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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 - 19 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted Jul 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane
EC Number:
300-346-5
EC Name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane
Cas Number:
93925-43-0
Molecular formula:
C4H8O2, C20H44O4SiSn, C24H52O6SiSn, C40H84O8SiSn2, C60H128O12Si2Sn3, C80H172O16Si3Sn4, C100H216O20Si4Sn5
IUPAC Name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

Method

Target gene:
his operon; trp operon
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 factor (S9 mix), prepared from the livers of male Sprague Dawley rats treated with Aroclor (500 mg/kg bw).
Test concentrations with justification for top dose:
Dose range finding test (TA100): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
First experiment (TA98, TA 1535, TA1537, WP2uvrA): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (5% (v/v) S9 mix) and without metabolic activation
Second experiment (all strains): 3, 10, 33, 100, 333, 1000, 3333 and 5000 µg/plate with metabolic activation (10% (v/v) S9 mix) and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (extra pure)
- Justification for choice of solvent/vehicle: Due to the reactivity to moisture of the test substance, ethanol was selected as the vehicle. At concentrations of 10 mg/mL and higher the test substance was suspended in ethanol. At concentrations of 3.33 mg/mL and lower the test substance was dissolved in ethanol.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: 2.5 µg/plate for TA1537 (-S9); 2-aminoanthracene: 1, 2.5, 5 or 10 µg/plate for all strains (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER
Justification for using S9-mix prepared from rats treated with different substances:
Since rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone and by a combination of Aroclor are recommended by the OECD guideline and the positive control data were within the laboratory historical range for each tester strain, this deviation of the S9 homogenate had no effect on the results of the study.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Mean values and standard deviations 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:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
reduction of the revertant colonies at concentrations of 3333 and 5000 µg/plate without metabolic activation; tested up to the limit dose concentration of 5000 μg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
other: but tested up to precipitating concentrations and up to the limit dose concentration of 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate with and without metabolic activation. This dose range finding test is reported as a part of the first experiment of the mutation test. Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA100 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The slight precipitate does not influence automated counting of the plate, the moderate precipitate required the plate to be hand counted.
Experiment I: Precipitation on the plates was observed at the start of the incubation period at concentrations of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentration of 333 μg/plate and above, except in tester strain TA98 where precipitation was observed at 1000 μg/plate and above. The precipitation of the test material at the dose level of 333 μg/plate contained a few particles only (please refer to Table 1 under "Any other information on results incl. tables").
Experiment II: Precipitation on the plates was observed at the start of the incubation period at the concentration of 1000 μg/plate and above. Precipitation on the plates was observed at the end of the incubation period at concentrations of 1000 μg/plate and above. Except in tester strain TA1535 (with and without metabolic activation) and TA98 (with metabolic activation) where a few particles of the test material were also observed at the dose level of 333 μg/ml (please refer to Table 2 under "Any other information on results incl. tables").

HISTORICAL CONTROL DATA
- Positive historical control data: The obtained data falls within the historical control data range (please refer to Table 4 under "Any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The obtained data falls within the historical control data range (please refer to Table 3 under "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Test results of Experiment I

With or without S9 Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 1535 TA100 WP2uvrA TA98 TA1537
Solvent control 13 ± 5 110 ± 26 23 ± 9 20 ± 6 10 ± 1
3  - 116 ± 19  -  -
10  - 129 ± 22  - -  -
33 13 ± 2 99 ± 14 22 ± 5 22 ± 2 5 ± 1
100 8 ± 3 94 ± 3 22 ± 6 28 ± 14 3 ± 1
333 4 ± 2 SP 92 ± 27 23 ± 3 SP 12 ± 4 3 ± 2 SP
1000 4 ± 1 SP 77 ± 8 19 ± 4 SP 11 ± 2 SP 4 ± 1 SP
3333 3 ± 1 SP 85 ± 11 22 ± 3 SP 8 ± 4 SP 5 ± 1 SP
5000 3 ± 2 SP 93 ± 14 26 ± 1 SP 9 ± 2 SP 4 ± 1 SP

Positive controls,

-S9

Name  SA MMS 4-NQO NF ICR-191
Concentrations (μg/plate) 5 650 10 10 2.5
Mean No. of colonies/plate (average of 3 ± SD) 728 ± 18 885 ± 27 925 ± 347 1067 ± 29 886 ± 10
+ Solvent control 16 ± 5 129 ± 17 25 ± 7 20 ± 5 9 ± 3
+ 3  - 127 ± 9
+ 10  - 146 ± 43 - -
+ 33 12 ± 3 118 ± 32 22 ± 6 26 ± 8 5 ± 1
+ 100 8 ± 1 89 ± 10 26 ± 3 17 ± 2 4 ± 1
+ 333 5 ± 2 SP 83 ± 12 25 ± 7 SP 24 ± 5 3 ± 1 SP
+ 1000 6 ± 1 SP 54 ± 9 25 ± 2 SP 18 ± 3 SP 4 ± 3 SP
+ 3333 5 ± 2 SP 57 ± 7 27 ± 4 SP 18 ± 2 SP 3 ± 1 SP
+ 5000 5 ± 2 SP 58 ± 3 24 ± 5 SP 19 ± 2 SP 2 ± 1 SP
Positive controls, +S9 (5% (v/v)) Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 1 10 1 2.5
Mean No. of colonies/plate (average of 3 ± SD) 197 ± 6 1009 ± 16 925 ± 347 1009 ± 16 415 ± 36

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 2: Test results of Experiment II

With or without S9-Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 1535 TA100 WP2uvrA TA98 TA1537
Solvent control 9 ± 1 120 ± 30 17 ± 4 16 ± 4 6 ± 1
100 4 ± 1 84 ± 5 20 ± 1 16 ± 3 8 ± 4
333 3 ± 1 SP 92 ± 12 18 ± 4 12 ± 4 5 ± 5
1000 3 ± 1 SP 89 ± 7 SP 24 ± 3 SP 16 ± 1 SP 2 ± 1 SP
3333 3 ± 2 SP 94 ± 2 SP 20 ± 4 SP 15 ± 3 SP 2 ± 1 SP
5000 3 ± 1 SP 89 ± 9 SP 19 ± 1 SP 13 ± 1 SP 2 ± 1 SP
Positive controls, –S9 Name  SA MMS 4-NQO NF ICR-191
Concentrations (μg/plate) 5 650 10 10 2.5
Mean No. of colonies/plate (average of 3 ± SD) 844 ± 14 838 ± 15 1232 ± 41 1081 ± 28 797 ± 35
+ Solvent control 11 ± 4 110 ± 2 22 ± 3 15 ± 3 7 ± 4
+ 100 8 ± 5 79 ± 15 18 ± 2 26 ± 8 1 ± 1
+ 333 4 ± 1 SP 93 ± 13 20 ± 2 19 ± 6 SP 5 ± 4
+ 1000 3 ± 2 SP 92 ± 12 SP 16 ± 2 SP 19 ± 7 SP 3 ± 1 SP
+ 3333 4 ± 1 SP 89 ± 10 SP 15 ± 4 SP 17 ± 3 SP 5 ± 1 SP
+ 5000 4 ± 2 SP 69 ± 6 SP 16 ± 6 SP 14 ± 6 MP 1 ± 2 SP
Positive controls, +S9 (10% (v/v)) Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 2.5 10 1 5
Mean No. of colonies/plate (average of 3 ± SD) 189 ± 18 1341 ± 68 387 ± 21 780 ± 54 267 ± 11

Solvent control: 0.1 mL ethanol

SA = sodium azide

MMS = methyl methane sulfa

4NQO = 4-nitroquinoline-N-oxide

NF = 2-nitrofluorene

2AA = 2-aminoanthracene

SP = slight precipitate

Table 3: Historical control data: Negative control

  TA 1535 TA1537 TA98 TA100 WP2uvrA
S9 mix - + - + - + - + - +
Range 3 - 25 3 - 31 2 - 19 2 - 16 11 - 49 12 - 59 61 - 195 58 - 179 8 - 45 6 - 46
Mean 10 8 5 5 19 24 106 88 22 23
SD 4 3 2 2 5 7 22 21 7 7
n 1208 1216 1073 1098 1191 1208 1224 1221 1090 1105

SD = Standard deviation

n = Number of observations

Table 4: Historical control data: Positive control

  TA 1535 TA1537 TA98 TA100 WP2uvrA
S9 mix - + - + - + - + - +
Range 24 - 1270 60 - 943 89 - 1086 82 - 677 401 - 1342 225 - 1656 348 - 1417 229 - 1752 138 - 1479 122 - 1248
Mean 887 265 314 374 1016 792 950 1103 1074 372
SD 112 111 146 104 133 286 117 261 212 137
n 1123 1150 1011 1039 1136 1157 1153 1170 1023 1047

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

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