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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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restriction

Data source

Reference
Reference Type:
publication
Title:
Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay: III. 72 coded chemicals.
Author:
McGregor DB, Brown A, Cattanach P, Edwards I, McBride D, Riach C and Caspary WJ.
Year:
1988
Bibliographic source:
Environ. mol. Mutag. 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:
one test with DMSO
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Other name = Ethylenethiourea (ETU)
Source : National Toxicology Program Chemical Repository, Radian Corporation, Austin, TX 78766.

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The tk+/tk-, 3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
10% Post-mitochondrial supernatant fractions of liver homogenates (rats aroclor 1254)
Test concentrations with justification for top dose:
0, 225, 450, 900, 1800, 3600 µg/ml
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 3-methylcholanthrene (with S9) and methyl-methanesulphonate (without S9)
Details on test system and experimental conditions:
Each experiment, other than the initial toxicity test, normally consisted of the following groups: vehicle control, four cultures; positive control, two cultures; at least five test compound concentrations, two cultures per concentration. With any chemical, the first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/ml unless a much lower concentration was indicated by the poor solubility of a compound.
Evaluation criteria:
See tables 1 and 2
Statistics:
yes

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Insolubility, rather than toxicity, restricted dosing to 3600 µg/ml, which, in the absence of S9 mix, failed to induce a mutagenic response in two experiments. In the presence of S9 mix statistically significant increases in mutant fraction occurred in two experiments. The LOED was 1800 µg/ml, where toxicity was either slight or not demonstrable. See tables 3-6.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 3 : Results of ETU without S9 (trial no.1)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

99

103

107

51

-

0

86

96

81

32

37

86

102

78

30

85

100

79

52

225

102

108

99

33

33

92

99

93

34

450

94

107

58

21

28

95

96

101

36

900

73

85

72

33

-

1800

71

67

86

40

46

63

80

97

51

3600

64

74

85

44

36

92

89

76

28

MMSb

35

24

132

126

154*

36

21

196

181

aMutation colonies per 10^6 clonable cells

bpositive control, Methymethanesulfonate, 2 assays, 15 µg/ml

* P<0.05

Table 4 : Results of ETU without S9 (trial no.2)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

79

99

69

29

-

0

69

93

65

31

26

69

92

40

19

80

116

54

21

225

90

104

71

26

21

83

111

38

15

450

82

109

48

20

19

90

1014

49

18

900

102

96

55

18

17

86

102

44

17

1800

86

96

41

16

15

90

98

36

15

3600

78

98

42

18

16

84

104

35

14

MMSb

44

35

172

130

126*

43

37

157

122

aMutation colonies per 10^6 clonable cells

bPositive control, Methymethanesulfonate, 2 assays, 15 µg/ml

* P<0.05

Table 5 : Results of ETU with S9 (trial no.1)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

63

107

28

15

-

0

77

101

46

20

17

61

81

36

20

71

111

27

13

225

61

87

53

29

25

63

97

39

21

450

73

109

35

16

17

65

104

37

19

900

68

68

51

25

26

82

92

66

27

1800

91

118

98

36

35*

73

97

73

35

3600

83

102

81

33

35*

85

98

94

37

MCAb

56

32

223

132

130*

51

32

195

128

aMutation colonies per 10^6 clonable cells

bPositive control, 3-mthylcholanthrene, 2 assays, 2.5 µg/ml

* P<0.05

Table 6 : Results of ETU with S9 (trial no.2)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

69

96

45

22

-

0

86

104

71

28

23

82

106

47

19

72

95

51

23

225

72

95

48

22

25

84

109

72

29

450

87

87

103

40

37*

84

89

88

35

900

75

77

97

43

40*

65

69

73

38

1800

78

71

103

44

46*

73

92

104

47

3600

73

80

124

56

55*

70

68

113

54

MCAb

69

29

490

238

269*

47

25

418

300

aMutation colonies per 10^6 clonable cells

bPositive control, 3-mthylcholanthrene, 2 assays, 2.5 µg/ml

* P<0.05


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

Positive result was only observed when using metabolic activation.
Executive summary:

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. The chemicals were tested at least twice. Significant responses were obtained with ETU with S9 (negative result without S9).