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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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: in vitro mutagenicity in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protect from moisture/water as well as heat and ignition sources
- Stability under test conditions: The test material was stable in dimethyl sulfoxide.
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide, to which molecular sieve (0.4 nm) beads were added to absorb the water.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in dimethyl sulfoxide, to which molecular sieve (0.4 nm) beads were added to absorb the water. Test item concentrations were used within 1 hour of preparation. The final concentration of the solvent in the exposure medium was 1.0% (v/v). The pH and the osmolarity of the culture medium containing the highest, non-precipitating concentration were recorded.
- Preliminary purification step (if any): No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
(2001)
- Suitability of cells: Recommended in Test Guideline
- Cell cycle length, doubling time or proliferation index: not stated
- 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: Stock cultures of the cells were stored in liquid nitrogen (-196°C)
- Modal number of chromosomes: not stated
- Normal (negative control) cell cycle time: not stated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: horse media
- 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:
Rat S9 homogenate, Sprague Dawley rats
Test concentrations with justification for top dose:
In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/ml for 3 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The test item was tested in the absence and presence of S9-mix. The highest tested concentration was 5000 µg/ml exposure medium.
Mutation experiment dose concentrations: 100, 1000, 2000, 3000, 3500, 4000, 4500, 5000 µg/ml without metabolic activation
Mutation experiment dose concentrations: 25, 50, 100, 250, 500, 1000, 3000 µg/ml with metabolic activation

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
ACTIVATION: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. The concentration of the S9-fraction in the exposure medium was 4% (v/v).

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours with or without metabolic activation at 37.0 ± 1.0 °C and 145 rpm
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): Incubation for 11 or 12 days with TFT-selective medium
- Fixation time (start of exposure up to fixation or harvest of cells): not specified

SELECTION AGENT (mutation assays): TFT-selective medium

NUMBER OF REPLICATIONS: single culture for test group and duplicate cultures for solvent and positive controls.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma).

NUMBER OF CELLS EVALUATED: 2000 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; relative cloning efficency; mutation frequency
- Any supplementary information relevant to cytotoxicity: not specified

OTHER EXAMINATIONS:
- Determination of polyploidy: not determined
- Determination of endoreplication: not determined

Evaluation criteria:
A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency of controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
Statistics:
A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9-mix, the dose levels of 100 to 4000 μg/ml showed no cytotoxicity. Cytotoxicity was observed at 4500 and 5000μg/ml.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 and 3000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the solubility test the pH at a concentration of 1250 μg/ml was 7.51.
- Effects of osmolality: In the solubility test the osmolarity at a concentration of 1250 μg/ml was 0.451 Osm/kg.
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: During the solubility test the test item precipitated in the exposure medium at concentrations of 2500 and 5000 μg/ml at the start of the treatment. After 3 hour treatment, the test item precipitated in the exposure medium at the concentration of 5000 μg/ml only. Based on these solubility findings, 5000 μg/ml was selected as the maximum final concentration for the dose range finding test. In the main test the test item precipitated in the exposure medium at test item concentrations 3500 µg/ml and above.
- Definition of acceptable cells for analysis: not specified
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: The relative suspension growth was 16% at the test item concentration of 5000 μg/ml compared to the relative suspension growth of the solvent control in the absence of metabolic activation. The relative suspension growth was 29% at the test item concentration of 5000 μg/ml compared to the relative suspension growth of the solvent control in the presence of metabolic activation.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In the absence of S9-mix, the dose levels of 100 to 4000 μg/ml showed no cytotoxicity and the relative total growth of the highest test item concentration was 37% compared to the total growth of the solvent controls. In the presence of metabolic activation the dose levels of 1000 to
3000 μg/ml showed similar cell growth delay. Therefore, the dose level of 2000 µg/ml was not regarded relevant for mutation frequency measurement. The dose levels of 4000 to 5000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The relative total growth of the highest test item concentration was 7% compared to the total growth of the solvent controls, see study plan deviation.

Any other information on results incl. tables

Table 1: Mutation Experiment: Cytotoxic and Mutagenic Response of the test item in the Mouse Lymphoma L5178Y Test System

dose

(µg/ml)

RSG

(%)

CE day2

(%)

RCE       

(%)

RTG

(%)

mutation frequency

per 106 survivors total (small large )

Without metabolic activation

 

3-hour treatment

 

 

 

SC1

100

 

118

 

100

100

96

SC2

 

 

131

 

 

 

77

100

 

137

118

95

130

77

1000

 

145

98

 

79

114

94

2000

 

150

127

 

102

153

96

3000

 

144

101

 

81

117

125

3500 (1)

 

111

116

 

94

104

69

4000 (1)

 

87

113

 

91

79

91

4500 (1)

 

73

120

 

96

70

88

5000 (1)

 

46

101

 

81

37

132

MMS

120

60

49

58

1201

With metabolic activation

 

3-hour treatment

 

 

 

SC1

 

100

86

100

100

53

SC2

 

 

58

 

 

90

25

88

78

108

95

83

50

65

97

134

87

64

100

60

79

109

66

146

250

57

85

118

67

347

500

30

64

89

27

681

1000

18

33

46

8

1318

3000

13

41

56

7

1230

CP

34

16

23

8

2860

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;

SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

(1)   = the test item precipitated in the exposure medium

Applicant's summary and conclusion

Conclusions:
Reaction mass of trimethoxy(aminoalkyl)silanes and modified alkylether oligomers has been tested for mutagenicity in Mouse lymphoma L5178Y cells according to OECD TG 490, and in compliance with GLP. No test-substance induced increase in the number of mutations was observed when the substance was test in the absence of metabolic activation. The test item induced an 18-fold and 9.5-fold dose related increase in the mutation frequency at concentrations of 1000 and 500 µg/ml, respectively, in the presence of metabolic activation. This increase was above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls). Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is positive for mutagenicity to mammalian cells under the conditions of the study.