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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 - 25 July 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
2 plates per concentration were used, no standard deviations were calculated; According to the guideline at least 5 analysable concentrations should be used. Due to the cytotoxicity only 4 concentrations were valid for several strains in experiment 1 and 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
yes
Remarks:
(2 plates per concentration were used, no standard deviations were calculated)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Refrigerated, shading- Solubility and stability of the test substance in the solvent/vehicle: No exothermic reaction nor generation of gas in DMSO, solubility in DMSO was 50 mg/mL and more.

Method

Target gene:
his operon (for S. typhimurium strains)trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/5,6-benzoflavone
Test concentrations with justification for top dose:
Pre-experiment: 1.2, 4 .9, 20, 78, 313, 1250 and 5000 μg/plate with and without metabolic activationMain Experiment:Experiment I: The pre-experiment served as Experiment I.Experiment II:10, 20, 39, 78, 156 and 313 μg/plate without metabolic activation10, 20, 39, 78, 156, 313, 625 and 1250 μg/plate with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
furylfuramide
other: -S9: ICR-191 (1.0 µg/plate) for TA1537; +S9: 2-Aminoanthracene (2-AA; 2.0 and 10.0 µg/plate) for TA1535 and WP2 uvrA, respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubationDURATION- Preincubation period: 20 min- Exposure duration: 48 hNUMBER OF REPLICATIONS: 2 replications each in 2 independent experimentsDETERMINATION OF CYTOTOXICITY- Method: growth inhibition
Evaluation criteria:
In the dose-finding test and the main test, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive.
Statistics:
Mean values were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: ≥ 313 µg/plate without metabolic activation; ≥ 1250 µg/plate with metabolic activation; Experiment 2: ≥ 313 µg/plate without metabolic activation; ≥ 625 µg/plate wit metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: Precipitation of the test substance on the plates was not observed either with or without metabolic activation.RANGE-FINDING/SCREENING STUDIES:The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

Any other information on results incl. tables

Table 1: Results of pre-experiment/Experiment I

With or without S9-Mix Test substance concentration
(μg/plate)
Average number of colonies of 2 plates
Base-pair substitution type Frameshift type
TA 100 TA1535 WP2uvrA TA98 TA1537
Solvent control (DMSO) 126 20 31 18 12
1.2 102 15 26 16 17
4.9 117 18 26 19 13
20 124 23 28 16 16
78 160 13 26 25 11
313 73* 8* 23* 9* 0*
1250 0* 0* 19* 0* 0*
5000 0* 0* 0* 0* 0*
Positive controls, –S9 Name  AF-2 SA AF-2 AF-2 ICR-191
Concentrations (μg/plate) 0.01 0.5 0.01 0.1 1.0
Average number of colonies of 2 plates 625 426 177 597 2361
+ Solvent control (DMSO) 130 14 36 31 18
+ 1.2 83 11 30 21 12
+ 4.9 112 11 17 29 24
+ 20 112 13 32 36 23
+ 78 119 12 29 32 29
+ 313 83* 11* 32 15* 5*
+ 1250 71* 11* 27* 13* 6*
+ 5000 0* 0* 0* 0* 0*
Positive controls, +S9 Name  B(a)P 2AA 2AA B(a)P B(a)P
Concentrations (μg/plate) 5.0 2.0 10.0 5.0 5.0
Average number of colonies of 2 plates 730 285 325 181 92

AF-2 = Furylfuramide

SA = Sodium azide

2AA = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene

* = Growth inhibition of the tested bacteria strain by the tested substance was observed

Table 2: Results of Experiment II

With or without S9-Mix Test substance concentration
(μg/plate)
Average number of colonies of 2 plates
Base-pair substitution type Frameshift type
TA 100 TA1535 WP2uvrA TA98 TA1537
Solvent control (DMSO) 101 18 28 27 11
10 111 15 26 20 12
20 91 19 27 26 12
39 113 21 20 30 9
78 121 22 20 21 10
156 100* 12* 21 17* 9*
313 73* 11* 21* 14*  3*
Positive controls, –S9 Name  AF-2 SA AF-2 AF-2 ICR-191
Concentrations (μg/plate) 0.01 0.5 0.01 0.1 1.0
Average number of colonies of 2 plates 552 451 162 594 2196
+ Solvent control (DMSO) 94 14 28 34 16
+ 10 104 15 NT 36 20
+ 20 89 14 NT 32 19
+ 39 97 16 20 32 20
+ 78 102 12 24 31 18
+ 156 109* 15* 21 30* 12*
+ 313 85* 9* 21 24* 6*
+ 625 NT NT 26* NT NT
+ 1250 NT NT 26* NT NT
Positive controls, +S9 Name  B(a)P 2AA 2AA B(a)P B(a)P
Concentrations (μg/plate) 5.0 2.0 10.0 5.0 5.0
Average number of colonies of 2 plates 785 330 359 230 93

AF-2 = Furylfuramide

SA = Sodium azide

2AA = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene

* = Growth inhibition of the tested bacteria strain by the tested substance was observed

NT = Not tested

Table 3: Historical control data (mean values)

Strain   Mean
-S9 mix +S9 mix
TA100 Solvent control 120 123
Positive control 626 713
TA1535 Solvent control 19 13
Positive control 442 312
WP2uvrA Solvent control 28 29
Positive control 153 383
TA98 Solvent control 22 30
Positive control 581 205
TA1537 Solvent control 12 20
Positive control 2334 78

Applicant's summary and conclusion