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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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste

Method

Target gene:
Thymidin kinase-locus
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented S9 mix prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I:
with S9 mix: 0.01, 0.05, 0.24, 0.28, 0.32, 0.36, 0.40, 0.44, 0.48 mM (incubation period: 4 h)
without S9 mix: 0.01, 0.02, 0.05, 0.10, 0.20, 0.24, 0.28, 0.32 mM (incubation period: 4 h)
Experiment II:
with S9 mix: 0.15, 0.35, 0.39, 0.43, 0.45, 0.47, 0.49, 0.53 mM (incubation period: 4 h)
without S9 mix: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.10, 0.15, 0.20, 0.25 mM(incubation period: 24 h)
Vehicle:
Based on a solubility test RPMI (+5% horse serum) was used as solvent.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS): 200 + 300 µg/mL; Methylmethanesulfonate (MMS): 10 µg/mL; Benzo(a)pyrene (BaP): 2.5 µg/mL
Details on test system and conditions:
Experiment I with and without S9 mix and Experiment II with S9 mix (incubation period: 4 h)
1 x 10E+7 cells were suspended in medium and exposed to the test item either in the presence or absence of metabolic activation in the mutation experiment for 4 h. Thereafter the test item was removed, the cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

Experiment II without S9 mix (incubation period: 24 h)
5 x 10E+6 cells were suspended in medium and exposed to the test item in the absence of metabolic activation. After 24 h the test item was removed, cells were washed and suspended in complete culture medium and grown for 2 days at 37 °C in humidified air. The cell density was determined each day and adjusted to 3 x 10E+5 cells/mL.

After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in selective medium with TFT. The plates were scored after an incubation period of approx. 14 days at 37 °C in humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. The mutant frequency is expressed as “mutants per 10E+6 viable cells”.

Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density.
The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE /100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation (GEF = 126 for the microwell method).
Evaluation criteria:
A mutation assay is considered acceptable if:
- At least 3/4 plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E+6 cells
- The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period or 32-180 fold increase during a 3 day growth period (long-term treatment).
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E+6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E+6 cells.
- The RTG is greater than 10%.

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 mutants per 10E+6 cells.
- A dose-dependent increase in mutant frequency is detected.
Statistics:
The Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. 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
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No precipitation was observed at any concentration. The pH-value detected in medium containing the test item was within the physiological range.

Any other information on results incl. tables

None of the experimental results met the criteria for considering the test substance mutagenic, except for the highest dose level in experiment I with metabolic activation. There a significantly increased number of mutants at 0.48 mM and a dose dependency were seen. In addition the Global Evaluation Factor of 126 was exceeded by the induced mutant frequency. This effect was only seen in the highly cytotoxic range (RTG below 10%) and was not seen in the verification experiment (experiment II with metabolic activation). Therefore this effect was considered to be of no biological relevance.

Table 1: Experiment I - 4 h exposure – With and without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

 

+S9 mix

Control 1

0

82.9

100

73.7

-

Control 2

0

70.6

92.8

Test Item

0.01

73.7

90.7

89.2

5.9

0.05

79.3

107.8

66.8

-16.5

0.24

71.7

97.2

93.8

10.6

0.28

80.5

93.1

98.1

14.9

0.32

72.7

78.0

88.4

5.1

0.36

80.5

60.1

70.2

-13.0

0.40

72.7

40.4

104.3

21.0

0.44

93.5

27.3

141.3 *

58.0

0.48

82.9

9.4

217.2 *

134.0

Benzo[a]pyrene

2.5

58.8

48.3

829.8 *

746.5

 

-S9 mix

Control 1

0

92.1

100.0

76.3

/

Control 2

0

84.1

83.3

/

Test Item

0.01

98.0

107.4

85.9

6.1

 

0.02

85.4

101.2

73.3

-6.5

 

0.05

82.9

109.0

82.8

2.9

 

0.10

96.5

110.7

87.0

7.2

 

0.20

110.1

79.6

70.8

-9.0

 

0.24

102.9

42.3

93.5

13.7

 

0.28

132.6

31.0

137.4*

57.6

 

0.32

114.0

23.6

126.4*

46.5

EMS

300

77.0

63.5

713.3*

633.5

MMS

10

79.3

64.3

447.5*

367.7

*: p <0.05

EMS   Ethyl methane sulphonate,       MMS Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 2: Experiment II - 4 h Exposure - With Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

68.7

100.0

115.5

/

Control 2

0

86.6

75.6

/

Test Item

0.15

93.5

121.1

89.6

-5.9

0.35

67.7

77.0

130.3

34.7

0.39

92.1

82.2

94.5

-1.0

0.43

93.5

60.7

118.5

22.9

0.45

116.0

60.8

102.5

7.0

0.47

108.2

36.1

148.6*

53.0

0.49

89.3

18.9

169.4*

73.9

0.53

89.3

11.1

220.6*

125.1

Benzo[a]pyrene

2.5

68.7

68.3

691.7*

596.1

*: p <0.05

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

 

Table 3: Experiment II - 24 h exposure - Without Metabolic Activation

Test group

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants/1E+06 surviving cells

Induced mutant frequency

Control 1

0

93.5

100.0

75.3

/ 

Control 2

0

95.0

108.4

/ 

Test Item

0.0005

118.1

124.7

77.4

-14.4

0.001

118.1

130.7

82.6

-9.2

0.005

89.3

98.1

74.9

-16.9

0.01

106.4

98.3

72.7

-19.1

0.05

127.4

94.1

69.3

-22.5

0.10

125.0

84.9

69.5

-22.4

0.15

89.3

43.7

92.0

0.2

0.20

92.1

26.0

113.6

21.8

0.25

80.5

7.9

188.6*

96.8

EMS

200

38.9

12.2

3002.5*

2910.7

MMS

10

34.2

10.1

2321.3*

2229.5

*: p <0.05

EMS   Ethyl methane sulphonate;      MMS  Methyl methane sulphonate

Induced mutant frequency = mutant frequency of Sample – mean value of mutant frequency in corresponding controls

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative