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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
Study period:
28 Jun - 23 Aug 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
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, München, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 1640 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphtoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Pre-Test:
Experiment 1:
- with and without metabolic activation: 0.5, 2, 4, 6, 8, 10 mM
Experiment 2:
- without metabolic activation. 0.005, 0.05, 0.2, 0.7, 1.3, 2.0 mM

Main Test:
Experiment 1:
- with metabolic activation: 0.70, 0.82, 0.94, 1.06, 1.18, 1.30, 1.42, 1.54 mM
- without metabolic activation. 0.22, 0.46, 0.58, 0.70, 0.82, 0.94, 1.06, 1.18 mM
Experiment 2:
- with metabolic activation: 1.0, 1.12, 1.24, 1.36, 1.48, 1.60, 1.72, 1.84 mM
- without metabolic activation. 0.0005, 0.001, 0.002, 0.005, 0.01, 0.06, 0.18, 0.3 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI cell culture medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
since medium was used as solvent, no further solvent control was necessary
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: methylmethanesulfonate (10 µg/mL, dissolved in 0.9% NaCl); ethylemethanesulphanate (200 and 500 µg/mL, dissolved in medium); +S9: benzo(a)pyrene (3.5 µg/mL, dissolved in DMSO (1% final concentration in medium))
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h (short-term exposure) with and without metabolic activation
Experiment 2: 4 h (short-term exposure) with metabolic activation and 24 h (long-term exposure) without metabolic activation
- Expression time (cells in growth medium): 3 days (short-term exposure) or 2 days (long-term exposure)
- Selection time (if incubation with a selection agent): 11 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 18 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; cloning efficiency; mitotic index

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations
Evaluation criteria:
There are several criteria for a positive result:
- clear and dose-related increase in the mutant frequency
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/Small colonies ratio (1.5 times the ratio of clastogenic control MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

The test substance is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

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:
at 1.54 mM with metabolic activation and at 1.18 mM without metabolic activation in experiment 1, respectively; at 1.84 mM with metabolic activation and at 0.30 mM without metabolic activataion in experiment 2, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: within the physiological range
- Effects of osmolality: within the physiological range
- Precipitation: in the pre-test with metabolic activation from concentrations of 4 mM and higher

RANGE-FINDING/SCREENING STUDIES:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)

COMPARISON WITH HISTORICAL CONTROL DATA:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5), is an indication for potential clastogenic effects and/or chromosomal aberrations.

Although in experiment 1 with metabolic activation an increased number of small colonies was noted at doses of 1.30 mM and 1.42 mM (26 and 31 small colonies, respectively, compared to 11 and 9 at control) all dose groups were considered as not clastogenic since no mutagenicity was found at these doses.

All other dose groups in the other experiments were also found not to be clastogenic, respectively.

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

86.77

1

3.66

0.7

96.63

95.51

95.86

1.10

--

0.82

104.12

92.71

88.56

1.02

--

0.94

109.36

95.60

76.43

0.88

--

1.06

110.11

78.73

78.58

0.91

--

1.18

109.36

60.06

86.55

1.00

--

1.30

116.10

40.14

100.88

1.16

1.77

1.42

112.36

31.61

121.24

1.40

1.55

1.54

96.63

9.84

139.92

1.61

2.78

B[a]P, 3.5 µg/mL

99.63

69.03

623.89

7.19

1.24

B[a]P:Benzo[a]pyrene

  

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation 

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

79.39

1

2.75

0.22

100.33

90.09

78.53

0.99

--

0.46

86.38

79.76

124.63

1.57

--

0.58

91.03

79.70

77.88

0.98

--

0.70

98.34

89.53

70.89

0.89

--

0.82

98.34

71.30

69.28

0.87

--

0.94

95.02

64.50

62.74

0.79

1.60

1.06

99.00

47.66

74.59

0.94

3.17

1.18

91.69

11.04

97.49

1.23

1.12

EMS, 500 µg/mL

86.38

62.27

1337.77

16.85

--

MMS, 10 µg/mL

85.71

66.62

841.03

10.59

0.69

EMS:Ethyl methane sulphonate

MMS:Methyl methane sulphonate

 Table 3: Experiment II - 4 h Exposure - With Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

82.64

1.00

2.07

1

96.97

85.91

82.23

1.00

--

1.12

101.68

89.08

61.54

0.74

--

1.24

98.99

86.20

86.92

1.05

--

1.36

99.66

83.65

75.75

0.92

--

1.48

90.91

64.04

118.05

1.43

--

1.60

96.07

35.51

70.23

0.85

2.38

1.72

91.58

38.25

84.77

1.03

2.13

1.84

98.99

19.98

98.81

1.20

4.73

B[a]P, 3.5 µg/mL

86.20

60.39

829.02

10.03

0.89

B[a]P:Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration
[mM]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 surviving cells

Mutation factor

Colony Sizing

Quotient Large/Small

0

100

100

104.66

1

2.61

0.0005

95.24

94.64

76.34

0.73

--

0.001

101.36

103.58

68.22

0.65

--

0.002

94.56

95.29

66.56

0.64

--

0.005

101.36

105.73

52.63

0.50

--

0.01

102.04

98.98

64.06

0.61

--

0.06

102.72

96.75

61.54

0.59

3.30

0.18

96.60

55.56

91.91

0.88

2.24

0.30

91.16

19.77

99.26

0.95

1.94

EMS, 200 µg/mL

70.75

34.84

2516.55

24.05

--

MMS, 10 µg/mL

59.18

27.33

2625.00

25.08

0.81

EMS: Ethyl methane sulphonate

MMS: Methyl methane sulphonate

Applicant's summary and conclusion

Conclusions:
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item decanoic acid is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.