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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
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1994
Reference Type:
secondary source
Title:
Chloroethane CAS: 75-00-3
Author:
OECD SIDS
Year:
2006
Bibliographic source:
SIDS Initial Assessment Report for SIAM 22

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroethane
EC Number:
200-830-5
EC Name:
Chloroethane
Cas Number:
75-00-3
Molecular formula:
C2H5Cl
IUPAC Name:
chloroethane
Details on test material:
- Name of test material (as cited in study report): chloroethane
- Physical state: gaseous
- Analytical purity: > 99% (Hüls AG, Marl, Germany)

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
Ham's F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 IU/mL penicillin and 50 µg/mL streptomycin
In the main study futher supplements were added to the media to reduce the number of cells harboring spontaneous mutations of the HPRT locus: 200 µM glycine, 5 µM thymidine, 10 µM hypoxanthine, 3.2 µM aminopterin)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from Aroclor-1254 induced livers derived from rats
Test concentrations with justification for top dose:
Trial I (-S9): 0.09, 0.38, 0.94, 1.36, 1.89 mg/mL (determined by GC-FID analyses)
Trial I (+S9): 0.10, 0.38, 0.96, 1.36, 2.34 mg/mL (determined by GC-FID analyses)
Trial II (-S9): 0.65, 0.91, 1.36, 1.72, 2.03 mg/mL (determined by GC-FID analyses)
Trial II (+S9): 1.02, 1.43, 1.80, 2.17, 2.48 mg/mL (determined by GC-FID analyses)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, which was used as solvent of the positive control substances
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methanesulfonate (without S9) and 3-methylcholanthrene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
CHO cells were seeded in gas-tight glass culture flasks equipped with a septum. After incubation at 37 °C for approx. 20 h, the medium was replaced by medium without FCS. The flasks were tightly closed and appropriate amounts of chloroethane were injected into the flasks through the septum.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 10 µg 6-thioguanine/mL

NUMBER OF REPLICATIONS: 2 independent experiments (each with duplicate exposure cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: After exposure period cells were harvested and seeded with 200 cells/dish in medium with FCS. After incubation for 7 days at 37 °C, colonies were fixed with methanol, stained with Giemsa and counted. Cell survival is expressed as the plating efficiency of treated cells relative to untreated cells.

Statistics:
The statistical significance of induced mutation frequencies was determined by means of t-test (two-sample analysis).

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING:
The concentrations used in the main studies (trial I and II) based on the results of the cytotoxicity assays.

Any other information on results incl. tables

Target and measured chloroethane concentrations in the HPRT test with CHO cells and corresponding plating efficiencies

Trial

S9 mix

chloroethane

target conc.

(mg/vial)

chloroethane

measured conc.

(mg/vial)

chloroethane

(mg/mL)

Absolute Plating efficiencies (%)

1

-

0

-

0

81

-

2.5

2.9

0.09

85

-

10.0

12.1

0.38

110

-

25.0

29.7

0.94

87

-

35.0

42.9

1.36

78

-

50.0

59.3

1.89

59

1

+

0

-

0

86

+

2.5

3.1

0.10

89

+

10.0

12.0

0.38

78

+

25.0

30.2

0.96

76

+

35.0

42.6

1.36

78

+

65.0

73.6

2.34

41

2

-

0

-

0

96

-

25.0

20.3

0.65

85

-

35.0

28.7

0.91

77

-

45.0

42.9

1.36

70

-

55.0

54.0

1.72

68

-

65.0

63.7

2.03

39

2

+

0

-

0

78

+

35.0

32.0

1.02

59

+

45.0

45.0

1.43

62

+

55.0

56.7

1.80

55

+

65.0

68.2

2.17

49

+

75.0

78.0

2.48

35

 

Induction of 6-thioguanine-resistent mutants in CHO cells after treatment with chloroethane for 4 h (without S9)

target dose (mg/vial) chloroethanea

relative survivalb

mean number of colonies per platec

mutant colonies per 106clonable cells

trial 1

trial 2

trial 1

trial 2

trial 1

trial 2

0

100

100

1 +/- 1d

0 +/- 1e

8 +/- 9

0 +/- 4

2.5

105

n.d.

0 +/- 1

n.d.

3+/- 4

n.d.

10

136

n.d.

0 +/- 0

n.d.

1+/- 3

n.d.

25

107

89

2 +/- 1

3 +/- 2

15 +/- 10

23 +/- 15**

35

96

80

2 +/- 1

2 +/- 1

13 +/- 9

15 +/- 8**

45

n.d.

73

n.d.

6 +/- 2

n.d.

48 +/- 16**

50

73

n.d.

6 +/- 2

n.d.

36 +/- 15**

n.d.

55

n.d.

71

n.d.

5 +/- 3

n.d.

46 +/- 28**

65

n.d.

51

n.d.

8 +/- 2

n.d.

74 +/- 19**

EMS

79

69

67 +/- 12

76 +/- 10

605 +/- 108**

585 +/- 77**

EMS: Ethylmethanesulfonate

n.d.: not determined

a: the actual chloroethane concentration were slighly different (see table above)

b: relative survival was calculated at the end of exposure as the plating efficiency of chloroethane-treated cells compared to negative control cells; 81% in Exp 1 (- S9) and 86% in Exp 2 (- S9)

c: in each trial, five flasks with 2E+5 cells/flask were plated with the exception of (d) [10 flasks] and (e) [15 flasks]

**: significantly higher than the negative control (p<0.01)

 

Induction of 6-thioguanine-resistent mutants in CHO cells after treatment with chloroethane for 4 h (with S9)

target dose (mg/vial) chloroethanea

relative survivalb

mean number of colonies per platec

mutant colonies per 106 clonable cells

trial 1

trial 2

trial 1

trial 2

trial 1

trial 2

0

100

100

0 +/- 1

3 +/- 1d

1 +/- 3

18 +/- 6

2.5

103

n.d.

2 +/- 2

n.d.

13 +/- 2

n.d.

10

91

n.d.

0 +/- 1

n.d.

1 +/- 3

n.d.

25

88

n.d.

1 +/- 1

n.d.

8 +/- 6

n.d.

35

91

76

2 +/- 3

3 +/- 1

12+/- 16

19 +/- 6

45

n.d.

80

n.d.

4 +/- 2

n.d.

27 +/- 14

55

n.d.

71

n.d.

4 +/- 2

n.d.

26 +/- 13

65

48

63

5 +/- 2

3 +/- 3

32 +/- 16**

24 +/- 24

75

n.d.

43

12 +/- 2

12 +/- 2

n.d.

74 +/- 12**

DMSO 1%

103

100

1 +/- 1

1 +/- 1

3+/- 3

7 +/- 7

MCA

99

97

59 +/- 10

47 +/- 11

348 +/- 55**

585 +/- 77**

MCA: 3-methylcholanthrene

n.d.: not determined

a: the actual chloroethane concentration were slighly different (see table above)

b: relative survival was calculated at the end of exposure as the plating efficiency of chloroethane-treated cells compared to negative control cells; 96% in Exp 1 (+ S9) and 78% in Exp 2 (+ S9)

c: in each trial, five flasks with 2E+5 cells/flask were plated with the exception of (d) [10 flasks]

**: significantly higher than the negative control (p<0.01)

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
ambiguous

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