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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HPRT locus
Species / strain
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium supplemented with 10% fetal bovine serum and 1% neomycin
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ß-naphtoflavone
Test concentrations with justification for top dose:
Experiment I
Without S9 mix: 0.25, 0.5, 1, 2 and 4 µg/mL (4 h)
With S9 mix: 4, 8, 16, 32 and 48 µg/mL (4 h)
Experiment II
Without S9 mix: 4, 8, 16, 64 and 96 µg/mL (24 h)
With S9 mix: 4, 8, 16, 48 and 56 µg/mL (4 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate, 150 µg/mL in nutrient medium, without S9; 7,12-dimethylbenz(a)anthracene, 1.1 µg/mL in DMSO, with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
4 h exposure with and without S9 mix
4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): The expression time in growth medium was 7 days. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL thioguanine.
- Selection time (if incubation with a selection agent): 2 days (experiment I) or 3 days (experiment II) after sub-cultivation stock cultures were trypsinized.
- Fixation time (start of exposure up to fixation or harvest of cells): 9-10 days

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine

STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 5 replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY: cloning efficiency and relative total growth
- Method: mitotic index
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 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 is < 0.05.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥4 µg/mL (experiment I, -S9), ≥ 48 µg/mL (experiment I,+ S9); 96 µg/mL (experiment II, -S9, 24 h), 56 µg/mL (experiment II, +S9, 4 h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In the preliminary test, the pH of the culture medium was adjusted at concentrations of 1900 and 3800 µg/mL with 2 N NaOH. In the main experiments no pH adjustment was necessary.
- Precipitation: precipitation occurred at 237.5 µg/mL an above after 24 h without metabolic activation. Following 4 h treatment no precipitation was noted.

RANGE-FINDING/SCREENING STUDIES: Concentrations between 29.7 and 3800 µg/mL were used to evaluate toxicity in the presence (4 h) and absence (4 h and 24 h treatment) of metabolic activation. After 24 h treatment without metabolic activation the cell growth was completely inhibited at 118.8 mg/L and above. Following 4 htreatment with and without metabolic activation the cell growth was already completely inhibited at a concentration of 29.7 µg/mL. Therefore, the preliminary experiment was repeated with concentrations of 0.23 to 30.0 µg/mL with and without metabolic activation. In the repeated preliminary experiment strong cytotoxic effects occurred only in the absence of metabolic activation at 7.5 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: All mutant frequencies were within the historical range of solvent controls. A single exception occurred in experiment I at 2.0 µg/mL. The induction factor (4.0) exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data is exceeded (44.0 mutant colonies/10E6 cells compared to 0.6 – 32.4 mutant colonies/10E6 cells of the historical solvent control data). However, the induction factor of the parallel culture remained below the threshold and this event is therefore considered to be not reproducible and of no biological relevance.

Any other information on results incl. tables

Table 1. Results of experiment I without metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Culture I

Culture II

0 (Acetone)

100

100

11

100

100

12,9

EMS 150

95,8

75,0

209,9

96,5

104,9

117,0

0,25

98,6

88,0

5,1

92,5

73,7

7,5

0,5

99,2

96,6

11,7

97,6

97,0

17,4

1

100

112,5

7,3

97,8

78,8

14,8

2

97,2

108,9

44,0

97,2

104,8

11,2

4

91,4

10,4

0,0

89,7

8,3

4,0

6

-*

-*

8

-*

-

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 2. Results of experiment I with metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Culture I

Culture II

0 (Acetone)

100

100

29,1

100

100

20,7

DMBA 1.1

80,7

80,2

665,9

86,6

95,5

411,3

2

96,0

116,6

**

98,0

105,4

**

4

92,7

98,2

28,1

95,0

100,3

10,5

8

88,5

110,7

5,2

89,4

118,5

22,5

16

90,6

141,1

11,6

94,5

100,3

18,4

32

87,3

95,0

13,0

85,3

88,8

23,6

48

41,3

40,7

29,8

12,1

12,5

16,8

64

-*

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 3. Results of experiment II without metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Without S9 mix

Without S9 mix

0 (Acetone)

100

100

14,6

100

100

13,5

EMS 150

81,0

133,5

224,7

110,0

129,3

260,1

2

102,3

98,9

**

109,7

107,6

**

4

102,8

136,3

9,7

111,8

82,0

11,1

8

103,1

115,3

17,9

111,3

93,5

16,3

16

101,9

114,3

17,3

100,9

104,9

17,8

64

91,2

164,5

11,8

102,6

60,1

9,5

96

24,1

32,9

15,0

25,5

23,9

22,1

128

-

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 4. Results of experiment II with metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

With S9 mix

With S9 mix

0 (Acetone)

100

100

10,5

100

100

10,2

DMBA 1.1

81,0

68,4

863,3

28,0

73,7

743,3

2

102,3

62,0

**

91,0

92,4

**

4

102,8

63,2

5,4

91,7

90,7

11,2

8

103,1

91,2

8,4

93,1

97,6

4,4

16

101,9

101,5

15,4

90,4

100,6

8,6

48

91,2

43,5

17,4

0

70,9

10,0

56

24,1

3,2

7,1

0

4,3

4,1

64

-*

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative