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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
Study period:
21 December 2012 to 15 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
from 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
from 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
from 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohexanol
EC Number:
203-630-6
EC Name:
Cyclohexanol
Cas Number:
108-93-0
Molecular formula:
C6H12O
IUPAC Name:
cyclohexanol
Test material form:
not specified
Details on test material:
Supplier: Univar Benelux N.V.

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: Complete culture medium: RPMI 1640 medium supplemented with 15 % horse serum, 100 U/ 100 µg/mL penicillin / streptomycin, 220 µg/mLsodium pyruvate and 0.5 - 0.75 U/mL amphotericin.Cloning medium: As for complete culture medium.Selective medium: Complete culture medium with the addition of 5 µg/mL TFT.- Properly maintained: Yes.- Periodically checked for Mycoplasma contamination: Yes- Periodically checked for karyotype stability:Yes- Periodically "cleansed" against high spontaneous background: YesPrior to mutagenicity testing, the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with 1.0E-04 M hypoxanthine, 2.0E-07 M aminopterin and 1.6E-05M thymidine. The incubation of the cells in HAT-medium was followed by a recovery period of two days in RPMI 1640 medium containing 1.0E-04 M hypoxanthine and 1.6E-05 thymidine. After this incubation the cells were returned to normal RPMI 1640 medium. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell back of Harlan CCR allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability.Thawed stock cultures are propagated in plastic flasks in RPMI 1640 complete culture medium. The cells are sub-cultured two times prior to treatment. The cell cultures were incubated at 37 ± 5 °C in humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
- Experiment I, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment I, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, without S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
- Experiment II, with S9 mix: 62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubilisation properties and its nontoxicity to the cells.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 U/mL Amphotericin B used as antifungal agent plus 5 µg/mL TFT.

NUMBER OF REPLICATIONS: 2NUMBER OF CELLS EVALUATED: 10^7

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growthIn the pre-test screen, 10^7 cells were exposed to each concentration of the test substance for four hours with and without metabolic activation. Following treatment the cells were washed twice by centrifugation (425 g, 10 mins) and re-suspended in “Saline G”. Subsequently the cells were re-suspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3E+05 cells/mL if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period.

- POSITIVE CONTROL: Methyl methane sulfonate in nutrient medium to a final concentration of 0.18 mM (19.5 µg) without metabolic activation. Cyclophosphamide in 0.9 % saline to a final concentration of 10.7 µM (3.0 µg/mL) and 16.1 µM (4.5 µg/mL) with metabolic activation.
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration. Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled. Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using a statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
All strains/cell types tested
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant test substance related effect on pH was noted.
- Effects of osmolality: No relevant test substance related effect on osmolarity was noted.
- Precipitation: No precipitation of the test item was noted in experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
- A pre-test was performed at doses of 7.8, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0 µg/mL in order to determine the concentration range of the mutagenicity experiments. Both pH value and osmolarity were determined at the maximum concentration of the test item and in the solvent control without metabolic activation. 10^7 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutation rates in the vehicle controls lie within the historical range.

OTHER:
- In both main experiments no precipitation was noted up to the maximum concentration with and without metabolic activation.No relevant cytotoxic effects indicated by a relative cloning efficiency 1 (survival) or a relative total growth of less than 50 % in both cultures occurred in experiment I and II with and without metabolic activation.No substantial and reproducible concentration dependent increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control was observed in the main experiments up to the maximum concentration tested with and without metabolic activation. A linear regression analysis (least squares) was performed to asses a possible concentration dependent increase of mutant frequencies using SYSTAT® 11 statistics software. No significant concentration dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.In this study, the range of the solvent control values was from 64 up to 158 mutant colonies per 10^6 cells; the range of the acceptable group values treated with the test substance was from 29 up to 226 mutant colonies per 10^6 cells. The highest value of 226 slightly exceeded the historical range of solvent controls (42 – 216 colonies per 10^6 cells) but there was no concentration dependent increase as indicated by the lack of statistical significance and the mutation frequency in the parallel culture remained well within the historical range of solvent controls under identical conditions, hence these data are considered acceptable.The positive controls showed a distinct increase in induced total mutant colonies at acceptable levels of cytotoxicity.

Any other information on results incl. tables

Summary of results, Experiment I and II

 

 

 

Relative

Relative

Mutant

 

Relative

Relative

Mutant

 

 

Conc. µg per mL

S9 mix

cloning efficiency

total growth

colonies/ 

106cells

 threshold

cloning efficiency

total growth

colonies/ 

106cells

  

thresholds

Column

1

2

3

4

5

6

7

8

9

10

Experiment I/ 4 h treatment

Culture I

Culture II

Solv. control with water

 

-

100.0

100.0

64

190

100.0

100.0

77

203

Pos. control with MMS

19.5

-

70.8

43.7

362

190

84.2

46.3

302

203

Test item

31.3

-

70.8

Culture was not continued #

88.9

Culture was not continued #

Test item

62.5

-

67.0

163.9

29

190

96.0

107.2

82

203

Test item

125.0

-

51.5

111.3

53

190

92.3

126.3

56

203

Test item

250.0

-

85.4

132.1

54

190

70.2

106.6

77

203

Test item

500.0

-

50.0

108.3

69

190

96.0

130.1

57

203

Test item

1000.0

-

61.4

88.9

88

190

88.9

73.1

97

203

 

 

 

 

 

 

 

 

 

 

 

Solv. control with water

 

 

100.0

100.0

81

207

100.0

100.0

74

200

Pos, control with CPA

3.0

+

29.4

27.1

665

207

18.6

26.6

928

200

Pos, control with CPA

4.5

+

25.5

14.4

1295

207

23.4

14.9

1355

200

Test item

31.3

+

124.2

Culture was not continued #

72.8

Culture was not continued #

Test item

62.5

+

91.6

80.8

95

207

97.8

94.0

103

200

Test item

125.0

+

104.6

137.8

92

207

75.3

101.4

87

200

Test item

250.0

+

103.0

84.8

109

207

80.6

116.4

88

200

Test item

500.0

+

82.9

54.4

142

207

74.0

103.6

56

200

Test item

1000.0

+

74.0

127.1

115

207

88.4

105.3

90

200

Experiment II / 4 h treatment

Culture I

Culture II

Solv. control with water

 

-

100.0

100.0

117

243

100.0

100.0

88

214

Pos. control with MMS

19.5

-

73.4

33.9

447

243

69.3

27.4

576

214

Test item

31.3

-

74.7

Culture was not continued #

89.6

Culture was not continued #

Test item

62.5

-

69.7

121.3

104

243

79.7

96.0

59

214

Test item

125.0

-

77.5

151.2

114

243

76.0

86.3

74

214

Test item

250.0

-

68.5

133.4

97

243

81.0

68.3

97

214

Test item

500.0

-

76.1

142.1

87

243

86.6

60.3

79

214

Test item

1000.0

-

70.9

133.6

106

243

77.2

51.0

125

214

 

 

 

 

 

 

 

 

 

 

 

Solv. control with water

 

 

100.0

100.0

150

276

100.0

100.0

158

284

Pos, control with CPA

3.0

+

51.0

29.2

744

276

41.7

34.8

1434

284

Pos, control with CPA

4.5

+

21.3

4.1

1652

276

12.8

4.7

3153

284

Test item

31.3

+

96.6

Culture was not continued #

98.1

Culture was not continued #

Test item

62.5

+

126.2

100.5

106

276

118.9

155.1

146

284

Test item

125.0

+

90.4

80.6

146

276

110.9

127.9

208

284

Test item

250.0

+

95.0

133.4

106

276

87.9

155.1

181

284

Test item

500.0

+

86.2

91.1

92

276

927

77.7

150

284

Test item

1000.0

+

67.8

62.2

129

276

86.4

83.2

226

284

threshold = number of mutant colonies per 10^6 cells of each solvent control plus 126

# culture was not continued since a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:
The test material did not induce mutations in the absence and presence of metabolic activation.
Executive summary:

The mutagenic potential of the test material was assessed according to OECD Guideline 476. This GLP-study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in three independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours.

The concentration range of the main experiments went up to approximately 10 mM. No cytotoxic effects occurred in the main experiments up to the maximum concentration in the absence and presence of metabolic activation. No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentrations tested with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item is considered to be non mutagenic in this mouse lymphoma assay.