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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:
weight of evidence
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:
2002
Report date:
2002

Materials and methods

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

Test material

Constituent 1
Reference substance name:
736150-63-3
EC Number:
616-005-1
Cas Number:
736150-63-3
Molecular formula:
C25H46 O6; C27H48O8
IUPAC Name:
736150-63-3

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: RPMI medium supplemented with 10% horse serum, 200 µg/mL sodium pyruvate and 50 µg/mL gentamycin
- Properly maintained: yes (stock cultures were kept in a liquid nitrogen tank to start new stock cultures periodically in which cells were diluted daily and kept at a density of about 2E+5 to 1.5E+6)
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
First experiment:
with and without metabolic activation: 625, 1250, 2500, 3600 and 5000 µg/mL

Second experiment:
without metabolic activation: 313, 625, 1250, 2500 and 3600 µg/mL
with metabolic activation: 156, 313, 625, 1250, 2500 and 3600 µg/mL

Repeat of second test:
without metabolic activation: 2.5, 5, 10, 20, 40, 80, 160 and 320 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitrosourea (50 µg/mL, -S9), 7,12-dimethyl-1,2-benzanthracene (3.3 µg/mL, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
First test:
- Exposure duration: +S9: 3 h, -S9: 4 h
Second main test:
- Exposure duration: +S9: 3 h, -S9: 24 h
Repeat of second main test:
- Exposure duration: -S9: 24 h
(In cell cultures with metabolic activation, the treatment period was limited to 3 h due to the cytotoxic effects induced by S9.)

- Expression time (cells in growth medium): 3 days (counting from the start of the experiment), cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates. The cell density was counted and adjusted to 3E+5 cells/mL daily.
- Selection time: approx. 10 days, cells were seeded in 2 microtitre plates with a density of 2000 cells/well in TFT selctive medium to determine the number of mutants.
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)


NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cytotoxicity corresponds to relative survival compared to the respective negative control values)

OTHER EXAMINATIONS:
Cloning efficiency was determined by seeding exposed cells in one microtiter plate with a density of 2 cells/well in medium without TFT.
Small and large colonies were differentiated as small colonies are capable to indicate chromosomal mutations.
Evaluation criteria:
The test item was considered as mutagenic when all of the following criteria were met:
- increases in the mutation frequency were observed in treated cultures compared to the corresponding negative control values at one or more test concentrations
- the increases showed a dose-response relationship
- the increases were reproducible between the replicates and the first and second test (when treatment conditions were the same)
- the increases were statistically significant
- the increases exceeded the historical negative control range
- the relative survival of the test groups was at least 15% at the end of the treatment period

When the above mentioned criteria were not met, the test item was considered as non-mutagenic.
Statistics:
Single values of the duplicates were determined from the examined parameters. Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA). In detail, mutation frequencies of treated samples were compared to the correpsonding negative controls with the analyis of variance test.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9: starting from 3600 µg/mL (first test (4 h exposure)) and 160 µg/mL (second test (24 h exposure)), with S9: starting from 2500 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the negative and positive controls were all within the range of the historical control data despite one positive control value which was slightly higher that the historical control value. Thus, the study was considered to be valid.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: First Experiment - 4 h exposure - Without Metabolic Activation

 

 

Concentration (µg/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutants per 1E+4 surviving cells

Mutation factor

 

Day 0

Day 3

 

0 (DMSO)

100

100

100

2.36

1

 

625

91

116

57

2.04

0.86

 

1250

108

94

65

2.44

1.03

 

2500

106

83

36

2.66

1.12

 

3600

89

76

11

0.30*

0.12

 

5000

74

83

8

0.04*

0.017

 

ENU (50)

53

62

45

#

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 2: First Experiment - 3 h exposure - With Metabolic Activation

 

 

Concentration (µg/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutants per 1E+4 surviving cells

Mutation factor

 

Day 0

Day 3

 

0 (DMSO)

100

100

100

2.39

1

 

625

75

81

65

2.59

1.08

 

1250

92

44

46

3.04

1.27

 

2500

93

30

9

0.9

0.38

 

3600

90

5

1

0.2

0.08

 

5000

84

4

1

0.47

0.2

 

DMBA (3.3)

24

50

29

#

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

Concentration (µg/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutants per 1E+4 surviving cells

Mutation factor

 

Day 0

Day 3

 

0 (DMSO)

100

100

100

not reported 

not reported 

 

313

82

9

2

not reported

not reported

 

625

73

4

1

not reported

not reported

 

1250

68

4

1

not reported

not reported

 

2500

38

4

1

not reported

not reported

 

3600

31

1

0

not reported

not reported

 

ENU (50)

53

35

9

not reported

 

not reported

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4: Second Experiment - 3 h exposure - With Metabolic Activation

 

 

Concentration (µg/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutants per 1E+4 surviving cells

Mutation factor

 

Day 0

Day 3

 

0 (DMSO)

100

100

100

2

1

 

156

83

95

62

1.95

0.98

 

313

84

101

70

1.84

0.92

 

625

82

105

65

1.79

0.9

 

1250

46

98.5

65

1.84

0.92

 

2500

38

88

37

1.93

0.97

 

3600

36

4

0

0

0

 

DMBA (3.3)

28

52

27

37.3

18.65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5: Repeat fo Second Experiment - 24 h exposure - Without Metabolic Activation

 

Concentration (µg/mL)

Cloning efficiency (%)

Relative Total Growth (%)

Mutants per 1E+4 surviving cells

Mutation factor

 

Day 0

Day 3

 

0 (DMSO)

100

100

100

2.17

1

 

`2.5

102

100

62

2.16

1

 

5

100

114

151

2.02

0.93

 

10

92

95

145

1.97

0.91

 

20

105

100

60

2.11

0.97

 

40

82

95

103

2.17

1

 

80

69

111

97

1.82

0.84

 

160

25

44

16

2.32

1.07

 

320

11

31

6

1.94

0.89

 

ENU (50)

16

31

9

51.8

23.87

 

 

ENU: N-ethyl-N-nitrosourea

DMBA: 7,12-dimethyl-1,2-benzanthracene

*: statistically significant with p < 0.01

#: could not be calculated as all wells contained mutant colonies

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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