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Genetic toxicity: in vitro

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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
Study period:
11 September 2012 - 5 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Clear orange/amber sticky high viscous liquid.
- Stability under storage conditions: Stable.
- Stability under test conditions: The stability of the test material was confirmed when used in the test system.
- Storage condition of test material: At room temperature in the dark.

Method

Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: Several different mediums were used; basic medium, growth medium, exposure medium, selective medium and non-selective. The compositions of which can be seen in the field "Any other information on materials and methods incl. tables".
- Properly maintained: Yes, Stock cultures of the cells were stored in liquid nitrogen (-196°C). Cell density was preferably kept below 1 x 106 cells/mL.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically "cleansed" against high spontaneous background: Yes.
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL.
Vehicle:
- Vehicle(s)/solvent(s) used: Ethanol
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
Based on the findings of a dose range finding test the definitive study was performed as two independent experiments.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
> Experiment 1: 3 hours with and without S9-mix (8% v/v).
> Experiment 2: 24 hours without S9-mix and 3 hours with S9-mix (12% v/v).
- Expression time: Cells were cultured for a further 2 days. During this culture period at least 4 x 10^6 cells (where possible) were subcultured every day in order to maintain log phase growth.
- Selection time: 11 or 12 days.

SELECTION AGENT: Selective medium (TFT-selection), see the field “Any other information on materials and methods incl. tables” for information on composition.
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). Cultures were stained for 2 hours.

NUMBER OF REPLICATIONS:
> Exposure cultures were performed as a single replicate.
> Solvent and positive controls were performed in duplicate.

INCUBATION CONDITIONS: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 45 – 98%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.2 – 37.9°C). Cultures for the 3 hour incubations (10^6 cells/mL) were places in 30 mL centrifuge tubes, and incubated in a shaking incubator at 145 rpm. The cell cultures for the 24 hour incubation (1.25 x 10^5 cells/mL) were places in sterile 72 cm² culture flasks.

DOSE RANGE FINDING TEST
- Method: The dose range finding test was conducted according to the same method employed for the definitive test, where cells were exposed to the test material for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix.
- Concentrations: 1, 3, 10, 33 and 100 µg/mL.
- Cytotoxicity determination: The surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted and subcultured again for another 24 hours, after which the cells were counted. The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 10^5 cells/mL were counted no subculture was performed.
- Determination of dosing range: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
Evaluation criteria:
DATA EVALUATION
> The test material is considered positive (mutagenic) in the mutation assay if it induces a MF (mutation frequency) of more than the control + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
> The test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
> The test material is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

ACCEPATBILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS (methylmethanesulfonate) should not be below 500 per 10^6 survivors, and for CP (cyclophosphamide) not below 700 per 10^6 survivors.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test material was tested beyond the limit of solubility to obtain adequate mutagenicity data.
- Precipitation: The test material precipitated in the exposure medium at concentrations of 33 μg/mL and above. It was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test material concentration for the dose range finding test was 100 μg/mL.

RANGE-FINDING/SCREENING STUDIES: No toxicity in the relative suspension growth was observed up to and including the highest concentration of 100 μg/mL compared to the suspension growth of the solvent control, in cultures exposed for 3 hours (with and without metabolic activation) and 24 hours (with metabolic activation).

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity was observed in any of the dose levels evaluated, both in the absence and presence of metabolic activation.

MUTAGENICITY IN THE DEFINITIVE STUDY
The numbers of small and large colonies in treated cultures were comparable to the numbers in the solvent controls.
Both in the presence and absence of metabolic activation (S9-mix), the test material did not induce a significant increase in the mutation frequency in the first experiment. These results were confirmed by the second experiment.

CONTROLS
- Solvent control: The growth rate over the two-day expression period for cultures treated with ethanol was between 14 and 21 (3 hours treatment) and 80 and 85 (24 hours treatment).
- Positive controls: Mutation frequencies in cultures treated with positive control chemicals were increased by 13- and 11-fold for MMS in the absence of S9-mix, and by 8.1- and 11-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay, with the exception of mutation frequency of the positive control in the presence of S9-mix (second experiment). However the value of 121% was just above the upper limit of the range (120%). Therefore this deviation in the absolute cloning efficiency had no effect on the results of the study.

ANALYSIS OF TEST SOLUTIONS
> Experiment 1 (with S9-mix): The concentrations analysed in the low and high formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Stability analysis suggested that formulations were not stable over a 4 hour period; therefore an additional experiment was performed.
No test substance was detected in the vehicle.
> Experiment 1 (without S9-mix): The concentrations analysed in the high formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
For the low formulation applied in experiment 1 (without S9-mix) the mean accuracy was below the target concentration (i.e. 89% of target). However this was just below the upper limit of the range (90%). Furthermore the high dose was in agreement with the nominal concentration, and this was used for the preparation of the lower dose level. Therefore this deviation is not considered to have affected the reliability of the test results.
Formulations at the entire range were determined to be stable when stored at room temperature under normal laboratory light conditions for at least 4 hours. Since the test solutions were added to the test system within one hour and the stability in the second measurement was stable after 0.5, 1 and 4 hours. It can be concluded that the test substance formulations were stable when used in the test system.
No test substance was detected in the vehicle.

Any other information on results incl. tables

Table 1: Experiment 1 – Cytotoxic and Mutagenic Response

Dose (µg/mL)

RSG (%)

CEday2 (%)

RSday2 (%)

RTG (%)

TotalMutation Frequency per 10^6 survivors (small/large)

3 hour exposure, without metabolic activation

SC1

100

121

100

100

50 (27/21)

SC2

 

102

 

 

61 (27/32)

0.03

121

108

97

118

64 (28/34)

0.1

107

125

112

119

60 (37/21)

0.3

111

116

104

115

67 (28/36)

1

119

116

104

123

41 (14/27)

3

131

102

91

120

56 (23/32)

10

127

115

102

130

48 (26/20)

33*

124

118

105

131

46 (20/25)

100*

123

135

120

148

40 (19/20)

MMS

80

78

70

56

705 (378/235)

3 hour exposure, with 8 % (v/v) metabolic activation

SC1

100

113

100

100

101 (29/68)

SC2

 

101

 

 

98 (25/69)

0.03

99

76

71

70

129 (48/74)

0.1

103

104

97

100

83 (23/57)

0.3

105

78

73

77

130 (40/84)

1

101

74

69

69

134 (41/88)

3

98

107

100

98

100 (21/75)

10

99

72

68

67

121 (28/89)

33*

95

97

90

86

92 (29/59)

100*

105

84

78

82

122 (29/88)

CP

45

49

46

20

811 (160/594)

 

Table 2: Experiment 2 – Cytotoxic and Mutagenic Response

Dose (µg/mL)

RSG (%)

CEday2 (%)

RSday2 (%)

RTG (%)

TotalMutation Frequency per 10^6 survivors (small/large)

24 hour exposure, without metabolic activation

SC1

100

86

100

100

54 (30/23)

SC2

 

95

 

 

52 (29/21)

0.03

107

80

88

95

46 (22/22)

0.1

101

89

98

99

58 (35/22)

0.3

100

97

106

107

58 (33/23)

1

99

104

114

113

47 (21/26)

3

141

89

98

138

62 (44/17)

10

169

95

105

178

55 (30/23)

33*

124

85

94

116

41 (20/20)

100*

104

90

99

104

40 (22/16)

MMS

108

70

77

84

605 (357/190)

3 hour exposure, with 12 % (v/v) metabolic activation

SC1

100

83

100

100

69 (31/36)

SC2

 

98

 

 

58 (27/30)

0.03

91

99

110

101

47 (24/22)

0.1

88

94

104

92

55 (26/27)

0.3

85

83

91

78

62 (40/21)

1

103

98

109

112

49 (25/23)

3

102

93

103

105

63 (29/32)

10

93

70

78

73

68 (29/38)

33*

93

95

105

98

53 (27/25)

100*

92

98

109

100

54 (34/18)

CP

66

52

58

38

699 (436/207)

 

Note all calculations were made without rounding off.

RSG = Relative Suspension Growth; CE = CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = ethanol; MMS = Methylmethanesulfonate; CP = Cyclophosphamide.

* = precipitated in the exposure medium.

Applicant's summary and conclusion

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

Under the conditions of the test, no significant increase in the mutation frequency at the TK Locus was observed after treatment with the test material. Cultures were exposed to the test material up to and including the precipitating concentration of 100 µg/mL, with and without metabolic activation (S9-mix). Accordingly the test material was determined to be non-mutagenic in the TK mutation test system.
Executive summary:

The genotoxic potential of the test material was determined in an in vitro gene mutation study using L5178Y mouse lymphoma cells. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and IWGT recommendations. Based on the findings of a dose range finding test the definitive study was performed as two independent experiments within the dose range 0.03 to 100 µg/mL. In experiment 1, cultures were exposed to the test material for 3 hours, with and without metabolic activation (8 % v/v S9-mix). In experiment 2, cultures were exposed for 3 hours without S9-mix, and for 24 hours with 12 % v/v S9-mix. The test material precipitated in the exposure medium at concentrations of 33 μg/mL and above. It was tested beyond the limit of the solubility to obtain adequate cytotoxicity and mutagenicity data.

The numbers of small and large colonies in treated cultures were comparable to the numbers in the solvent controls. Both in the presence and absence of metabolic activation (S9-mix), the test material did not induce a significant increase in the mutation frequency in the first experiment, results that were confirmed by the second experiment. Therefore under the conditions of the test, the test material was determined to be non-mutagenic.