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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-03-09 to 2015-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

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

Method

Target gene:
TK-locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: complete culture medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced S9 mix
Test concentrations with justification for top dose:
Experiment I:
0.0010, 0.0025, 0.005, 0.010, 0.025, 0.05, 0.10, 0.25 and 0.5 μL/mL, with and without metabolic activation
Experiment II:
0.0015, 0.003, 0.008, 0.015, 0.03, 0.08, 0.15 and 0.5 μL/mL, with metabolic activation
0.0025, 0.005, 0.010, 0.025, 0.05, 0.10, 0.25 and 0.5 μL/mL, without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: 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:
ethanol
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:
ethanol
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:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: benzo[a]pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
ACTIVATION SYSTEM: S9 cofactor solution included S9 at a concentration of 0.75 mg/ml in the cultures. Cofactors added to the S9 mix were: 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 complete culture medium

DURATION
- Exposure duration:
Experiment I: 4 hours, with and without metabolic activation
Experiment II: 4 hours, with metabolic activation; 24 hours, without metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days
- Determination of cloning efficiency: cells were incubated for 6 days to determine cloning efficiency

SELECTION AGENT (mutation assays): selective medium with TFT

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item was considered mutagenic if the following criteria were met:
- The induced mutant frequency meets or exceeded the Global Evaluation factor (GEF) of 126
mutants per 106 cells
- A dose-dependent increase in mutant frequency was detected.
Statistics:
Mean values and t-test.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
PRECIPITATION: Precipitation was observed at 0.5 μL/mL in experiment I and II, with and without metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY: No growth inhibition was observed in experiment I and II, with and without metabolic activation
Remarks on result:
other:
Remarks:
No mutagenic potential observed

Any other information on results incl. tables

Table 1. Experiment I, without metabolic activation

 

Test Group

Concentrations

µL/ml

RCE %

RTG %

MF

(mutants/106cells)

IMF (mutants/106cells)

GEF exceeded

Statistical Significant Increase

 

 

 

 

 

Experiment I: without metabolic activation

C1

0

103.0

97.2

88.6

/

/

/

C2

0

115.2

105.1

88.6

/

/

/

S1

0

100.0

100.0

73.9

/

/

/

S2

0

100.0

100.0

73.9

/

/

/

2

0.0010

119.0

108.5

65.8

-8.2

-

-

3

0.0025

95.5

85.5

97.9

23.9

-

+

4

0.005

104.7

90.9

60.5

-13.4

-

-

5

0.010

99.9

87.1

76.5

2.6

-

-

6

0.025

103.0

99.7

67.9

-6.1

-

-

7

0.05

106.3

87.3

54.9

-19.0

-

-

8

0.10

113.3

104.2

61.8

-12.1

-

-

9

0.25

123.2

106.9

78.6

4.7

-

-

10

0.5

137.2

137.8

41.6

-32.4

-

+

EMS

300 μg/mL

76.8

52.6

967.7

893.8

+

+

MMS

10 μg/mL

80.2

58.7

582.5

508.5

+

+

Table 2. Experiment II, without metabolic activation

 

Test Group

Concentrations

µL/ml

RCE %

RTG %

MF

(mutants/106cells)

IMF (mutants/106cells)

GEF exceeded

Statistical Significant Increase

 

 

 

 

 

Experiment II: without metabolic activation

C1

0

88.8

97.6

68.0

/

/

/

C2

0

90.1

105.3

68.0

/

/

/

S1

0

100.0

100.0

86.0

/

/

/

S2

0

100.0

100.0

86.0

/

/

/

3

0.0025

113.9

99.1

72.1

-13.9

-

-

4

0.005

88.8

76.7

89.1

3.2

-

-

5

0.010

122.9

122.0

57.2

-28.8

-

-

6

0.025

88.8

77.6

97.6

11.6

-

-

7

0.05

100.8

103.9

91.8

5.9

-

-

8

0.10

93.0

91.1

90.0

4.1

-

-

9

0.25

90.1

90.5

97.8

11.9

-

-

10

0.5

106.1

104.7

92.1

6.1

-

-

EMS

200 μg/mL

52.7

31.0

2366.9

2281.0

+

+

MMS

8 μg/mL

58.3

36.1

1081.3

995.4

+

+

Table 3. Experiment I, with metabolic activation

 

Test Group

Concentrations

µL/ml

RCE %

RTG %

MF

(mutants/106cells)

IMF (mutants/106cells)

GEF exceeded

Statistical Significant Increase

 

 

 

 

 

Experiment I: with metabolic activation

C1

0

102.1

86.1

73.6

/

/

/

C2

0

125.6

122.4

73.6

/

/

/

S1

0

100.0

100.0

82.0

/

/

/

S2

0

100.0

100.0

82.0

/

/

/

2

0.0010

121.4

140.4

63.9

-18.2

-

-

3

0.0025

117.5

120.0

75.3

-6.8

-

-

4

0.005

105.2

116.7

64.4

-17.7

-

-

5

0.010

113.8

119.6

60.9

-21.2

-

-

6

0.025

108.5

117.5

93.0

11.0

-

-

7

0.05

112.0

123.1

67.8

-14.3

-

-

8

0.10

119.4

137.3

69.4

-12.6

-

-

9

0.25

134.7

146.4

70.7

-11.3

-

-

10

0.5

113.8

123.8

77.3

-4.7

-

-

B[a]P

3.5 μg/mL

68.2

23.3

656.5

574.5

+

+

Table 4. Experiment II, with metabolic activation

 

Test Group

Concentrations

µL/ml

RCE %

RTG %

MF

(mutants/106cells)

IMF (mutants/106cells)

GEF exceeded

Statistical Significant Increase

 

 

 

 

 

Experiment II: with metabolic activation

C1

0

102.7

91.1

64.6

/

/

/

C2

0

91.1

92.7

64.6

/

/

/

S1

0

100.0

100.0

66.1

/

/

/

S2

0

100.0

100.0

66.1

/

/

/

3

0.0015

104.3

102.0

71.0

4.8

-

-

4

0.003

93.8

87.5

70.2

4.0

-

-

5

0.008

109.3

105.0

61.6

-4.5

-

-

6

0.015

120.5

113.6

49.0

-17.1

-

-

7

0.03

111.0

115.1

59.4

-6.8

-

-

8

0.08

96.6

102.9

47.7

-18.4

-

-

9

0.15

105.9

104.6

57.4

-8.7

-

-

10

0.5

91.1

89.5

79.7

13.6

-

-

B[a]P

3.5 μg/mL

99.6

62.4

576.2

510.1

+

+

C: Negative Controls

S: Solvent Controls

Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100]

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

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

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

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

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

Statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test , p<0.05).

+: significant; -not significant

EMS: Ethylmethanesulfonate [200 μg/mL and 300 μg/mL]

MMS: Methylmethanesulfonate [8 μg/mL and 10 μg/mL]

B[a]P: Benzo[a]pyrene [3.5 μg/mL]

Applicant's summary and conclusion

Conclusions:
Tetradecamethylhexasiloxane has been tested in a valid study according to OECD 476 and under GLP. No statistically and biologically significant increase in the mutant frequency was observed with or without metabolic activation when tested up to precipitating concentrations in mouse lymphoma L5178Y cells. 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 test.