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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-21 to 2012-03-20
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: 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:
Pre-expt I: 0.2, 0.5, 2.5, 5.0, 7.5 and 10.0 mM (+/-MA); Pre-expt II: 0.01, 0.1, 1.0, 2.0, 5.0, 10.0 mM (-MA, 24 h exp); Expt I: 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 mM (+/-MA); Expt II: 0.15, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM (+MA); 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.10 and 0.20 mM (-MA)




Vehicle / solvent:
THF was used as solvent (0.35% THF v/v in the samples). To reach a final concentration of 0.35% THF v/v in the samples the test item stock solution was diluted in RPMI + 5% HS for short-term exposure or RPMI + 7.5% HS for long-term exposure. After adding the THF stock solution to cell culture medium precipitate formed.
The solvent was compatible with the survival of the cells and the activity of the S9 mix.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation: 3.5 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation: 200 µg/mL and 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in THF (0.35% THF v/v in the samples)
DURATION: 4 h (short-term exposure), 24 h (long-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/ml trifluorothymidine
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors, and sufficient S9 to give a final protein concentration in the cultures of 0.75 mg/ml
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 10^6 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative /solvent controls.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG of 14.6% and 9.7% without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Pre-experiment for toxicity with and without metabolic activation

Concentration (mM)

Number of cells 4 h after treatment

Number of cells 24 h after treatment

Number of cells 48 h after treatment

Suspension growth

Relative suspension growth

 

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

Negative control

271000

278000

946000

948000

1600000

1570000

15.1

14.9

89.8

103.6

307000

356000

1050000

1150000

1510000

1540000

15.9

17.7

94.1

123.3

Solvent control

307000

256000

104000

807000

1560000

1570000

16.2

12.7

100.0

100.0

339000

313000

112000

101000

1560000

1590000

17.5

16.1

0.2

284000

271000

77000

593000

1540000

1480000

11.9

8.8

70.4

61.1

0.5

307000

252000

876000

377000

1490000

1190000

13.1

4.5

77.5

31.2

2.5

289000

220000

728000

186000

1620000

637000

11.8

1.9

70.0

13.3

5.0

316000

225000

798000

137000

1550000

286000

12.4

0.9

73.4

6.0

7.5 (P)

303000

271000

650000

239000

1590000

850000

10.3

2.6

61.3

17.8

10.0 (P)

305000

244000

728000

183000

1580000

481000

11.5

1.4

68.3

10.0

Summary: Experiment 1 and 2 with metabolic activation

 

Treatment (mM)

RTG (%)

MF (mutants/106cells)

IMF (mutants/106cells)

Precipitate

 

Negative control

103.5

125.6

/

-

 

98.1

/

-

 

Solvent control

100.0

135.2

/

-

 

/

-

 

0.1

72.3

142.3

7.2

-

Exp 1

0.2

73.5

143.3

8.1

-

 

0.5

84.9

97.1

-38.1

-

 

1.0

63.7

182.8

47.6

-

 

2.5

67.1

144.4

9.2

-

 

5.0

87.2

117.6

-17.6

-

 

7.5

78.1

154.8

19.6

-

 

10.0

74.3

130.3

-4.8

+

 

Positive control

38.4

918.4

783.2

-

 

 

 

 

 

 

 

Treatment (mM)

RTG (%)

MF (mutants/106cells)

IMF (mutants/106cells)

Precipitate

 

Negative control

98.9

116.5

/

-

 

111.8

/

-

 

Solvent control

100.0

116.7

/

-

 

/

-

 

0.15

111.4

105.1

-11.6

-

Exp 2

0.3

96.2

131.1

14.4

-

 

0.7

75.7

128.5

11.8

-

 

2.0

42.3

220.5

103.8

-

 

4.0

72.7

138.8

22.2

-

 

6.0

58.9

170.6

53.9

-

 

8.0

74.1

134.6

17.9

+

 

10.0

76.4

154.9

38.2

+

 

Positive control

50.5

1141.0

1024.3

-

MF - mutant frequency

IMF = induced mutant frequency

RTG = relative total growth

Summary: Experiment 1 and 2 without metabolic activation

 

Treatment (mM)

RTG (%)

MF (mutants/106cells)

IMF (mutants/106cells)

Precipitate

 

Negative control

122.0

142.0

/

-

 

129.6

/

-

 

Solvent control

100.0

144.3

/

-

 

/

-

 

0.1

100.3

177.2

32.9

-

Exp 1

0.2

87.3

157.4

13.0

-

 

0.5

98.4

186.0

41.6

-

 

1.0

49.2

190.1

45.7

-

 

2.5

37.8*

179.4

35.1

-

 

5.0

37.2

179.3

35.0

-

 

7.5

17.1

224.1

79.8

+

 

10.0

14.6

264.7

120.4

+

 

Positive control 1

75.1

817.6

673.2

-

 

Positive control 2

85.2

564.1

419.7

-

 

 

 

 

 

 

 

Treatment (mM)

RTG (%)

MF (mutants/106cells)

IMF (mutants/106cells)

Precipitate

 

Negative control

145.4

110.6

/

-

 

122.8

/

-

 

Solvent control

100.0

109.3

/

-

 

/

-

 

0.001

114.6

108.6

-0.8

-

Exp 2

0.002

99.3

94.7

-14.6

-

 

0.005

103.4

120.0

10.6

-

 

0.01

62.1

121.8

12.5

-

 

0.02

82.5

160.0

50.6

-

 

0.05

98.6

108.4

-0.9

-

 

0.10

16.8

173.0

63.6

-

 

0.20

0.9

119.1

9.7

-

 

Positive control 1

21.3

2373.6

2264.2

-

 

Positive control 2

14.7

1826.6

1717.3

-

Applicant's summary and conclusion

Conclusions:
Triethoxyoctylsilane has been tested for mutagenicity to mammalian cells in a reliable study conducted according to OECD TG 476 and in compliance with GLP. No biologically relevant increase in mutation rate was found in mouse lymphoma L5178Y cells with or without metabolic activation in either the initial or the repeat experiment, up to limit and cytotoxic concentrations. The global evaluation factor was not exceeded at any concentration. In addition, colony sizing showed no clastogenic effects in either experiment. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Executive summary:

The test item Triethoxyoctylsilane was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In Experiment I 10.0 mM (with and without metabolic activation) was selected as the highest concentration. In Experiment II 10.0 mM (with metabolic activation) and 0.20 mM (without metabolic activation) were selected as the highest concentrations. Experiment I with and without metabolic activation and Experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

THF was used as solvent (0.35% THF v/v in the samples). After adding the THF stock solution to cell culture medium precipitate formed.

The test item was investigated at the following concentrations:

Experiment I

with and without metabolic activation:

0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 mM

Experiment II

with metabolic activation:

0.15, 0.3, 0.7, 2.0, 4.0, 6.0, 8.0 and 10.0 mM

and without metabolic activation:

0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.10 and 0.20 mM

Precipitation of the test item was noted in Pre-experiment Iwith and without metabolic activation, Pre-experiment II without metabolic activation, Experiment I with and without metabolic activation and in Experiment II with metabolic activation.

In Experiment I with metabolic activation the relative total growth (RTG) was 74.3% for the highest concentration (10.0 mM) evaluated. At two concentrations (1.0 mM and 2.5 mM) growth inhibition was observed with a RTG of 63.7% and 67.1%, respectively. However, considering all evaluated concentrations this effect showed no concentration relationship. The highest concentration evaluated without metabolic activation was 10.0 mM with a RTG of 14.6%. The Experiment without metabolic activation displayed a concentration related decrease in RTG starting at 1.0 mM with a RTG of 49.2%.

In Experiment II with metabolic activation the relative total growth (RTG) was 76.4% for the highest concentration (10.0 mM) evaluated. At two concentrations (2.0 mM and 6.0 mM) growth inhibition was observed with a RTG of 42.3% and 58.9%, respectively. However, considering all evaluated concentrations this effect showed no concentration relationship. The highest concentration evaluated without metabolic activation was 0.20 mM with a RTG of 0.9%. Due to high cytotoxicity the highest concentration was not considered for evaluation of mutagenicity. Experiment II without metabolic activation displayed a concentration related decrease in RTG starting at 0.1 mM with a RTG of 16.8%.

In Experiments I and II no biologically relevant increase of mutants were found after treatment with the test item (with and without metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed in Experiment I with metabolic activation and in Experiment II with and without metabolic activation. In Experiment I without metabolic activation a dose-response relationship was observed in the higher concentrations.

Additionally, in Experiments I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

This study is classified as acceptable. This study satisifes the requirements for Test Guidelines OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward mutation) data.