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Toxicological information

Genetic toxicity: in vitro

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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:
2007-02-01 to 2007-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
yes
Remarks:
2-AA was the sole positive control for S9 mix
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BAYERISCHES LANDESAMT FÜR ARBEITSSCHUTZ, ARBEITSMEDIZIN UND SICHERHEITSTECHNIK, Munich, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Diethoxymethylsilane; SEMICONSIL M2E; Silane, diethoxymethyl-
- Physical state: colourless liquid
- Lot/batch No.: LC 11/05 Pos.2
- Expiration date of the lot/batch: 12 January 2008
- Storage condition of test material: at room temperature, protected from light and humidity

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: all strains except TA102 carry a mutation of the uvrB gene coding for the DNA excision repair system (uvrB-), all strains carry the deep rough mutation (rfa), TA102, TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital and ß-Naphthoflavone
Test concentrations with justification for top dose:
- Cytotoxicity pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate
- Experiment I: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (with and without metabolic activation - plate incorporation)
- Experiment II: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (with and without metabolic activation - pre-incubation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties of the test article, compatibility with the S9 activity, and relative non-toxicity to bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
aqua dest.
Positive controls:
yes
Positive control substance:
other: sodium azide (NaN3, TA100, TA1535-10µg/plate, -S9); 4-Nitro-o-phenylendiamine (4-NOPD, TA98-10µg/platem TA1537-40µg/plate, -S9); methyl methane sulfonate (MMS, TA102-1µL/plate, -S9); 2-aminoanthracene (2-AA, TA102-10µg/plate, all others-2.5µg/plate, +S9)
Remarks:
TA 100, TA 1535 (without activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 1 h
- All plates with bacteria were incubated at 37°C for 48 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- A cytotoxicity pre-experiment was carried out with the tester strains TA 98 and A 100 to determine the non-toxic concentrations for the main genotoxicity experiments.
- Method: Background lawn assessment / revertant colony counts
Evaluation criteria:
A test system is considered as mutagenic if
- there is a clear and dose-related increase in the number of revertants and/or a
- biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 100 and TA 102 and 3-fold of the solvent control for TA 98, TA 1535 and TA 1537.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Any other information on results incl. tables

Table 1: Dose range-finding study. Number of revertants per plate (2 plates per strain)

TA 98

TA 100

Concentration (μg/Plate)

Plate 1

- MA

Plate 2

+ MA

Cytotoxic (Yes/No)

Plate 1

- MA

Plate 2

+ MA

Cytotoxic (Yes/No)

DMSO

1

1

No

1

1

No

3.16

0.8

1.3

No

1

0.9

No

10

0.9

1.3

No

0.9

0.9

No

31.6

1.1

1.3

No

1

0.9

No

100

1

1.2

No

1

0.9

No

316

0.7

1.3

No

0.9

0.8

No

1000

0.7

1.1

No

0.9

1

No

2500

0.7

1.3

No

0.9

0.9

No

5000

0.9

1.3

No

0.9

0.9

No

Positive control

21.5

113.3

No

8

20.1

No

Table2: Test results of Experiment I (plate incorporation)

 with or without S9 -Mix  Test substance  Mean number of revertant colonies per plate (average of 3 plates ± SD)          
   concentration  Base-pair substitution type        Frameshift type   
   [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  DMSO  111 ± 3.6  10 ± 2.6  150 ± 4.0  22 ± 2.3  7 ± 0.6
 -  31.6  107 ± 6.2  7 ± 3.0  128 ± 25.0  24 ± 5.2  15 ± 3.5
 -  100  109 ± 11.7  11 ± 3.6  153 ± 16.5  22 ± 6.1  16 ± 5.1
 -  316  100 ± 7.1  8 ± 1.7  138 ± 12.5  15 ± 3.5  11 ± 1.0
 -  1000  98 ± 8.9  7 ± 1.5  148 ± 7.8  16 ± 4.6  8 ± 2.5
 -  2500  99 ± 7.5  9 ± 2.0  127 ± 21.9  16 ± 2.9  11 ± 2.6
 -  5000  96 ± 4.6  9 ± 1.5  135 ± 19.5  20 ± 2.5  15 ± 6.8
 Negative control  Aqua dest.  108 ± 8.2  10 ±2.1  188 ± 8.1  19 ± 2.6  13 ± 2.1
 Positive controls  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
   concentrations [µg/plate]  10  10  1µL  10  40
   Mean No. of colonies/plate (average of 3 ± SD)  893 ± 47.4  828 ± 38.9  1611 ± 208.4 481 ± 13.5   129 ± 10.6
 +  DMSO  127 ± 5.3  9 ± 4.5  192 ± 14.4  26 ± 2.6  10 ± 4.0
 +  31.6  114 ± 15.8  9 ± 2.1  149 ± 19.9  35 ± 1.0  11 ± 3.2
 +  100  119 ± 6.8  6 ± 3.1  182 ± 10.3  31 ± 6.0  12 ± 0.6
 +  316  103 ± 6.2  10 ± 0.6  188 ± 11.5  34 ± 3.6  11 ± 2.6
 +  1000  128 ± 15.7  8 ± 1.2  212 ± 19.3  28 ± 5.0  12 ± 0.6
 +  2500  120 ± 10.2  12 ± 1.0  201 ± 24.8  35 ± 6.0  10 ± 3.5
 +  5000  112 ± 6.4  9 ± 3.1  167 ± 23.5  34 ± 2.5  14 ± 1.5
 Negative control  Aqua dest. 124 ± 11.0   7 ± 2.0  211 ± 9.7  35 ± 3.6  13 ± 4.7
 Positive controls  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
   concentrations [µg/plate]  2.5  2.5  10  2.5  2.5
   Mean No. of colonies/plate (average of 3 ± SD)  2547 ± 100.3  102 ± 6.9  1156 ± 49.0  2945 ± 191.1  358 ± 12.0

Table2: Test results of Experiment II (pre-incubation)

 with or without S9 -Mix  Test substance  Mean number of revertant colonies per plate (average of 3 plates ± SD)          
   concentration  Base-pair substitution type        Frameshift type   
   [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  DMSO  95 ± 6.8  10 ± 3.1  178 ± 9.2  22 ± 3.5  11 ± 3.0
 -  31.6  89 ± 22.7  7 ± 4.5  104 ± 21.6  19 ± 5.6  12 ± 2.0
 -  100  101 ± 8.2  9 ± 1.0  146 ± 11.1  21 ± 1.7  10 ± 3.0
 -  316  97 ± 10.5  6 ± 1.2  157 ± 7.2  21 ± 1.0  7 ± 2.1
 -  1000  74 ± 13.2  6 ± 1.0  178 ± 14.8  18 ± 1.5  7 ± 2.5
 -  2500  58 ± 10.6  4 ± 1.5  158 ± 1.5  25 ± 4.7  9 ± 2.5
 -  5000  70 ± 7.5  7 ± 2.6  130 ± 39.5  23 ± 0.6  12 ± 2.0
 Negative control  Aqua dest.  103 ± 14.4  7 ± 1.0  186 ± 12.1  27 ± 3.1  9 ± 1.2
 Positive controls  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
   concentrations [µg/plate]  10  10  1µL  10  40
   Mean No. of colonies/plate (average of 3 ± SD)  701 ± 14.5  842 ± 30.0  1716 ± 117.7 472 ± 53.1   141 ± 9.3
 +  DMSO  77 ± 4.5  9 ± 3.0  269 ± 14.4  36 ± 3.2  10 ± 3.1
 +  31.6  84 ± 2.5  7 ± 2.0  143 ± 38.9  30 ± 2.5  15 ± 2.3
 +  100  88 ± 14.6  6 ± 1.2  208 ± 26.0  42 ± 13.3  16 ± 3.1
 +  316  92 ± 11.0  7 ± 4.0  226 ± 10.6  41 ± 6.9  13 ± 2.3
 +  1000  99 ± 6.0  7 ± 1.2  240 ± 22.9  39 ± 7.6  9 ± 1.5
 +  2500  75 ± 0.6  9 ± 0.6  206 ± 32.4  33 ± 4.2  12 ± 1.5
 +  5000  86 ± 3.2  7 ± 2.1  191 ± 44.5  43 ± 8.0  11 ± 1.5
 Negative control  Aqua dest. 100 ± 5.8   8 ± 3.1  307 ± 13.8  37 ± 5.5  13 ± 1.7
 Positive controls  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
   concentrations [µg/plate]  2.5  2.5  10  2.5  2.5
   Mean No. of colonies/plate (average of 3 ± SD)  872 ± 112.1  39 ± 7.5  1386 ± 138.1  1762 ± 40.7  120 ± 3.5

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

The test item was investigated for mutagenicicty to bacteria according to the OECD TG 471, and in compliance with GLP. In two independent experiments (plate incorporation and preincubation) the test material was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 102 up to limit concentrations with and without a metabolic activation system. No significant increase in the number of revertants was observered in any of the tester strain with and without metabolic activation. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.