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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
Study period:
09 March 2017 to 31 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro gene mutation in mammalian cells

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-dithiobisethanol
EC Number:
217-576-6
EC Name:
2,2'-dithiobisethanol
Cas Number:
1892-29-1
Molecular formula:
C4H10O2S2
IUPAC Name:
2-[(2-hydroxyethyl)disulfanyl]ethan-1-ol
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: No assay of test item stability, nor its concentration and homogeneity in solvent were undertaken.
- Solubility and stability of the test substance in the solvent/vehicle: The test item was found to be soluble in culture medium at the maximum concentration of
50mg/mL based on solubility test.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in culture medium
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Method

Target gene:
TK locus (TK+/−)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, Rockville, Maryland
- Suitability of cells: Recommended in Test Guideline
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Modal number of chromosomes: not specified
- Normal (negative control) cell cycle time: not specified

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- 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:
Phenobarbital – 5,6-Benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1, 3-hour treatment with metabolic activation: 385, 193, 96.3, 48.1, 24.1 and 12.0 μg/mL
Experiment 1, 3-hour treatment without metabolic activation: 385, 193, 96.3, 48.1, 24.1, 12.0 and 6.02 μg/mL
Experiment 2, 24-hour treatment without metabolic activation: 50.0, 40.0, 32.0, 25.6, 20.5, and 16.4 μg/mL
The top dose was selected based on toxicity observed in the preliminary toxicity assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The solvent used in this study was RPMI Minimal medium A
- Justification for choice of solvent/vehicle: based on solubility test
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
complete medium
Negative solvent / vehicle controls:
yes
Remarks:
minimal medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
complete medium
Negative solvent / vehicle controls:
yes
Remarks:
minimal medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): not specified

ACTIVATION: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL): S9 tissue fraction 0.408mL, NADP (30 mM) 0.204mL, G-6-P (590 mM) 0.204mL, KCl (150 mM) 0.204mL, Complete medium (5%) 8.98mL

DURATION
- Preincubation period:
- Exposure duration: experiment 1 3 hours with and without metabolic activation; experiment 2 24 hours without metabolic activation
- Expression time (cells in growth medium): two days after treatment at 37°C in a 5% CO2 atmosphere
- Selection time (if incubation with a selection agent): one to two weeks incubated at 37°C in a 5% CO2 atmosphere
- Fixation time (start of exposure up to fixation or harvest of cells): not specified

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures apart from positive control

NUMBER OF CELLS EVALUATED: not specified

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency; relative total growth
- Any supplementary information relevant to cytotoxicity: none

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Rationale for test conditions:
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Evaluation criteria:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Statistics:
Statistical analysis was performed according to UKEMS guidelines:
- Test for consistency between plates;
- Heterogeneity factors for replicate cultures;
- Test for overall consistency;
- Updated heterogeneity factors;
- Comparison of each treatment with the control;
-Test for linear trend

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
3-hour treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity was noted at the two highest dose levels reducing RS below 10%
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
24-hour treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity was noted at the two highest dose levels reducing RS below 10%
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
3-hour treatment
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
severe toxicity was noted at the three highest dose levels
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the test item solution did not have any obvious effect on the pH of the treatment medium.
- Effects of osmolality: the test item solution did not have any obvious effect on the osmolality of the treatment medium.
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of the treatment period.
- Definition of acceptable cells for analysis:
- Other confounding effects: not specified

RANGE-FINDING/SCREENING STUDIES: Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1540µg/mL and at a wide range of lower dose levels: 770, 385, 193, 96.3, 48.1, 24.1, 12.0 and 6.02µg/mL. No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of the treatment period.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the ranges of positive control data
- Negative (solvent/vehicle) historical control data: within the ranges of negative control data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC
- Other observations when applicable: NONE

Any other information on results incl. tables

Table 1: Experiment 1: 3 HOURS - WITHOUT S9, Cytotoxicity test

Dose-level (µg/mL)

 106cells

/ mL Cell count Post treatment factor

 

106cells

/ mL Cell count Post treatment factor

Total wells

           

Plate counts #

Plate counts #

Cloning efficiency

Relative survival

0.00

6.02

12.0

24.1

48.1

96.3 193

385

770

1540

0.56

0.55

0.58

0.42

0.38

0.36

0.28

0.25

0.26

0.23

1.00

0.98

1.04

0.75

0.68

0.64

0.50

0.45

0.46

0.41

192

192

192

192

192

192

192

192

192

192

70

71

66

68

67

45

47

25

8

0

72

73

62

72

65

46

45

28

9

0

0.84

0.87

0.69

0.82

0.73

0.40

0.41

0.20

0.06

0.00

100%

101%

85%

73%

59%

31%

24%

11%

3%

0%

Table 2: Experiment 2: 24 HOURS - WITHOUT S9, Cytotoxicity test

Dose-level (µg/mL)

 

106

cells / mL Cell count Post treatment factor

 

106

cells / mL Cell count Post treatment factor

Total wells

           

Plate counts #

 

Plate counts #

Cloning efficiency

Relative survival

0.00

6.02

12.0

24.1

48.1

96.3 193

385

770

1540

0.53

0.58

0.45

0.47

0.14

0.03

0.03

0.01

0.00

0.00

1.00

1.09

0.85

0.89

0.26

0.06

0.06

0.02

0.00

0.00

192

192

192

192

192

0

0

0

0

0

81

77

80

64

39

£

£

£

£

£

79

80

78

68

35

£

£

£

£

£

1.12

1.06

1.08

0.73

0.30

£

£

£

£

£

100%

104%

82%

58%

7%

0%

0%

0%

0%

0%

# = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival £ = Insufficient number of cells recovered after treatment period

Table 3: Experiment 1: 3 HOURS - WITH S9, cytotoxicity test

Dose-level (µg/mL)

 

106

cells / mL Cell

 

106

cells / mL Cell

Total wells

Plate counts #

Plate counts #

Cloning efficiency

Relative survival

 

count Post treatment factor

count Post treatment factor

           

 

 

 

 

0.00

6.02

12.0

24.1

48.1

96.3 193

385

770

1540

0.40

0.40

0.39

0.41

0.36

0.37

0.35

0.39

0.36

0.38

1.00

1.00

0.98

1.03

0.90

0.93

0.88

0.98

0.90

0.95

192

192

192

192

192

192

192

192

192

192

76

80

79

70

60

44

37

16

1

0

77

77

75

71

55

49

37

12

1

0

1.00

1.06

1.01

0.83

0.57

0.41

0.30

0.10

0.01

0.00

100%

107%

99%

85%

52%

38%

27%

10%

1%

0%

# = Wells with clones/plate - 1.6 cells in each well of two 96-well plates plated for survival £ = Insufficient number of cells recovered after treatment period

 Table 4:

SUMMARY TABLE, MAIN ASSAY I - TREATMENT: 3 HOURS - WITH S9

Dose-level (µg/mL)

RTG

MF §

P

IMF

Proportion

Precipitation

 

 

 

 

§

small colony mutants

 

0.00

100%

74.1

-

-

0.12

-

12.0

39%

70.2

NS

-

-

-

24.1

78%

73.2

NS

-

-

-

48.1

37%

98.2

NS

24.03

-

-

96.3

18%

182.3

**

108.14

-

-

193

15%

298.9

**

224.8@

0.33

-

385

6%

698.9

$

624.8@

-

-

B(a)P 2.00

38%

1140.4

-

1066.2@

0.20

-

Linear trend

 

 

***

 

 

 

Table 5: SUMMARY TABLE, EXPERIMENT 1 - TREATMENT: 3 HOURS - WITHOUT S9

Dose-level (µg/mL)

RTG

MF §

P

IMF

Proportion

Precipitation

 

 

 

 

§

small colony mutants

 

0.00

100%

65.6

-

-

0.19

-

6.02

99%

70.5

NS

4.95

-

-

12.0

81%

66.5

NS

0.98

-

-

24.1

55%

61.7

NS

-

-

-

48.1

37%

73.1

NS

7.50

-

-

96.3

19%

100.0

NS

34.43

-

-

193

7%

317.7

$

252.1@

-

-

385

2%

445.5

$

379.9@

-

-

Linear trend

 

 

*

 

 

 

Table 6: SUMMARY TABLE, EXPERIMENT 2 - TREATMENT: 24 HOURS - WITHOUT S9

Dose-level (µg/mL)

RTG

MF §

P

IMF

Proportion

Precipitation

 

 

 

 

§

small colony mutants

 

0.00

100%

77.2

-

-

0.19

-

16.4

61%

96.2

NS

19.03

-

-

20.5

56%

91.2

NS

14.03

-

-

25.6

43%

69.3

NS

-

-

-

32.0

29%

75.0

NS

-

-

-

40.0

3%

134.5

$

57.28

-

-

50.0

0%

125.1

$

47.84

-

-

MMS

5.00

53%

628.1

-

550.8@

0.29

-

Linear trend

 

 

NS

 

 

 

§

=

per 10 viable cells

NS

=

Not statistically significant

*

=

Statistically significant at p < 5%

**

=

Statistically significant at p < 1%

***

=

Statistically significant at p < 0.1%

+

=

Opacity of treatment medium

++

=

Precipitation

$

=

Treatment excluded from test statistics due to excessive toxicity

@

=

Induced mutant frequency (IMF) > global evaluation factor (GEF = 126 x 10-6)

Applicant's summary and conclusion

Conclusions:
Di-2-hydroxyethyl disulfide has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD TG 490, and under GLP (RTC, 2017). No test-substance induced increase in the number of mutations was observed when treated without metabolic activation. However, a test substance induced increase in the induced mutant frequency (IMF) relative to the global evaluation frequency (GEF) was observed when treated with metabolic activation. This increase occurred only at the highest dose of 193 μg/mL, at which the RTG = 15%. At the next lower dose, 96.3 μg/mL, RTG value 18%, the IMF was lower than the GEF. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in the absence of S9 metabolic activation. In the presence of S9 metabolic activation, under the conditions of the study, it is not possible to conclude whether the substance is negative or positive for mutagenicity to mammalian cells.