Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A pKM 101 and WP2 pKM101 (OECD TG 471) (Dow Corning Corporation, 1995a).
Cytogenicity in mammalian cells: negative in Chinese hamster V79 cells (OECD TG 473) (BSL Bioservice, 2013).
Mutagenicity in mammalian cells: negative in L5178Y mouse lymphoma cells (OECD TG 476) (Dow Corning Corporation, 1995b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-02-06 - 1995-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD not actually specified but mentioned in 'Statement of Compliance'.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
E. coli, other: WP2 pKM101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Sponsor's request and compatibility with the target cells.
NOTE: the solvent is described as acetone in the method section but in most of the tables of results it is reported to be DMSO. It is thought by the reviewer that this is a typographical error: if DMSO was used rather than acetone the study would still be valid.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 1.0 µg/plate
Remarks:
TA98, TA100, TA1535, TA1537 with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 10 µg/plate
Remarks:
WP2 uvrA (pKM101) with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sterigmatocystin 100 µg/plate
Remarks:
WP2 (pKM101) with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 without metabolic activation 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535 without metabolic activation 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without metabolic activation 75 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (pKM101), WP2 (pKM101) without metabolic activation 1,000 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors. Concentration of S9 in the mix was 10%. 0.5mlk of S9 mix were added to a total volume of 2.65ml, giving a final concentration of approximately 2% S9.

DURATION
- Preincubation period: 60 minutes +/- 2 minutes at 37ºC
- Exposure duration: 48 - 72 hours at 37ºC


SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn



Evaluation criteria:
For the test to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. TA1535 and TA1537 were judged positive if the increase in mean revertants is equal to or greater than three times the mean vehicle control value. TA98, TA100, WP2 uvrA (pKM101) and WP2 (pKM101) were judged positive in the increase in mean revertants is equal to or greater than two times the mean vehicle control value.
Statistics:
None stated in report
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: WP2 (pKM101)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Precipitation was observed at 333 µg/plate and above

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was not evident at any concentration
Remarks on result:
other: No mutagenic potential

Summary of results – Experiment B1 revertants per plate (mean of 3 plates)

Dose µg/plate

+/-

metabolic activation

Average revertants per plate

TA98

TA100

TA1535

TA1537

WP2 uvrA (pKM101)

WP2 (pKM101)

Solvent control

-

21

143

11

7

210

44

100

-

19

158

11

6

212

52

333

-

24

141

11

7

199

50

1000

-

20

136

15

6

232

48

3333

-

20

142

14

4

163

53

5000

-

22

153

14

6

169

46

Positive control

-

804

675

467

156

1963

205

Solvent control

+

28

157

15

9

276

58

100

+

27

159

15

9

294

61

333

+

29

188

10

8

242

50

1000

+

30

182

12

8

254

52

3333

+

30

157

14

7

287

55

5000

+

28

180

12

7

270

58

Positive control

+

1363

1241

94

179

1395

2018

NOTE: the solvent is described as acetone in the method section but in most of the tables of results it is reported to be DMSO. It is thought by the reviewer that this is a typographical error, however if DMSO was used rather than acetone the study would still be valid.

Conclusions:
Silsesquioxanes, phenyl has been tested for mutagenicity to bacteria, in a study which was conducted according to a protocol that was similar to OECD 471, and in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without activation in the experiment. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-22 to 2012-09-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
A pre-experiment with THF as solvent
with and without metabolic activation: 0.008, 0.016, 0.03, 0.06, 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 µl/ml

A pre-experiment using Acetone as solvent
with and without metabolic activation: 0.004, 0.008, 0.016, 0.03, 0.06, 0.13, 0.25, 0.5, 1.0 and 2.0 µl/ml
Experiment I:
without metabolic activation: 0.05, 2.0, 4.0 and 5.0 µl/ml
with metabolic activation: 0.16, 2.0, 4.0 and 5.0 µl/ml

Experiment II:
without metabolic activation: 0.16, 4.0 and 5.0 µl/ml
with metabolic activation: 0.47, 4.5 and 5.0 µl/ml
Vehicle / solvent:
-Vehicle (s)/solvent(s) used: THF and Acetone
-Justification for choice of solvent/vehicle: A solubility test was performed up to a maximum concentration of 800 mg/ml using acetone and tetrahydrofurane (THF) as vehicle. The solvent was compatible with the survival of the cells and the S9 activity. For preparing the test concentrations the THF and acetone solutions were diluted with MEM, both formed precipitations (oily droplets). After treatment with ultrasound for around 5 minutes the test item was well suspended in both solvents. The final concentration of THF in the samples was 0.5% v/v. The final concentration of acetone in the samples was 0.25% v/v. As with THF it was possible to reach higher final concentrations of the test item and on sponsor’s request THF was used as solvent in the main experiments.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofurane (THF)
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 400 and 900 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofurane (THF)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation 0.83 µg/mL
Details on test system and experimental conditions:
TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SEEDED: 1 x 10E+04 - 5 x 10E+04 cells
NUMBER OF CULTURES: two cultures per concentration. Reviewers note, it is not clear from the study report if there were actually two cultures per concentrations, or whether, as indicated in the executive summary prepared by the test laboratory, only one culture was used, but the four slides per culture were divided into two sets for counting.
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture)
Except experiment I with metabolic activation : 4.0 µl/ml (500 cells), 5.0 µl/ml (300 cells)
experiment II without metabolic activation: µl/ml (400 cells)
experiment II with metabolic activation: µl/ml (400 cells)

DETERMINATION OF CYTOTOXICITY: Mitotic index, cell density

ACTIVATION: Phenobarbital (80 mg/kg bw) and β naphthoflavone (100 mg/kg bw) induced rat liver S9 was included in the S9 mix to a final protein concentration of 0.75 mg/ml. Cofactors were added to the following concentrations: 8 mM MgCl2; 33 mM KCl; 5 mM Glucose-6-phosphate; 5 mM NADP.
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).


Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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

Results of chromosome analysis
without metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I without metabolic activation                             
negative control 200 - 2 3 0 0 0 0 1 0 102 99 1 3.0 2.0
solvent control 200 - 2 0 0 0 1 0 0 1 100 100 0 2.0 0.5
0.016 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 98 n.d. n.d. n.d. n.d.
0.05 µL/mL 200  no 2 1 0 0 0 0 0 0 94 99 1 1.5 0.5
0.16 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 101 n.d. n.d. n.d. n.d.
0.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 108 n.d. n.d. n.d. n.d.
1.0 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 110 n.d. n.d. n.d. n.d.
2.0 µL/mL 200 no 2 2 1 1 0 0 0 0 105 101 1 3.0 2.0
4.0 µL/mL 200  no 3 0 0 1 0 0 1 0 102 104 0 2.0 1.0
5.0 µL/mL 200  no 3 2 0 0 0 0 1 0 112 100 1 2.5 1.5
EMS 900 µg/mL 200 - 4 10 6 0 0 0 1 0 96 95 0 9.0 8.0
Experiment II  without metabolic activation                                
negative control 200 - 4 5 0 0 0 0 0 0 86 102 1 4.5 2.5
solvent control 200 - 1 1 0 0 0 0 1 0 100 100 0 1.0 1.0
0.016 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 96 n.d. n.d. n.d. n.d.
0.05 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 101 n.d. n.d. n.d. n.d.
0.16 µL/mL 400  no 1 2 3 1 1 0 1 0 104 102 2 2.3 1.8
0.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 91 n.d. n.d. n.d. n.d.
1.0 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 93 n.d. n.d. n.d. n.d.
2.0 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 95 n.d. n.d. n.d. n.d.
4.0 µL/mL 200  no 1 3 0 0 0 0 0 0 107 115 1 1.5 1.5
5.0 µL/mL 200 no 1 0 0 0 0 0 0 0 102 107 1 0.5 0.0
EMS 400 µg/mL 200 - 4 13 3 2 0 1 1 0 83 103 0 10.0 9.0
Results of chromosome analysis
with metabolic activation
     Cytotoxicity Chromatid aberrations         Isochromatid aberrations       rel. Mitotic index (%) rel. Cell density (%)  Poly-ploidy mean % aberrant cells
Scored cells  gaps breaks  inter-changes  other  gaps breaks  inter-changes  other incl. Gaps excl. Gaps
Experiment I  with metabolic activation                            
negative control 200 - 3 1 0 0 1 0 0 0 95 104 0 2.5 0.5
solvent control 200 - 0 0 0 0 0 0 0 3 100 100 2 0.5 0.5
0.016 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 104 n.d. n.d. n.d. n.d.
0.05 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 105 n.d. n.d. n.d. n.d.
0.16 µL/mL 200 no 4 1 0 1 1 0 0 0 110 95 0 3.0 1.0
0.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 109 n.d. n.d. n.d. n.d.
1.0 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 116 n.d. n.d. n.d. n.d.
2.0 µL/mL 200 no 5 0 0 0 0 0 0 0 100 99 1 2.5 0.0
4.0 µL/mL 500  no 9 4 5 0 0 0 4 0 97 99 4 3.8 2.6
5.0 µL/mL 300  no 9 6 0 1 0 0 0 1 118 105 1 5.3 2.7
CPA 0.83 µg/mL 200 - 6 13 6 1 0 0 1 0 91 92 0 12.0 9.5
Experiment II with metabolic activation                                 
negative control 200 - 6 3 0 0 1 0 0 0 105 98 2 5.0 1.5
solvent control 200 - 3 5 1 1 1 0 0 0 100 100 2 4.5 3.0
0.047 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 97 n.d. n.d. n.d. n.d.
0.15 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 88 n.d. n.d. n.d. n.d.
0.47 µL/mL 400  no 10 5 2 2 0 0 1 0 104 87 3 5.5 2.5
1.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 91 n.d. n.d. n.d. n.d.
2.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 100 n.d. n.d. n.d. n.d.
3.5 µL/mL -  no n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 104 n.d. n.d. n.d. n.d.
4.5 µL/mL 200 no 4 3 0 1 0 0 0 0 92 81 0 4.0 2.0
5.0 µL/mL 200  no 6 2 0 0 0 0 0 0 106 88 0 3.5 1.0
CPA 0.83 µg/mL 200 - 8 20 6 0 0 1 0 0 77 77 1 14.0 10.5

n.d. not determined

Conclusions:
Silsesquioxanes, phenyl has been tested in a reliable study which was conducted according to OECD 473 (1997) and in compliance with GLP. No evidence for a potential to cause structural or numerical damage to chromosomes was observed with or without activation when tested up to limit concentrations in Chinese hamster V79 cells in either the initial or the repeat experiment. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for cytogenicity to mammalian cells under the conditions of the test.
Executive summary:

A solubility test was performed up to a maximum concentration of 800 mg/ml using Acetone and Tetrahydrofurane (THF) as vehicle.For preparing the test concentrations the THF solution was diluted with MEM where precipitate was formed (oily droplets). After treatment with ultrasound for around 5 minutes the test item was well suspended. The final concentration of THF in the samples was 0.5% v/v. As the test item formed precipitate upon addition to the samples, cells were incubated with a dispersed test item suspension. The vehicle was compatible with the survival of the cells and the S9 activity. However, to decide which solvent should be used and on sponsor’s request the test item was also dissolved in acetone. The acetone solution was then diluted with MEM where also precipitate was formed (oily droplets). After treatment with ultrasound for around 5 minutes the test item was well suspended. The final concentration of acetone in the samples was 0.25% v/v. The vehicle was compatible with the survival of the cells and the S9 activity.

Pre-experiments with both vehicles were performed.

As with THF it was possible to reach higher final concentrations of the test item and on sponsor’s request THF was used as solvent in the main experiments.

To investigate the potential of Silsesquioxanes, Phenyl to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in Experiment I. In Experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. For each concentration, one Quadriperm dish containing four microscopic slides was seeded. At the end of the 20 hour preparation interval, the slides were divided into sets of two. At least one slide from each set was counted. Generally at least 100 metaphases per set were scored for structural chromosomal aberrations (for exceptions, see Tables).

The following concentrations were selected for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 0.05, 2.0, 4.0 and 5.0 µl/ml

with metabolic activation: 0.16, 2.0, 4.0 and 5.0 µl/ml

Experiment II:

without metabolic activation: 0.16, 4.0 and 5.0 µl/ml

with metabolic activation: 0.47, 4.5 and 5.0 µl/ml

 

In Experiment I precipitation of the test item was noted with and without metabolic activation at concentrations of 2.0 µl/ml and higher. In Experiment II precipitation of the test item was seen without metabolic activation at concentrations of 4.0 µl/ml and higher, with metabolic activation at concentrations of 4.5 µl/ml and higher.

In Experiments I and II with and without metabolic activation no cytotoxic effects of the test item were noted at the evaluated concentrations.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the solvent control. However, there were 3.5% exchanges found on one slide at a concentration of 4 µl/ml in the first experiment with metabolic activation. Exchanges are not always detected with Giemsa staining (depending on their size), therefore the induction of exchanges should be evaluated additionally for example by using Fluorescence in situ hybridisation (FISH) analysis. However, these analyses were not performed, because that method is not established at BSL at this time.

In the Experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the solvent controls.

EMS (400 and 900 µg/ml) and CPA (0.83 µg/ml) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

The positive controls induced the appropriate response.

There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5375; OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-02-17 - 1995-03-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus of L5178Y
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's Medium for Leukemic Cells of Mice
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
3000, 3500, 4000, 4500 and 5000 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Chosen by the sponsor. The test substance was soluble in acetone at a maximum concentration of approx 500 mg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 0.25 and 0.50 µg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation 2.5 and 5.0 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 24 - 48 hours after treatment
- Selection time (if incubation with a selection agent): 10 - 12 days

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: 2 flasks per culture, 3 replicate dishes

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: other: % viability

ACTIVATION: S9 mix contained 250 μl S9, and NADP as co-factor in 750 μl medium. Final concentration of S9 is not clearly presented in the study report.
Evaluation criteria:
The mutant frequency of the positive controls must be at least twice that of the appropriate solvent controls. The spontaneous mutant frequency of the solvent controls must be between 20 and 100 TFT-resistant mutants per 10x6 surviving cells. The cloning efficiency of the solvent controls must be greater than 50%.
Statistics:
None in report
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: Some globules of the test article stuck to the side of the culture tubes or floated in the treatment medium. The discrepancy in precipitation profile could be result of being inadvertently not documented during preliminary toxicity assay.


Remarks on result:
other: No mutagenic potential

Cloning data in the absence and presence of metabolic activation

Dose level µg/ml

+/- metabolic activation

Mutant Frequency

% Total Growth

Solvent control 1

-

38

-

Solvent control 2

-

33

-

3000 A

-

25

100

3000 B

-

49

74

3500 A

-

26

92

3500 B

-

43

96

4000 A

-

28

105

4000 B

-

27

92

4500 A

-

27

91

4500 B

-

28

92

5000 A

-

25

98

5000 B

-

33

110

DMSO 1

-

27

-

DMSO 2

-

29

-

Positive control 1

-

400

66

Positive control 2

-

817

26

Solvent control 1

+

25

-

Solvent control 2

+

32

-

3000 A

+

38

93

3000 B

+

33

89

3500 A

+

34

101

3500 B

+

46

100

4000 A

+

33

110

4000 B

+

30

113

4500 A

+

29

107

4500 B

+

39

116

5000 A

+

41

94

5000 B

+

26

110

DMSO 1

+

24

-

DMSO 2

+

26

-

Positive control 1

+

203

25

Positive control 2

+

++

++

++ = too toxic to clone

Conclusions:
Silsesquioxanes, phenyl was tested in a L5178Y/TK+/- mouse lymphoma mutagenesis assay, in a study which was conducted according to OECD 476 and in compliance with GLP. No evidence of a test substance related increase in mutant frequency was detected at any concentration in the presence or absence of metabolic activation. Appropriate solvent and positive controls were concluded and gave expected results. It is concluded that the test substance is negative for the induction of mutation in L5178Y cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The selected in vitro studies are the only available cytogenicity and bacterial and mammalian mutagenicity studies for the substance. The bacterial study was conducted according to a protocol that is equivalent to an appropriate OECD guideline; the studies in mammalian cells were conducted according to appropriate OECD guidelines. All studies were conducted in compliance with GLP.

Information is available for phenyl silsesquioxanes from reliable in vitro studies on mutagenicity to bacterial and mammalian cells, and cytogenicity to mammalian cells. The results of all the studies were in agreement: no evidence for mutagenicity was obtained. It is therefore considered that in vivo testing is not required.

Phenyl silsesquioxanes has been tested for mutagenicity to bacteria, in a study which was conducted according to a protocol that was similar to OECD Test Guideline 471, and in compliance with GLP (Dow Corning Corporation, 1995a). No evidence of a test substance related increase in the number of revertants was observed with or without activation in the experiment, which tested Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A pKM 101 and WP2 pKM101up to limit concentrations. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Information on the potential of phenyl silsesquioxanes to cause structural or numerical damage to chromosomes is available from a reliable study which was conducted according to OECD Test Guideline 473 (1997) and in compliance with GLP (BSL Bioservice, 2013). No evidence for a potential to cause structural or numerical damage to chromosomes was observed with or without activation when tested up to limit concentrations in Chinese hamster V79 cells. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for cytogenicity to mammalian cells under the conditions of the test.

Phenyl silsesquioxanes was tested in a L5178Y/TK+/- mouse lymphoma mutagenesis assay, in a study which was conducted according to OECD Test Guideline 476 and in compliance with GLP (Dow Corning Corporation, 1995b). No evidence of a test substance related increase in mutant frequency was detected at any concentration in the presence or absence of metabolic activation. Appropriate solvent and positive controls were concluded and gave expected results. It is concluded that the test substance is negative for the induction of mutation in L5178Y cells under the conditions of the test.


Justification for classification or non-classification

Based on the available in vitro data, phenyl silsesquioxanes does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.