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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:
07 February 2018 to 25 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: gene mutation in mammalian cells using the thymidine kinase locus

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years.
- Cell cycle length, doubling time or proliferation index: 10-12 hours
- ff Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich.
- Modal number of chromosomes: near diploid karyotype (40 +/- 2 chromosomes)
- Normal (negative control) cell cycle time: 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: not applicable
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital / β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations:
without metabolic activation:
50, 100, 150, 200, 250 and 300 µg/mL
and with metabolic activation:
20, 50, 100, 200, 400 and 600 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on a pre-experiment for solubility. DMSO was used as solvent and 2 mg/mL was used as the highest concentration. The test item was dissolved in anhydrous DMSO shortly (less than 1 hour) before treatment. From the highest test item stock solution of 200 mg/mL separate dosing solutions were prepared for each of the concentrations by serial dilution with DMSO. 1% (v/v) of each dosing solution will be added directly to the cell culture medium. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 469 mOsm/kg. The solvent was compatible with the survival of the cells and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.


METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1 x 10⁷ cells

DURATION
- Exposure duration: short-term 4-hour exposure
- Expression time (cells in growth medium): 2 days following 4-hour exposure
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): selective medium

NUMBER OF REPLICATIONS: Single cultures. Negative and solvent controls are tested in duplicate.

NUMBER OF CELLS EVALUATED: 2000 cells/well in 200 µL selective medium

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; Relative Total Growth, RTG
- Any supplementary information relevant to cytotoxicity: not specified

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Colony sizing colony characterization by colony sizing was carried out in case the test was positive

Rationale for test conditions:
Based on pre-experiment.
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- 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 of the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
Statistical significance

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: 10.6% at 300 µg/mL without metabolic activation; 11.5% at 600 µg/mL with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: No precipitation of the test item was noted in the pre-experiment / main experiment.
- Definition of acceptable cells for analysis: not specified
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Six concentrations [25, 50, 150, 450, 1000 and 2000 µg/mL] were tested without and with metabolic activation. The experimental conditions in these pre-experiment were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.
- Negative (solvent/vehicle) historical control data: All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Growth inhibition was observed in the experiment without and with metabolic activation. The relative total growth (RTG) was 10.6% (without metabolic activation) and 11.5% (with metabolic activation) for the highest concentration evaluated.

Remarks on result:
other: See "Remarks"
Remarks:
The mutant frequencies induced by the test item did not show any biologically relevant increase. The GEF of 126 was not exceeded in any of the dose groups. A statistical analysis displayed that one of the mutant frequencies (with metabolic activation at a test concentration of 50µL/ML) was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship. Therefore this effect was considered as not biologically relevant.

Any other information on results incl. tables

Table1: Summary: Main Experiment, without and with metabolic activation

Test Group

Conc.

[µg/mL]

RCEa[%]

RTGb[%]

MFc[mutants/ 106cells]

IMFd[mutants/ 106cells]

GEFeexceeded

Statistical

Significant Increasef

Precipitate

 

Exp without S9

 

 

 

 

 

 

 

 

C1

0

114.4

112.8

117.5

-

-

C2

0

123.6

121.5

117.5

-

-

S1

0

100.0

100.0

126.8

/

-

S2

0

100.0

100.0

126.8

/

-

1

50

121.7

114.7

109.6

-17.1

-

-

-

2

100

123.6

115.7

122.7

-4.1

-

-

-

3

150

125.6

119.1

136.9

10.1

-

-

-

4

200

123.6

75.7

99.9

-26.9

-

-

-

5

250

127.7

39.1

80.9

-45.9

-

-

-

6

300

98.5

10.6

122.9

-3.9

-

-

-

EMS

300

90.4

77.5

814.0

687.2

+

+

-

MMS

10

93.0

77.0

891.9

765.1

+

+

-

 

Exp with S9

 

 

 

 

 

 

 

 

C1

0

93.1

99.8

113.8

/

/

-

-

C2

0

91.6

94.4

113.8

/

/

-

-

S1

0

100.0

100.0

108.2

/

/

/

-

S2

0

100.0

100.0

108.2

/

/

/

-

1

20

88.9

90.1

132.1

23.9

-

-

-

2

50

90.3

96.5

158.5

50.4

-

+

-

3

100

93.1

97.3

111.8

3.6

-

-

-

4

200

82.5

85.5

129.6

21.4

-

-

-

5

400

79.0

30.6

121.3

13.1

-

-

-

6

600

70.4

11.5

137.9

29.8

-

-

-

B[a]P

2.5 µg/mL

52.2

25.3

560.9

452.7

+

+

-

C: Negative controls

S: Solvent controls (1% DMSO; v/v)

a:  Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test, p<0.05).
+: significant; -not significant

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

B[a]P: Benzo[a]pyrene

Applicant's summary and conclusion

Conclusions:
3-(Triacetoxysilyl)propyl methacrylate has been tested for mutagenicity to mammalian cells in a study conducted according to OECD TG 490, and in compliance with GLP using mouse lymphoma L5178Y cells. No test-substance induced increase in the number of mutations was observed when tested with and without metabolic activation. Appropriate solvent, negative (treatment medium) 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 study.