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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to OECD guideline and GLP

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
substrain K3
Metabolic activation:
with and without
Metabolic activation system:
S9 rat
Test concentrations with justification for top dose:
1st Experiment

without S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)
0; 6.3; 12.5; 25.0; 50.0; 100.0; 150.0; 200.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period)
0; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL

with S9 mix (4-hour exposure period)
0; 12.5; 25.0; 50.0; 100.0; 150.0; 175.0; 200.0 μg/mL
Vehicle / solvent:
Due to the insolubility of the test substance in water, dimethylsulfoxide (DMSO) was selected
as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for
which historical control data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
other: methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium: Ham's F12 + 10%FCS

DURATION
- Preincubation period: 1 week, elimination of spontaneous HPRT-deficient mutants by pretreatment with "HAT" medium
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): about one week
- Selection time (if incubation with a selection agent): about one week
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) L-glutamine (200 mM), and 10% (v/v) fetal calf serum (FCS)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: six flasks from every treatment group

NUMBER OF CELLS EVALUATED:3*10^5 cells seeded per flask at beginning of selection period

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;
Evaluation criteria:
Cytotoxicity
The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle/negative control

The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies were
calculated. Based on these values the relative cloning efficiencies of the test groups were calculated and given in percentage compared with the
respective CEabsolute value of the corresponding vehicle/negative control (vehicle/negative control = 100%).

Mutant frequency
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10^6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 106
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest conc. of 200 µg/ml each
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MUTANT FREQUENCY
- no relevant increase in the number of mutant colonies either without S9 mix or after addition of S9 in both experiments (4 and 24 hours treatment)
- positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies

CYTOTOXICITY
- Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in both experiments in the absence and presence of S9 mix in the highest applied concentration of 200 μg/mL each.

CELL MORPHOLOGY
- In both experiments, in the absence and presence of S9 mix after 4 and 24 hours treatment the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration.

TREATMENT CONDITIONS
- Osmolarity and pH values were not influenced by test substance treatment.
- In this study, in the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.

Any other information on results incl. tables

Summary of results

Exp.

Exposure period

Test groups

S9 mix

Prec.*

Genotoxicity** MFcorr.
[per 10^6
cells]

Cytotoxicity***

CE1[%]

CE2[%]

1

4 hrs

Vehicle control1

-

-

8.17

100.0

100.0

 

 

6.3 μg/mL

-

-

(-)

109.5

(-)

 

 

12.5 μg/mL

-

-

1.67

108.9

104.0

 

 

25.0 μg/mL

-

-

2.95

114.7

105.4

 

 

50.0 μg/mL

-

-

1.69

102.4

97.1

 

 

100.0 μg/mL

-

-

2.65

99.9

102.7

 

 

150.0 μg/mL

-

-

1.45

42.3

93.9

 

 

200.0 μg/mL

-

-

-

0.0

-

 

 

Positive control2

-

-

164.63

112.5

77.8

2

24 hrs

Vehicle control1

-

-

3.01

100.0

100.0

 

 

25.0 μg/mL

-

-

3.24

105.8

99.8

 

 

50.0 μg/mL

-

-

5.89

88.6

98.0

 

 

100.0 μg/mL

-

-

1.82

72.7

87.1

 

 

150.0 μg/mL

-

-

7.35

49.7

83.1

 

 

175.0 μg/mL

-

-

0.69

30.6

97.1

 

 

200.0 μg/mL

-

-

-

1.6

-

 

 

Positive control2

-

-

682.83

55.2

59.8

1

4 hrs

Vehicle control1

+

-

3.59

100.0

100.0

 

 

6.3 μg/mL

+

-

(-)

91.0

(-)

 

 

12.5 μg/mL

+

-

(-)

95.3

(-)

 

 

25.0 μg/mL

+

-

1.02

86.3

108.2

 

 

50.0 μg/mL

+

-

7.31

71.7

106.2

 

 

100.0 μg/mL

+

-

2.04

94.9

105.2

 

 

150.0 μg/mL

+

-

4.35

71.4

100.8

 

 

200.0 μg/mL

+

-

3.60

5.0

118.6

 

 

Positive control3

+

-

75.54

96.7

102.1

2

4 hrs

Vehicle control1

+

-

7.11

100.0

100.0

 

 

12.5 μg/mL

+

-

(-)

83.5

(-)

 

 

25.0 μg/mL

+

-

3.53

85.3

90.3

 

 

50.0 μg/mL

+

-

4.04

79.2

87.6

 

 

100.0 μg/mL

+

-

2.42

100.7

90.8

 

 

150.0 μg/mL

+

-

7.77

76.9

88.2

 

 

175.0 μg/mL

+

-

3.86

34.0

94.0

 

 

200.0 μg/mL

+

-

-

0.0

-

 

 

Positive control3

+

-

157.17

86.2

96.5

 

*    Precipitation in culture medium at the end of exposure period

**  Mutant frequency MFcorr.: number of mutant colonies per 10^6 cells corrected with the CE2 value

*** Cloning efficiency related to the respective negative/vehicle control

-    Due to strong cytotoxicity the cultures were not continued

(-)  Culture was not continued since a minimum of four concentrations is required by the guidelines

1DMSO 1% (v/v)              2EMS 300 μg/mL         3MCA 20 μg/mL

According to the results of the present in vitro study, the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each

other. The mutant frequencies at any concentration were within the range of the concurrent vehicle control values and within the range of our historical negative control data.

The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the authors, under the experimental conditions chosen here, the conclusion is drawn that the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.