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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

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, D.B. et al.
Year:
1988
Bibliographic source:
Environmental and Molecular Mutagenesis 12:85-154
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
lack of details on test substance
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium dodecyl sulphate
EC Number:
205-788-1
EC Name:
Sodium dodecyl sulphate
Cas Number:
151-21-3
Molecular formula:
C12H26O4S.Na
IUPAC Name:
sodium dodecyl sulfate

Method

Target gene:
Thymidine kinase locus (tk)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fisher’s medium supplemented with 2 mM L-glutamine, sodium pyruvate, 110 µg/mL 0.05% pluronic F68, antbiotics and 10% heat-inactivated donor horse serum (v/v)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Fischer 344 rats, intraperitoneally induced with Arochlor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Experiments 1-5: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60,65, 70, 80 and 100 µg/mL
Experiments 6-8: +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: methylmethanesulfonate (MMS), 15 µg/mL; +S9: 3-methylcholanthrene (3-MCA), 2.5 µg/mL
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without S9 mix
- Expression time: 2 days
- Selection time: 11-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-16 days

SELECTION AGENT: 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: four cultures for vehicle control; two cultures for positive controls and each test substance concentration

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
Four response categories for evaluation of results were defined (see below).

Response Categories for Experiments:
Positive response (+): The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
Negative response (-) Two categories were used. In both there was
a) no dose-related trend,
b) no statistically significant response at any dose,
c) an acceptable positive control response.
Nontoxic, negative response ( = )
There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable due to intrinsic properties of either the compound or the system.
Toxic, negative response (-)
There was either an RTG of <30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 x a lower concentration at which the RTG was >30%.
Inconclusive (i)
There was
a) no dose-related trend and a statistically significant dose was any other than one of the highest three doses,
b) a response which would have been negative, but the lowest RTG acceptable doses was >35%,
c) a response which would have been negative, but there were no acceptable positive controls.
Questionable (?)
There was either
a) no dose-related trend, but a statistically significant response occurred at one of the highest three doses, or
b) a statistically significant dose-related trend, but none of the acceptable doses was statistically significant on its own.

Primary judgments were made at the level of individual experiments, but judgment on the mutagenic potential of a chemical was made on a basis of consensus of all valid experimental results (see "Any other information on materials and methods inlc. tables").
Statistics:
The statistical analysis was based upon the mathematical model proposed for this system and consisted of a dose-trend test and a variance analysis of pair-wise comparisons of each dose against the vehicle control. Significant differences from concurrent vehicle control values are indicated at the 5% 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:
-S9: 70, 80 and 90 µg/mL; +S9: 95 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

RESULTS OF EXPERIMENTS 1-8

Eight acceptable experiments were conducted, five in the absence of S9 mix.

In the first of these, statistically significant increases in mutant fraction were observed at three dose levels: 6.25, 25, and 50 µg/mL; 100 µg/mL was a lethal concentration in cells (see Table 1). Over the nonlethal range, there were generally elevated mutant fractors, the highest being 1.9-fold the control level at 25 µg/mL. Although these increases in mutant fraction were significant, the lack of an obvious dose-related response with a relatively soluble chemical over a dose range which was not toxic encouraged speculation that the increases were not due to treatment with the test material.

 

Table 1. Experiment 1 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]

Cloning efficiency

Relative Total Growth

Mutants per 1E+06 surviving cells

Mutation factor

Average Mutation factor

DMSO (NC)

62

95

56

30

43

68

98

90

44

65

95

66

34

80

112

153

64

3.125

77

106

84

36

61

59

97

151

85

6.25

71

119

176

83

78*

73

107

160

74

12.5

90

145

133

49

65

67

133

160

80

25

84

90

247

99

83*

65

88

130

67

50

86

82

192

75

69*

76

98

145

64

100

lethal

lethal

n.a.

n.a.

n.a.

n.a.

n.a.

MMS (15 µg/mL) PC

27

21

135

167

232*

25

23

219

298

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

In the second experiment without S9 mix, there was a clearly significant response at 60 µg/mL, but at no other concentration. The RTG was about 22%

Table 2. Experiment II - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]

Cloning efficiency

Relative Total Growth

Mutants per 1E+06 surviving cells

Mutation factor

Average Mutation factor

DMSO (NC)

80

100

115

48

48

74

108

119

53

86

106

115

45

61

87

83

45

10

75

112

107

47

58

66

94

137

69

20

67

89

119

59

52

53

76

70

44

30

71

70

98

46

60

64

86

144

75

40

77

77

126

54

n.a.

50

94

61

191

68

74

68

56

163

80

60

81

27

365

150

203*

77

16

595

256

70

lethal

lethal

n.a.

n.a.

n.a.

n.a.

n.a.

MMS (15 µg/mL) PC

34

30

683

666

664*

29

27

573

662

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

Experiment 3 gave a statistically significant response (1.7-fold increase) at 60 µg/mL, but not at the next higher concentration of 65 µg/mL. The mutant fraction at 70 µg/ml was only 44/106 survivors, so this single culture result supported the view that the statistically significant result at the lower dose level was a chance event. Thus, this experiment was judged to be questionable.

 

Table 3. Experiment 3 - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]

Cloning efficiency

Relative Total Growth

Mutants per 1E+06 surviving cells

Mutation factor

Average Mutation factor

DMSO (NC)

76

103

84

37

34

71

104

73

34

82

97

98

40

74

96

58

26

50

60

71

65

36

36

75

75

82

36

55

68

44

68

33

38

53

71

69

44

60

72

63

107

50

56*

72

74

134

62

65

64

71

87

45

42

79

68

93

39

70

80

83

107

44

n.a.

MMS (15 µg/mL) PC

36

26

127

119

158*

26

23

156

197

MMS = methylmethanesulfonate; NC = negative control; PC = positive control; *p < 0.05; n.a. = not applicable

 

However, the succeeding experiments 4 and 5 without S9 mix were unambiguously negative; therefore the test substance was considered to be non-mutagenic in the absence of S9 mix.

Two experiments (6 and 7) were performed in the presence of S9 mix, showing unambiguously negative results. The last experiment with S9 mix was inconclusive because the cloning efficiency at 80 µg/mL was about 86% and there was no indication of a mutagenic response. However, based on the two experiments with S9 mix showing clearly negative results, the test substance was considered to be not mutagenic in the presence of S9 mix.

Applicant's summary and conclusion

Conclusions:
negative

The test item was not considered to be mutagenic, neither in the presence nor in the absence of metabolic activation.
Executive summary:

No enhanced mutation rate in the S9 treated or untreated cells was observed in this mouse lymphoma assay. Therefore, the test substance was not considered to be mutagenic.