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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:
29 June to 27 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
only 2-aminoanthracene was used as positive control for metabolic activation, however, in pre-tests of S9 mix benzo(a)pyrene was also used to establish functionality of S9 mix
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Landesinstitut fuer Arbeitsschutz und Produktsicherheit, 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): (Chloromethyl)triethoxysilane
- Physical state: colourless liquid
- Lot/batch No.: KH09561
- Expiration date of the lot/batch: 29. April 2013
- Stability under test conditions: stable
- Storage condition of test material: at room temperature, protected from light

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: deep rough mutation (rfa), defect DNA nucleotide excision repair (uvrB-), TA100 and TA98 contain pkM101 plasmid to detect weak mutagens due to enhancing an error-prone DNA repair system
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: deep rough mutation (rfa), defect DNA nucleotide excision repair (uvrB-), contain pkM101 plasmid to detect weak mutagens due to enhancing an error-prone DNA repair system
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitale and ß-Naphthoflavone
Test concentrations with justification for top dose:
pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
experiment I: 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
experiment II: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
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-phenylene-diamine (4-NOPD, TA98 10µg/plate, TA1537 40µg/plate, -S9); methylmethanesulfonate (MMS, TA102 1µL/plate, -S9); 2-aminoanthracene (2-AA, TA102 10µg/plate, all other - 2.5µg/plate, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency & relative total growth

Evaluation criteria:
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs 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 biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
than the revision rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp.I: for all strains: at 316 µg/plate and higher (-S9) and 1000µg/plate and higher (+S9); exp.II: 500 µg/plate and higher (-S9) and 1580 µg/plate and higher (+S9), each time depending on particular tester strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp.I: for all strains: at 316 µg/plate and higher (-S9) and 1000µg/plate and higher (+S9); exp.II: 500 µg/plate and higher (-S9) and 1580 µg/plate and higher (+S9), each time depending on particular tester strain
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was done with strains TA98 and TA100. Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately equal or less then 0.5 in relation to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
the resulted are in range with the historical control data (see tables 1-4 under "Any other information on meterials and methods including tables")

Any other information on results incl. tables

Table 5: Results of experiment I

 with or without  Test substance  Mean number of revertant colonies per plate(average of 3 plates Standard deviation)
  S9 mix   concentration       Base pair substitutiontype    cross linking type  Frameshift type
    [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  acetone  102±12.5  10±4.7  188±6.2  30±10.1  7±1.0
 -  10  97±13.0  10±3.1  185±17.2  19±4.7  10±4.5
 -  31.6  93±3.2  11±3.8  184±14.6  26±1.7  12±1.5
 -  100  96±4.0  6±1.2  188±11.6  27±10.2  8±2.1
 -  316  68±4.4 B  8±2.9  152±7.9  21±2.5  8±4.0
 -  1000  67±4.2 B  3±1.5 B  89±11.4 B  18±8.7 B  6±1.5 B
 -  2500  51±8.5 B  5±1.7 B  89±17.6 B  14±2.5 B  5±1.5 B
-  5000  63±17.7 B  4±2.6 B  82±11.9 B  15±3.2 B  5±1.0 B
 neg. control  aqua dest.  113±17.5  11±1.5  195±2.1  21±1.5  17±3.2
pos. control  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
  µg/plate  10  10  1 µL  10  40
   Mean No. of colonies/plate(average of 3±SD)  974±40.6  984±13.2  1282±141.0  396±20.8  131±8.9
 + acetone  102±11.5  7±2.6  253±14.5  30±6.1  18±3.8
 +  10  95±7.5  9±1.7  288±32.0  35±5.3  12±2.0
 +  31.6  102±9.5  8±1.5  286±5.1  32±3.8  12±1.0
 +  100  105±8.4  8±2.5  261±12.9  44±5.6  20±2.1
 +  316  112±16.9  8±2.9  252±10.5  28±7.6  11±2.3
 +  1000  75±7.0 B  7±2.5 B  248±41.6  26±8.1 B  6±3.5 B
+ 2500  62±5.6 B  4±1.5 B  235±14.5  24±5.3 B  6±0.6 B
+ 5000  70±12.3 B  4±0.6 B  253±7.5  21±7.6 B  9±4.4 B
neg. control  aqua dest.  93±7.1  9±5.1  212±9.6  34±7.5  15±5.6
 pos. control  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
  µg/plate  2.5  2.5  10  2.5  2.5
  Mean No. of colonies/plate(average of 3±SD)  1985±133.2  144±5.9  738±74.1  2469±122.3  389±18.6

B:       Background lawn reduced

Table 6: Results of experiment II

 with or without  Test substance  Mean number of revertant colonies per plate (average of 3 plates Standard deviation)
  S9 mix   concentration   Base pair substitutiontype      cross linkingtype  Frameshifttype   
    [µg/plate]  TA100  TA1535  TA102  TA98  TA1537
 -  acetone  107 ± 16.1  12 ± 4.9  272 ± 14.0  19 ± 8.7  10 ± 4.4
 -  5  119±16.5  13±3.6  266±10.4  19±8.2  9±4.0
 -  15.8  112±2.5  15±2.6  235±22.1  22±11.5  7±3.2
 -  50  104±7.2  14±4.5  247±8.1  27±5.7  7±2.6
 -  158  106±7.6  12±3.5  245±37.9  23±9.8  6±2.1
 -  500  96±15.3  8±3.6 B  282±21.7  21±2.1  3±2.3 B
 -  1580  51±8.5 B  6±2.6 B  231±22.5  21±3.1  3±2.3 B
-  5000  88±4.6 B  5±0.6 B  189±30.9  21±3.5  3±2.6 B
 neg. control  aqua dest.  80±24.4  15±3.5  212±15.7  19±4.0  9±3.1
pos. control  Name  NaN3  NaN3  MMS  4 -NOPD  4 -NOPD
  µg/plate  10  10  1 µL  10  40
   Mean No. of colonies/plate(average of 3±SD)  898±144.4  1289±70.5  1486±379.3  612±122.5  133±4.4
 + acetone  120±13.0  10±3.0  246±18.5  28±5.9  10±2.1
 +  5  130±17.2  12±2.0  397±23.2  27±4.6  15±4.0
 +  15.8  123±13.0  8±3.1  384±21.2  29±5.5  13±4.0
 +  50  134±12.3  8±6.4  317±34.1  35±2.0  14±3.2
 +  158  122±9.5  11±2.1  288±46.5  32±4.2  14±5.8
 +  500  103±9.9  8±3.1  294±31.3  26±6.9  8±3.2
+ 1580  86±21.0 B  4±0.6 B  307±13.2  22±10.0 B  9±0.6 B
+ 5000  67±6.1 B  4±1.2 B  286±26.1  21±7.4 B  6±0.6 B
neg. control  aqua dest.  122±3.2  10±1.0  229±18.3  32±4.2  8±1.5
 pos. control  Name  2 -AA  2 -AA  2 -AA  2 -AA  2 -AA
  µg/plate  2.5  2.5  10  2.5  2.5
  Mean No. of colonies/plate(average of 3±SD)  2136±261.4  107±14.0  525±7.4  1892±52.5  332±56.6

B: Background lawn reduced

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

In conclusion, it can be stated that during the described mutagenicity test, conducted according to OECD 471 and under GLP, and under the experimental conditions reported, (Chloromethyl)triethoxysilane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, (Chloromethyl)triethoxysilane is considered to be non-mutagenic in this bacterial reverse mutation assay.