7 17 11,7 14,3 35,3 38
333 43 45,7 118 89 17,3 22,7 10,3 14 32,3 42,7
1000 34,7 41 99,3 93,3 13,3 17 10 15,7 31,3 44,3
2500 36,7 42,7 105,7 107,3 12,3 17,3 8,7 16 37 40,7
5000 34,7 46,7 108,3 110 12,3 21 12,7 16,7 28,7 46,7
Untreated controls 41,3 40 149 111 12,7 20 14 20,3 37,3 46,3
Positive control 527 1008,7 1924,3 725,3 1862 190,3 89 178,3 632,7 248,3

Results with pre-incubation method (mean of 3 replicates):

  TA 98 TA 100 TA 1535 TA 1537 WP2 uvrA
Conc. without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix without S9 mix with S9 mix
µg/plate
0 37 44,7 131 126,3 18,3 18 11,3 15 38,7 43,3
3 33,7 49,7 112,3 111 14,3 17,3 12 16,7 39,3 51
10 40 47 135,3 125,3 13,3 19,3 13 14,7 43,3 44,7
33 36,7 50,3 139 149 15,3 19,7 13 15,7 40,3 45,7
100 41,3 44,3 137 135,7 15 15,3 9,3 13,7 33,7 45,7
333 34,7 54,3 135,3 123,7 15,3 19,7 11,3 18,3 41,3 51,3
1000 38,7 48,3 143,3 135,3 10,3 20,7 13 14,3 26 51
2500 43,3 49 131,7 130,3 15,7 15,7 10,7 13,7 27,3 54
5000 34,3 45,3 139,7 137,7 8 16,7 10,3 15,3 31,7 44,7
Untreated controls 39,7 53,3 120,7 126 17,7 15,3 10,3 16,3 37 48,7
Positive control 549 922,3 2344 1105,3 2064 278,3 115,7 121,3 456,3 322,7

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item TFSIH was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment 11) 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 (active ingredient): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with TFSIH 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.