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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:
27 Aug - 24 Oct 2012
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline S2 (R1): 'Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (EMA/CHMP/ICH/126642/2008)'
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2-dimethyl-1,3-propanediyl dioleate
- Analytical purity: 100%
- Physical state: light yellowish, clear liquid
- Batch No.: OE10124A
- Storage condition of test material: at room temperature, in a tightly closed container; kept away from heat, sparks, flames and direct sunlight

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: growth medium: RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% v/v);
treatment medium: growth medium without sodium pyruvate, gentamycin and fungizone;
cleaning medium: growth medium supplemented with approx. 4.0E-05 M thymidine, 1.2E-04 M hypoxanthine, 3.3E-05 M glycine and 7.2E-07 M methotrexate;
recovery medium: growth medium supplemented with approx. 4.0E-05 M thymidine, 1.2E-04 M hypoxanthine and 3.3E-05 M glycine;
selection medium: growth medium containing 3 µg/mL trifluorothymidine (TFT)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary experiment: 25, 100, 250, 1000, 2500 and 5000 µg/mL
- 24 h treatment: without metabolic activation
- 4 h treatment: with metabolic activation

Main assay: 312.5, 625, 1250, 2500 and 5000 µg/mL
- Experiment I - 3 h treatment: with and without metabolic activation
- Experiment II – 24 h treatment: without metabolic activation
- Experiment II – 3 h treatment: with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was completely dissolved in acetone, but not soluble in water or DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: methylmethanesulfonate, 10 and 15 g/mL; +S9: 3-methylcholanthrene, 2.5 and 4 µg/mL
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 3 h (± S9); Experiment II: 24 h (-S9) and 3 h (+S9)
- Expression time (cells in growth medium): after the end of treatment, cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture was placed in 2 different 96 well microtiter plates (192 wells, averaging 1.6 cells/well) and incubated for 1 week, whereas the rest of the cells was incubated for 2 days for the expression period (plating efficiency step 1). The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 10E+6 cells per mL and a minimum of 4 concentration levels plus positive and negative control were selected for 5-trifluoro-thymidine (TFT) resistance. At the end of the second expression period the selected cultures were diluted to 1E+4 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency step 2). The plating for survival was identical to the above described method (plating efficiency step 1 in 192 wells with average 1.6 cells/well). For the plating for TFT resistance 3 µg/mL TFT (final concentration) was added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (384 wells, averaging 2E+3 cells/well). The plates were incubated for 11-14 days.
- Selection time (if incubation with a selection agent): 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 11-15 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: single treated cultures and duplicate solvent control cultures each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: suspension growth, relative suspension growth, plating efficiency and relative survival

OTHER EXAMINATIONS:
- Other: small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

OTHER:
- Concentration selection for the main experiment: the lowest separation factor of 2 as recommended by the guidelines was used and 5 concentrations were selected. Since no increase in the mutant frequency was observed in the preliminary experiment, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.
Evaluation criteria:
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment was a mutant frequency ≥ 2 compared to the concurrent background mutant frequency. The observation of a mutant frequency that met the minimum criterion for a single treated culture within a range of assayed concentrations was not considdered as sufficient to evaluate a test item as a mutagen.
The test results were positive, if the following criteria were met:
- a concentration-related or toxicity-related increase in mutant frequency preferably for at least 3 concentrations (depending on the concentration steps chosen and toxicity at which mutagenic activity appeared).
- the mutant frequency for a single concentration at or near the highest testable toxicity was ≥ 4 times the concurrent background mutant frequency. Smaller increases at a single concentration near the highest testable toxicity needed to be confirmed by a repeat assay.
- either parameter (concentration or toxicity (percent relative growth)) was used to establish whether the increase in mutant frequency was related to an increase in effective treatment.
- treatments inducing less than 10% relative growth were included in the assay, but not used as primary evidence for mutagenicity.
The test item was considered as negative in this assay if neither of the above criteria were met. A test item was evaluated as non-mutagenic in a single assay if the minimum increase in mutant frequency was not observed for a range of applied concentrations that extended toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.
Statistics:
The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003. For microwell assays, the GEF is defined as 126 mutants per 1E+6 viable cells. For valid data, the test item was considered to be mutagenic in this assay if the mutant frequency (MF) of any test concentration exceeded the sum of the mean control mutant frequency plus GEF and the linear trend test was positive. This would indicate a positive, biologically relevant response.

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:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: in a preliminary cytotoxicity test with concentrations ranging from 25 to 5000 µg/mL, no signs of cytotoxicity were noted in the experiment without and with metabolic activation (24-h or 4-h exposure, respectively) at any of the concentrations tested. Hence, the highest concentration of the test substance selected for treatment in the main study was 5000 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: mutant frequencies of the positive and negative controls were within the respective historical ranges. The mutant frequencies in treated colonies were within the range of the negative control values.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Experiment I - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]

Plating efficiency 2

Relative survival [%]

Mutants per 1E+06 surviving cells

% large colonies of total colonies

% small colonies of total colonies

ratio small/large colonies

SC*

1.1193

100

57.93

45.7

54.3

1.19

312.5

0.8796

79

79.30

48.0

52.0

1.08

625

0.7270

65

81.71

51.2

48.8

0.95

1250

0.9208

82

62.88

42.9

57.1

1.33

2500

1.0124

90

57.19

50.0

50.0

1.00

5000

0.9208

82

69.29

52.2

47.8

0.92

3-MCA, 2.5

0.3096

28

693.15

43.3

56.7

1.31

3-MCA, 4.0

0.3596

32

653.50

52.1

47.9

0.92

3-MCA = 3-methylcholanthrene; SC = solvent control (acetone)

* mean value of 2 parallel cultures

Table 2. Experiment I - 3 h exposure - Without Metabolic Activation

Concentration [µg/mL]

Plating efficiency 2

Relative survival [%]

Mutants per 1E+06 surviving cells

% large colonies of total colonies

% small colonies of total colonies

ratio small/large colonies

SC*

0.8866

100

72.27

54.3

45.7

0.84

312.5

0.9649

109

66.12

45.7

54.3

1.19

625

0.9499

107

71.85

46.9

53.1

1.13

1250

0.9804

111

65.08

45.7

54.3

1.19

2500

1.2033

136

59.12

58.8

41.2

0.70

5000

1.1602

131

60.12

52.0

48.0

0.92

MMS, 10

0.1627

18

1782.42

27.8

72.2

2.60

MMS, 15

0.2248

25

1919.48

37.8

62.2

1.64

MMS = methylmethanesulfonate; SC = solvent control (acetone)

* mean value of 2 parallel cultures

Table 3. Experiment II - 3 h exposure - With Metabolic Activation

Concentration [µg/mL]

Plating efficiency 2

Relative survival [%]

Mutants per 1E+06 surviving cells

% large colonies of total colonies

% small colonies of total colonies

ratio small/large colonies

SC*

0.9070

100

53.48

47.9

52.1

1.09

312.5

0.8664

96

50.21

43.8

56.3

1.29

625

0.9208

102

55.01

51.4

48.6

0.95

1250

0.8164

90

67.37

45.0

55.0

1.22

2500

0.6769

75

62.20

41.9

58.1

1.38

5000

0.8286

91

66.38

45.0

55.0

1.22

3-MCA, 2.5

0.4203

46

894.72

48.3

51.7

1.07

3-MCA, 4.0

0.4598

51

776.64

52.0

48.0

0.92

3-MCA = 3-methylcholanthrene; SC = solvent control (acetone)

* mean value of 2 parallel cultures

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

Concentration [µg/mL]

Plating efficiency 2

Relative survival [%]

Mutants per 1E+06 surviving cells

% large colonies of total colonies

% small colonies of total colonies

ratio small/large colonies

SC*

0.7274

100

55.94

48.3

51.7

1.07

312.5

0.6674

92

54.61

51.9

48.1

0.93

625

0.7166

99

74.73

51.3

48.7

0.95

1250

0.6866

94

61.32

51.6

48.4

0.94

2500

0.8045

111

61.16

47.2

52.8

1.12

5000

0.7065

97

55.56

55.2

44.8

0.81

MMS, 10

0.3654

50

1139.30

39.2

60.8

1.55

MMS, 15

0.2537

35

2005.13

37.6

62.4

1.66

MMS = methylmethanesulfonate; SC = solvent control (acetone)

* mean value of 2 parallel cultures

RATIO OF SMALL TO LARGE COLONIES

No change was observed in the ratio of small to large mutant colonies between treated and solvent control cultures.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative