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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A well reported non-GLP study conducted to sound scientific principles with the read across substance, bis(2-ethylhexyl) adipate.

Data source

Reference
Reference Type:
publication
Title:
Responses of the L8178Y tk+/tk- Mouse Lymphoma Cells Forward Mutation Assay: III. 72 Coded Chemicals
Author:
McGregor D B, Brown A, Cattanach P, Edwards I, McBride D, Riach C, Casary W J
Year:
1988
Bibliographic source:
Environmental and Molecular Mutagenesis 12:85-154

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
(study lacked a longer exposure time)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): di(2-ethylhexyl)adipate

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The tissue culture medium was maintained in Fischer’s medium. Fischer’s medium was supplemented with 2 mM L-glutamine, 110 µg/mL sodium pyruvate, 0.05 % pluronic F68 antibiotics, and 10 % heat-inactivated donor horse serum (v/v).
- Properly maintained: Yes. L5178Y TK+/- cells were stored in a liquid nitrogen freezer. Following removal from liquid nitrogen, the cultures were kept at 37 °C on gyratory tables
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, 5000 µg/mL - Trial 1 (without metabolic activation)
0, 1800, 2600, 3400, 4200, 5000 µg/mL - Trial 2 (without metabolic activation)
0, 1000, 2000, 3000, 4000, 5000 µg/mL - Trials 1 and 2 (with metabiolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHODOLOGY
Each exposed culture consisted of 6 x 10^6 cells in a final volume of 10 mL Fischer's medium in a 30 mL plastic tube. The tube was incubated for 4 hours on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time, the cells were sedimented by centrifugation at 500 g.av. for 10 minutes, washed and resuspended in 20 mL Fischer's medium. the cell suspensions (3 x 10^5 cells/mL) were incubated for a 2 day expression period, the cell population density being adjusted back to 20 mL of 3 x 10^5 cells/mL after 24 hours. After 48 hours, the cell population densities were estimated and culture volumes containing 3 x 10^6 cells adjusted to 15 mL with Fischer's medium, giving a cell population density of 2 x 10^5 cells/mL.

CLONING EFICIENCY
A 0.1 mL sample of the cell suspension was withdrawn and diluted 1:100. Three 0.1 mL samples (200 cells) of the diluted cultures were transferred to 30 mL tubes, mixed with 25 mL Fischer's medium containing 20 % heat-inactivated horse serum containing 0.35 % Noble agar and poured into 90 mm Petri plates.

MUTANT SELECTION
Three aliquots (each containing 10^6 cells) of the remaining culture were distributed to 30 mL tubes, mixed with 20 mL cloning medium to give final concentrations of 0.35 % Noble agar and 3µg trifluorothymidine/mL, then poured into 90 mm Petri plates.

INCUBATION
The agar was gelled at 4 °C for 5 - 10 minutes, then the plates were incubated for 11 - 14 days in 5 % CO2: 95 % air at 37 °C.

NUMBER OF REPLICATIONS
Vehicle controls were performed in quadruplicate, positive controls in duplicate and test concentrations in duplicate

COLONY COUNTING
Colonies were counted using at Artek 880 Automates Colony Counter.

CALCULATIONS
Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG) which was determined as follows:

RTG = (total suspension growth x cloning efficiency) in dosed culture / (total suspension growth x cloning efficiency) in control culture

Mutant fraction (MF) was calculated as follows:

MF = 200 x (mutant clones per plate / total clones per plate)

= mutants / 10^6 clonable cells
Evaluation criteria:
CRITERIA FOR A POSITIVE RESPONSE
A positive response is obtained when there is a statistically significant dose-related increase at one of the three highest acceptable doses.

CRITERIA FOR A NEGATIVE RESPONSE
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency.
Statistics:
The statistical analysis consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. The significance level was set at 5 %.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation of the test material was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The Lowest Observed Effect Dose in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results (without metabolic activation)

Without S9 Trial 1

Without S9 Trial 2

Conc (µg/mL)

CE

RTG

MC

MF

Mean MF

Conc (µg/mL)

CE

RTG

MC

MF

Mean MF

0

78

96

60

26

24

0

92

100

75

27

22

84

85

49

19

63

97

31

16

91

105

65

24

103

104

61

20

85

114

67

26

84

98

63

25

312.5

75

74

64

28

26

1800

88

26

41

16

18

80

80

57

24

74

25

47

21

625

88

75

35

13

20

2600

73

24

51

23

18

80

70

64

27

88

23

36

14

1250

69

25

58

28

23

3400

97

20

48

17

16

75

38

39

17

93

15

44

16

2500

79

13

50

21

27

4200

78

15

35

15

20

70

16

68

32

69

13

50

24

5000

87r

13

48

18

5000

74

12

37

17

17

LETHAL

72

13

36

17

15 (MMS)

32

25

112

119

118*

15 (MMS)

44

29

110

83

83*

44

24

156

118

39

34

97

84

Table 2: Results (with metabolic activation)

With S9 Trial 1

With S9 Trial 2

Conc (µg/mL)

CE

RTG

MC

MF

Mean MF

Conc (µg/mL)

CE

RTG

MC

MF

Mean MF

0

80

98

72

30

33

0

102

116

82

27

37

76

109

55

24

109

101

114

35

70

98

88

42

100

101

111

37

73

94

78

36

68

82

102

50

1000

65

39

47

24

32

1000

93

70

116

42

40

75

38

90

40

100

67

115

38

2000

50

27

53

36

33

2000

111

28

294

88

73*

48

22

43

30

101

32

176

58

3000

59

18

55

31

34

3000

93

23

226

81

85*

59

19

65

37

91

21

242

89

4000

56

16

61

37

39

4000

78

20

187

80

80*

53

22

66

41

100

26

238

79

5000

40

16

37

31

34

5000

94

28

240

85

81*

48

13

53

37

77

27

177

77

2.5 (MCA)

29

16

187

214

222*

2.5 (MCA)

55

11

603

364

363*

32

21

223

231

55

11

592

361

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the experiment it was concluded that the test material was not mutagenic in this system.
Executive summary:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in a study which was conducted to a methodology which was similar to that outlines in the standardised guideline OECD 476. L51788Y TK+/-mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary metabolic system (S9 mix) in two independent experiments. Mutant colonies were scored for all cultures in each experiment. The test material was tested up to a maximum concentration of 5000 µg/mL in the presence and absence of metabolic activation. This concentration is approximately equivalent to the limit concentration for this assay.

Under the conditions of the study precipitation of the test material was observed at 1000 µg/mL, but testing was continued up to 5000 µg/mL. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occurred in a second experiment. The Lowest Observed Effect Dose in this experiment was 2000 µg/mL, higher than the precipitation concentration. Significant toxicity was observed in all experiments. It was therefore concluded that the test material was not mutagenic in this system.