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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:
Feb 07, 2012 - May 25, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Expiration Date: October 27, 2019
Physical state, appearance: Solid, yellow
Molecular weight: 916.65 g/mol
Storage conditions: Room temperature

Specific details on test material used for the study:
- Lot/batch No.: 0004038273
- Expiration date of the lot/batch: October 27, 2019
- Storage condition of test material: room temperature

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- 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/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: 6.6, 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL
Experiment II: 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: the test substance was diluted in medium
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
EMS: without S9 mix; DMBA: with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Experiment I: 4 hours without and 4 hours with S9 mix
Experiment II: 24 hours without and 4 hours with S9 mix
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1500000 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 300000 - 500000 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were 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 and statistical significance was considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment precipitation was observed at 105.0 µg/mL and above in the absence and at 26.3 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation was observed at 210.0 µg/mL in the absence and at 52.5 µg/mL and above in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Summary of results

conc.

P

S9 mix

releativ cloning efficiancy I

relative cell density

releativ cloning efficiancy II

mutant colonies/106cells

induction factor

releativ cloning efficiancy I

relative cell density

releativ cloning efficiancy II

mutant colonies/106cells

induction factor

µg/mL

culture I

culture II

Experiment I / 4 h treatment

solvent control in medium

-

100

100

100

12.9

1

100

100

100

9.7

1

Positive control (EMS)

150

-

96

84.9

91.5

70.2

5.4

93.4

127.8

86.1

50.7

5.2

Test item

6.6

-

96.4

culture was not continued#

98.8

culture was not continued#

Test item

13.1

-

104

96.6

98.7

20.6

1.6

104.5

104.8

98.5

4.7

0.5

Test item

26.3

-

95.1

94.2

90.7

10.7

0.8

99

98.1

97.2

12.1

1.2

Test item

52.5

-

94.8

83.2

94.6

9.8

0.8

105.1

94.9

106.8

6.6

0.7

Test item

105

P

-

97

89.4

91

10.8

0.8

96

131.6

107.3

6.4

0.7

Test item

210

P

-

99

68.2

91

21.1

1.6

98.2

126.2

101.6

29.4

3

solvent control in medium

+

100

100

100

23.9

1

100

100

100

9.2

1

Positive control (DMBA)

1.1

+

50.6

76.3

94

446

18.6

58.2

107.2

86

376.5

41.1

Test item

6.6

+

97.2

96

112.7

12.6

0.5

100.6

113.8

100.8

12.7

1.4

Test item

13.1

+

94.7

95.1

122.7

17

0.7

103.1

117.2

95.4

9.5

1

Test item

26.3

P

+

90.1

97.2

118.1

11.7

0.5

94.3

112.1

96.3

19.6

2.1

Test item

52.5

P

+

94.8

culture was not continued##

94.9

culture was not continued##

Test item

105

P

+

93.3

87.6

110.3

14

0.6

101.6

111.5

109.2

11.8

1.3

Test item

210

P

+

89.7

87.3

123.3

11

0.5

101.5

115.1

92.4

10.9

1.2

Experiment II / 24 h treatment

 

solvent control in medium

-

100

100

100

6

1

100

100

100

11

1

Positive control (EMS)

150

-

95.8

95.2

92.9

405.8

67.2

78.5

81.6

63.2

524.4

47.8

Test item

6.6

-

98.4

culture was not continued#

93.3

culture was not continued#

Test item

13.1

-

100.3

97.5

100.2

6.1

1

98.6

93

80.2

15.4

1.4

Test item

26.3

-

100

99.5

103.4

15.7

2.6

95.2

73.7

78.1

12.6

1.1

Test item

52.5

-

98.7

108

100.7

11.7

1.9

100

99.9

84.5

7.5

0.7

Test item

105

-

100.1

102

98.8

9.5

1.6

98.1

81.9

72.4

18.1

1.6

Test item

210

P

-

96.8

82.5

90

29.5

4.9

97.8

76.5

57.9

14.9

1.4

Experiment II / 4 h treatment

solvent control in medium

+

100

100

100

8.2

1

100

100

100

6.1

1

Positive control (DMBA)

1.1

+

86.6

112.7

99.5

369.1

44.8

88.2

51

63.4

495.3

81.6

Test item

3.3

+

101.7

culture was not continued#

104.3

culture was not continued#

Test item

6.6

+

100.3

142.2

105.1

15.1

1.8

102.4

90.9

102.3

7.9

1.3

Test item

13.1

+

100.9

204

94.7

13.1

1.6

106.2

121.7

94.2

6.5

1.1

Test item

26.3

+

102

186.1

104.1

19.5

2.4

101.6

75.6

97.1

15.3

2.5

Test item

52.5

P

+

100.3

195

81.2

11

1.3

97.6

71.3

96.2

5.8

1

Test item

105

P

+

99.9

290.9

92

9.9

1.2

91.6

83.2

87.6

7.8

1.3

P = Precipitation

# cutlture was not continued as a minimum of only four concentrations is required

## culture was not continued to avoid analysis of too many precipitating concentrations

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor reaching or exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment without metabolic activation at 210 µg/mL and in the first culture of the second experiment without metabolic activation at 210 µg/mL. However, the increases were based on a rather low mutation frequency of the corresponding solvent controls of just 9.7 and 6.0 colonies per 106cells, respectively. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increases of the induction factor were judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment at culture I without metabolic activation. However, the trend was judged as biologically irrelevant as all of the values of the mutation frequency remained within the historical range of solvent controls.

Applicant's summary and conclusion

Conclusions:
negative