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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:
03 February 2010 and 06 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 15 September 2009. Date of signature: 26 November 2009
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

Method

Target gene:
Thymidine kinase operon (tk)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 with 20% horse serum (R20) and without serum (R0)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
-Others: the cells were originally obtained from urroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen aty that time. The cell line was obtained form the MRC Cell Mutation Unit at the University of Sussex, Brighton, United Kingdom.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
20% S9-mix (2% final concentration of S9, prepared in-house with phenobarbital and beta-naphthoflavone induced rat liver, S9) was added to the cultures of the preliminary toxicity test and of experiment 1.
Test concentrations with justification for top dose:
Prelim. test: 8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 1140, 2280 µg/ml; Exp 1: 1.13, 2.25, 4.5, 9, 18, 24, 30, 36 µg/ml (-MA), 2.25, 4.5, 9, 18, 36, 48, 60, 72 µg/ml (+S9); Expt 2: 2.25, 4.5, 9, 18, 27, 36, 54, 72 µg/ml (-MA), 2.25, 4.5, 9, 18, 36, 48, 60, 72 µg/ml (+S9).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: formed a solution suitable for dosing that was compatible with the test material at the required concentration.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: (Preliminary test) 4 hours with or without S9-mix, 24 hours without S9-mix. (Experiment 1) 4 hours with or without S9-mix, (Experiment 2) 4 hours with S9-mix, 24 hours without S9-mix.
- Expression time (cells in growth medium): Incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/ml.
- Selection time: 10 - 14 days.

SELECTION AGENT: 5 trifluorothymidine.

STAIN: MTT

NUMBER OF REPLICATIONS: (Main test) Duplicate culturese, experiment repeated.

NUMBER OF CELLS EVALUATED: (Preliminary test) 5 x 10E+05 cells/ml for 4 hours of exposure, 1.5 x 10E+05 cells/ml for 24 hours of exposure (Experiment 1) 1 x 10E+06 cells/ml in 10 ml aliquots, (Experiment 2) 1 x 10E+06 cells/ml in 10 ml aliquots for 4 hours of exposure, 0.3 x 10E+06 cells/ml in 10 ml for 24 hours of exposure.

DETERMINATION OF CYTOTOXICITY:
- Method: Cell viability, relative suspension growth and relative total growth. Magnifying mirror box was used for plate scoring.

OTHER EXAMINATIONS:
- Other: Determination of mutagenicity: Mutation frequency.
Evaluation criteria:
This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequence value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF values and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
UKEMS statistical package.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
yes (at 35.63 μg/ml and above in 4-hour exposure group without S9 mix, at 8.91 μg/ml and above in 4- hour exposure group with S9 mix)
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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 18 µg/ml in 4-hour exposure group without S9 mix, at and above 36 µg/ml in 4-hour exposure group with S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY:
See Table 1 for the results.
The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in the absence or presence of metabolic activation. In Experiment 2, a very modest statistically significant dose related (linear-trend) increase in mutant frequency was observed in the presence of metabolic activation. However, a statistically significant increase in mutant frequency was only observed at one individual dose level that was at the limit of acceptable toxicity, the GEF was not exceeded at any of the dose levels, there were no marked increases in absolute numbers of mutant colonies, and the mutant frequency values observed were within the acceptable range for vehicle controls. The response was therefore considered to be spurious and of no toxicological significance.

CYTOTOXICITY:
There was evidence of marked toxicity following exposure to the test material in both the presence and absence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any marked dose-related reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. In Experiment 1, near optimum levels of toxicity were achieved in the absence of metabolic activation and optimum levels of toxicity were achieved in the presence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the absence of metabolic activation, it was considered that with no evidence of any toxicologically significant response at any of the dose levels in either the first or second experiment, including the 24-hour exposure group in the absence of metabolic activation where optimum levels of toxicity were achieved in the second experiment, the test material has been adequately tested.
In near Experiment 2, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 72 µg/ml in both the absence and presence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a modest effect on the toxicity of the test material.

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects on pH: There was no marked change in pH when the test material was dosed into media.
- Effects on osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: No precipitate of test material was observed at any of the dose levels.

RANGE-FINDING/SCREENING STUDIES:
See Table 2 for the results.
In all three of the exposure groups there were marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve of the test material was steep in all three of the exposure groups. A greasy/oily precipitate of the test material was observed at the end of the exposure period at and above 570 µg/ml in the 4-hour exposure groups, and at and above 285 µg/ml in the 24 hour exposure group. In the subsequent mutagenicity experiments the maximum dose was limited by test material-induced toxicity.

Any other information on results incl. tables

Table 1: Summary of results (Experiments 1 and 2)

Experiment 1

Treatment 4 Hours -S9

Treatment 4 Hours +S9

(µg/ml)

%

RTG

MF§

(µg/ml)

%

RTG

MF§

0

100

1.00

56.05

0

100

1.00

63.38

1.13

108

1.11

53.03

2.25

95

1.18

71.29

2.25

117

1.18

53.98

4.5

109

1.15

66.11

4.5

109

1.08

61.39

9

110

1.06

81.23

9

107

1.21

61.74

18

97

1.20

70.33

18

77

0.77

56.55

36

73

0.90

76.08

24

56

0.63

46.37

48

52

0.69

52.12

30

44

0.54

47.59

60

44

0.54

66.15

36

36

0.30

65.17

72

16

0.16

83.95

Linear trend

NS

Linear trend

NS

EMS

 

 

 

CP

 

 

 

400

76

0.55

597.25

2

60

0.32

676.68

 

Experiment 2

Treatment 24 Hours -S9

Treatment 4 Hours +S9

(µg/ml)

%

RTG

MF§

(µg/ml)

%

RTG

MF§

0

100

1.00

80.27

0

100

1.00

75.13

2.25 Ø

111

 

 

2.25 Ø

102

 

 

4.5

118

1.00

84.56

4.5

107

0.98

70.84

9

98

0.94

82.63

9

101

0.94

100.26

18

71

0.69

74.53

18

98

0.97

73.71

27

61

0.58

85.32

36

81

0.77

70.94

36

35

0.25

76.81

48

50

0.39

105.74

54

25

0.17

80.09

60

11

0.13

132.15*

72 Ø

0

 

 

72 Ø

6

 

 

Linear trend

NS

Linear trend

*

EMS

 

 

 

CP

 

 

 

150

71

0.51

992.76

2

65

0.43

498.65

 

Table 2: Results of Preliminary Toxicity Test

Dose

%(-S9)

%(+S9)

%(-S9)

(µg/ml)

4 Hour Exposure

4 Hour Exposure

24 Hour Exposure

0

100

100

100

8.91

88

83

84

17.81

102

87

65

35.63

2

86

28

71.25

0

1

0

142.5

0

0

0

285

0

0

0

570

0

0

0

1140

0

0

0

2280

0

0

0

 

Applicant's summary and conclusion

Conclusions:
Tri(isopropyl)silyl acrylate has been tested in a reliable study conducted in accordance with OECD 476 and in compliance with GLP. No evidence of a biologically significant test-substance induced increase in mutant frequency was observed when L5178Y cells were tested with and without metabolic activation up to cytotoxic concentrations in an initial experiment with 4 hours exposure. The results were confirmed in a repeat experiment where cells were exposed for 4 hours with metabolic activation and for 24 hours without metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is considered that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4 -hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 -hour ecposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. For the first experiment the dose range was 1.13 to 36 µg/ml in the absence of metabolic activation, and 2.25 to 72 µg/ml in the presence of metabolic activation. For the second experiment the dose range was 2.25 to 72 µg/ml in both the absence and presence of metabolic activation.

Results. The maximum dose level used was limited by test material induced toxicity. No precipitate of test material was observed at any of the dose levels in the mutagenicity test.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satidfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.