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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria:
Ames, S. typhimurium TA 100, TA 1535, E. coli WP2 uvr A, +/- S9: positive (Araki et al., 1994, E.I. Dupont de Nemours and Company, 1978; NTP, 1989)
Ames, S. typhimurium TA 98, TA 1537, +/- S9: negative (Araki et al., 1994, E.I. Dupont de Nemours and Company, 1978; NTP, 1989)
Mutagenicity in mammalian cells:

HPRT-test in Chinese hamster ovary cells, +/- S9: ambiguous (Ebert et al., 1994)

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose for cross-reference:
reference to same study
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
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene
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)
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).
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.

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)

 

Conclusions:
Interpretation of results:
ambiguous
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon (S. typhimurium strains), Trp operon (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
range: 1-17%
Vehicle / solvent:
air
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Bacterial plates were prepared by the agar overlay method with tester strain and S9 mix or phosphate buffer solution and exposed to the test substance gas using a gas sampling bag as exposure vessel. The exposure volume of gas was 500 mL per plate. HEPA-filtered air was used as dilution gas; the concentration of the test substance was determined using gas chromatography.


DURATION
- Exposure duration: 48 hours at 37 °C
Species / strain:
other: TA 1535, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at a concentration range of 1-17%
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: TA 98 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Interpretation of results:
positive in S. typhmurium strains TA 100 and TA 1535 and E. coli WP2 uvr A
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no bacterial strain tested to detect crosslinking mutagens, only four different test concentrations
GLP compliance:
no
Remarks:
test conducted before the implementation of GLP
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction from Aroclor-1254 induced livers derived from rats
Test concentrations with justification for top dose:
Trial I: 0%, 7%, 11%, 14%, and 18%
Trial II: 0%, 9%, 14%, 19%, and 24%
Trial III: 0%, 9%, 14%, 21%, and 24% (only strain TA 1535)

Vehicle / solvent:
air
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
air
True negative controls:
no
Positive controls:
yes
Remarks:
in the tester strains TA 1535 and TA 100
Positive control substance:
other: chloroethylene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The minimal plates with the bacteria overlay (with or without activation system) are exposed to the test agent in 9-liter glass chambers. The test gas was mixed with filtered air from a compressed air line and introduced into the chambers through a flow-meter system. After the gas-air mixture flowed through the exposure chamber for 5 volume changes, the chambers were closed and placed in a 37 °C incubator for 48 hours. At the end of the exposure period the chambers were flushed with air for 5 volume changes, the plates removed and revertant frequencies determined.
Concentrations of the test gas in the chambers were determined 2 times (2-3 h after starting exposure and just before the end of treatment) by GC-analysis.
The concentrations of gas that were averaged were calculated with the use of a standard (i.e., the stated concentrations are not the flow-meter readings).

DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
In trials I and II: activated assay - average of 3 plates; non-activated assay - average of 2 plates
In trial III: activated assay - average of 5 plates; non-activated assay - average of 5 plates
Evaluation criteria:
A chemical is classified as mutagenic if a concentration related increase in reversion frequency and a reversion frequency greater than three times the spontaneous frequency is observed.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
with activation: TA 100 ranged from 4.6 to 6.1 times the spontaneous frequency; without activation: TA 100 ranged from 5.8 to 6.8 times the spontaneous frequency
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
with activation: TA 1535 ranged from 16.9 to 19.8 times the spontaneous frequency; without activation: TA 1535 ranged from 9.4 to 11.4 times the spontaneous frequency
Cytotoxicity / choice of top concentrations:
other: While a clear toxic level for TA1535 in the presence of an activation system was not found, an inhibition of growth, as noted by reduced colony size, was found at a concentration of about 24% in the presence and the absence of an activation system.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium strain TA 98 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The cytotoxicity of the test sample in the presence and absence of an activation system, as measured in strain TA1535, was the basis for selecting concentrations to be used in the mutagenesis experiment. The protocol used to determine the cytotoxicity was identical to the mutagenesis protocol except that 10E+07 rather than 10E+08 bacteria were used per plate and a nonlimiting concentration of histidine was present. Concentrations of test sample that were nontoxic and, if possible, slightly toxic were selected for the mutagenesis assay.
While a clear toxic level for TA1535 in the presence of an activation system was not found, an inhibition of growth, as noted by reduced colony size, was found at a concentration of about 24%. This inhibition was noted both in the presence and the absence of an activation system.

Trial I with activation (Histidine revertants per plate)

Chloroethane

TA 1535

TA 1537

TA 98

TA 100

0%

-

13

33

158

7%

-

10

32

537

11%

-

4

26

694

14%

-

3

14

960

18%

-

0

22

763

Choroethylene 10%

-

 

 

482

 

Trial I without activation (Histidine revertants per plate)

Chloroethane

TA 1535

TA 1537

TA 98

TA 100

0%

-

7

24

153

7%

-

10

16

374

11%

-

12

16

574

14%

-

6

14

881

18%

-

0

17

674

Choroethylene 10%

-

 

 

285

 

 

Trial II with activation (Histidine revertants per plate)

Chloroethane

TA 1535

TA 1537

TA 98

TA 100

0%

26

7

44

168

9%

142

6

44

581

14%

246

3

32

765

19%

473

2

24

766

24%

192

3

27

617

Choroethylene 10%

270

 

 

461

 

 Trial II without activation (Histidine revertants per plate)

Chloroethane

TA 1535

TA 1537

TA 98

TA 100

0%

33

6

23

117

9%

115

11

20

359

14%

233

7

17

557

19%

305

5

16

799

24%

311

0

18

510

Choroethylene 10%

198

 

 

305

 

 

Trial III with activation (Histidine revertants per plate)

Chloroethane

TA 1535

0%

21

9%

196

14%

332

21%

416

24%

304

Choroethylene 10%

256

 

Trial III without activation (Histidine revertants per plate)

Chloroethane

TA 1535

0%

34

9%

130

14%

289

21%

387

24%

286

Choroethylene 10%

212

Conclusions:
Interpretation of results:
positive in S. typhimurium strains TA 100 and TA 1535
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 3 strains used; no bacterial strain tested to detect cross-linking mutagens; only two different concentrations
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100 and TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Sprague-Dawley rats or Syrian hamsters induced with Aroclor 1254
Test concentrations with justification for top dose:
10, 20 and 42 µg/plate
Vehicle / solvent:
air
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene used in all strains in the presence of S9; 4-nitro-o-phenylenediamine was used with TA 98 and sodium azide was used with TA 100 and TA 1535.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Chloroethane was equilibrated with air and introduced through valves into sealed desiccators containing minimal glucose agar plates with the Salmonella typhimurium tester strains alone or S9 mix. The entire apparatus was incubated at 37 °C for 48 hours.

NUMBER OF REPLICATIONS: TA 98: one trial; TA 100 and 1535 with metabolic activation: three trials; TA 100 and TA 1535 without metabolic activation two trials; each trial consisted of triplicate plates;
Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as a low-level increase in revertants. A response was considered negative if no increase in revertant colonies was observed after chemical treatment.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest concentration (42 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
positive in the presence of rat liver S9; an increase in revertant colonies was observed in strain TA 100 when exposure occurred in the presence of hamster S9, but this was of insufficient magnitude for a positive call
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest concentration (42 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest concentration (42 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest concentration (42 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

TA 100

- S9

+ 30% S9 (hamster)

+ 30% S9 (rat)

Trial 1

Trial 2

Trial 3

Trial 1

Trial 2

Trial 3

Trial 1

Trial 2

Trial 3

0

128 +/- 6.8

131 +/- 4.9

112 +/- 7.6

122 +/- 9.2

138 +/- 5.2

117 +/- 7.7

139 +/- 3.7

136 +/- 7.9

115 +/- 2.6

10 µg/plate

146 +/- 5.0

133 +/- 10.3

--

128 +/- 7.3

234 +/- 8.5

--

368 +/- 8.5

209 +/- 6.4

--

20 µg/plate

--

--

103 +/- 21.1

--

--

168 +/- 44.3

--

--

275 +/- 9.5

Sodium azid (-S9)

2-aminoanthracene (+S9)

738 +/- 20

452 +/- 28.4

647 +/- 6.5

919 +/- 14.8

862 +/- 53.8

879 +/- 5.6

1236 +/- 28.2

1164 +/- 80.5

919 +/- 23.1

 

TA 1535

- S9

+ 30% S9 (hamster)

+ 30% S9 (rat)

Trial 1

Trial 2

Trial 1

Trial 2

Trial 3

Trial 1

Trial 2

Trial 3

0

14 +/- 1.5

21 +/- 1.7

15 +/- 3.2

12 +/- 2.3

14 +/- 0.7

18 +/- 1.8

15 +/- 0.9

14 +/- 0.6

10 µg/plate

133 +/- 3.6

--

102 +/- 2.9

251+/- 12.2

--

375 +/- 11.1

203 +/- 7.8

--

20 µg/plate

--

200 +/- 17.6

--

--

346 +/- 43.7

--

--

287 +/- 6.0

Sodium azid (-S9)

2-aminoanthracene (+S9)

142 +/- 31.5

199 +/- 10.4

290 +/- 13.1

228 +/- 18.3

242 +/- 16.4

206 +/- 18

344 +/- 8.3

475 +/- 15.2

 

TA 98

- S9

+ 30% S9 (rat)

0

19 +/- 1.7

21 +/- 2.5

10 µg/plate

21 +/- 1.5

34 +/- 1.2

4-nitro-o-phenylenediamine (-S9)

2-aminoanthracene (+S9)

170 +/- 4.1

617 +/- 233.3

 

Conclusions:
Interpretation of results:
positive
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Cytogenicity:
micronucleus assay: mouse, 65961 mg/m³ inhalative: negative (Ebert et al., 1994)

DNA damage and/or repair:
UDS: mouse, 65961 mg/m³ inhalative: negative (Ebert et al., 1994)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
only one test concentration
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
yes
Remarks:
only one test concentration
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga, Germany
- Weight at study initiation: 16-22 g
- Assigned to test groups randomly: yes, under following basis: individual group weights differed from the mean by no more than 5%
- Housing: in groups of no more than two animals in steel mesh cages with wire mesh lids and floors.
- Diet: ad libitum laboratory chow diet (Special Diet Services Ltd, RM1.(E).SQC.)
- Water: ad libitum tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-25
- Humidity (%): 40-59
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: gas
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

Duration of treatment / exposure:
6 h
Frequency of treatment:
3 consecutive days
Post exposure period:
no
Dose / conc.:
25 000 ppm (nominal)
Remarks:
corresponding to 65961 mg/m³
Dose / conc.:
25 045 ppm (analytical)
Remarks:
experiment 1
Dose / conc.:
25 071 ppm (analytical)
Remarks:
experiment 2
No. of animals per sex per dose:
10 females for chloroethane exposure and control group; 5 females for each positive control group
Control animals:
yes, sham-exposed
Positive control(s):
dimethylnitrosamine (DMN) in corn oil for the 12 - 14 h experiment and fast garnet GBC (also known as solvent yellow 3 or o-aminoazotoluene) in distilled water for the 2 - 4 h experiment

- Route of administration: oral
- Doses / concentrations: 200 mg/kg DMN and 10 mg/kg fast garnet GBC (both compounds were administered at 10 ml/kg)
Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Approx. 12-14 h (experiment 1) or 2-4 h (experiment 2) after dosing, animals were anesthetized with ether and maintained under deep anaesthesia to prevent recovery.

DETAILS OF SLIDE PREPARATION:
Hepatocytes were isolated using a two step perfusion protocol. Livers were flushed with calcium free buffer 1 and hepatocytes freed using buffer 2 supplemented to 3.85 mM with calcium chloride and with 0.25 mg/ml collagenase. Where possible, up to 4.5 x 10e5 viable cells were seeded, in Williams E Medium Complete, onto 25 mm thermanox coverslips in 6 well dishes. Cells were allowed to attach for approx. 90 min.

METHOD OF ANALYSIS:
For UDS analysis, cultures were exposed for 4 h to 10 µCi/ml [3H]thymidine in Williams E Medium Incomplete (WE 1, as WE C but without FCS), chased with WE 1 containing 0.25 mM thymidine and incubated overnight at 37°C. After 19.5-22.5 h, UDS cultures were rinsed, fixed and dried, then coated with Ilford K2 emulsion. Following approx. 14 days refrigeration, slides were developed, stained and mounted with coverslips. Grain counting was performed blind with coded slides, using a microscope and video camera connected to a Biotran or Artek colony counter and a computer programmed for automatic data capture. Single nuclear and one (manual) or three (automatic) separate adjacent areas of cytoplasm were scored for autoradiographic grains. For in vivo treatments, 100 cells per animal, from at least two coverslips, were scored. Mean ± SD of triplicate coverslips, up to 50 cells in each case, were calculated and reported for in vitro positive controls. For S phase analysis, up to 2000 cells were scored on cultures from test article treated and negative control animals, using at least two slides where possible. Positive control slides were not scored.

Evaluation criteria:
A treatment was considered clearly positive, if it yielded a net grain count of 5 or more with 20% or more cells in repair. Values greater than 0 would be suspect. S phase proportions greater than the negative control and more than 1% would be regarded as evidence of induction of proliferation.
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Assessment of unscheduled DNA synthesis (UDS) in hepatocytes of female B6C3F1 mice treated in vivo with chloroethane

Agent

Dose

Timea (h)

nb

NGc

(mean +/- SD)

% cells in repair (NG > 5) (mean +/- SD)

NG of cells in repair

(mean +/- SD)

air

-

2-4

5

-0.5 +/- 0.2

2.1 +/- 1.9

6.0 +/- 0.6

-

12-14

5

-0.4 +/- 0.8

0.8 +/- 0.8

7.0 +/- 1.5

chloroethane

25045 ppm

2-4

5

-1.2 +/- 1.3

2.0 +/- 2.4

5.9 +/- 0.3

25071 ppm

12-14

5

-0.1 +/- 1.2

1.8 +/- 1.5

7.1 +/- 1.7

Dimethylnitrosamine

10 mg/kg

2-4

5

6.7 +/- 2.6

58.5 +/- 19.8

9.2 +/- 2.7

Fast garnet GBC

200 mg/kg

12-14

5

5.9 +/- 1.3

59.0 +/- 14.1

8.4 +/- 0.4

a: Time of sacrifice after the final treatment
b: Number animals assayed per dose group
c: Mean and standard deviation of individual net grain (NG) counts. 100 cells were analyzed per animal, normally using 2 slides
S-phase analysis in hepatocytes of female B6C3F1 mice treated in vivo with chloroethane

Agent

Dose

Timea (h)

Animal

nb

% cells in S-phase (animal)

(mean +/- SD)

% cells in S-phase

(dose group)

(mean +/- SD)

Air

-

2-4

1

1981

0.8 +/- 0.5

0.3 +/- 0.5

2

2000

0.0 +/- 0.1

3

2000

0

4

*c

*c

5

*c

*c

12-14

1

2215

0.5 +/- 0.7

0.4 +/- 0.4

2

2077

1.0 +/- 1.0

3

3291

0.1 +/- 0.1

4

2000

0.2 +/- 0.3

5

2000

0.1 +/- 0.1

chloroethane

25045 ppm

2-4

1

2000

0.4 +/- 0.5

0.4 +/- 0.6

2

2543

0

3

2000

0.1 +/- 0.1

4

2000

0

5

558

1.5 +/- 1.8

25071ppm

12-14

1

2000

0

6 +/- 11

2

2000

0.9 +/- 0.3

3

2002

0.3 +/- 0.4

4

2206

26 +/- 8.4

5

2045

3.4 +/- 0.3

a: Time of sacrifice after the final treatment
b: Number of cells scored per animal
c: Very few cells, not scored for S-phase

Conclusions:
Interpretation of results: negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga, Germany
- Weight at study initiation: 20-24 g for males and 17-20 g for females
- Assigned to test groups randomly: yes, under following basis: group weights differed from the mean by no more than 5%
- Housing: in groups of no more than three animals of the same sex in stainless steel cages
- Diet: ad libitum Special Services Ltd, RM1.(E).SQC. pellets
- Water: ad libitum tap water



ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-25
- Humidity (%): 40-50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: gas
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only in a continuous flow system

TEST ATMOSPHERE
- Brief description of analytical method used: samples taken continuously and recorded half-hourly using an infrared spectrophotometer
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
3 consecutive days
Post exposure period:
no
Dose / conc.:
25 000 ppm (nominal)
Remarks:
corresponding to 65961 mg/m³
Dose / conc.:
25 071 ppm (analytical)
Remarks:
corresponding to 66148 mg/m³
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Positive control(s):
cyclophosphamide (in distilled water)
- Route of administration: oral
- Doses / concentrations: 80 mg/kg (24 h prior to sacrifice)
Tissues and cell types examined:
bone marrow cells (erythrocytes) from both femurs
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
An initial range finding study with 19000 ppm chloroethane was conducted using groups of male and female mice to determine mortality. All animals survived. In the main study, the dose was therefore increased to 25000 ppm which represents approximately 66% of the flammability limit and the highest dose which could be safely administered.

DETAILS OF SLIDE PREPARATION:
Cells were pelletted by centrifugation (1250xg for 2-3 min) and smears prepared on microscope slides. Preparations were stained using a modification of the method of Gollapudi and Kamra (1979). Following fixation for 5 min in methanol, slides were rinsed in water and stained for 10 min in filtered Giemsa stain diluted 1:6 in water. When dry, slides were cleared in xylene and mounted.

METHOD OF ANALYSIS:
For each animal scored, at least 1000 polychromatic (PCE) and normochromatic (NCE) erythrocytes were analyzed to obtain a PCE/NCE ratio. At least 2000 PCE per animal were scored for micronuclei. The PCE/NCE ratio for each sex and the mean per treatment group were calculated as a potential indicator of bone marrow toxicity. The sex and group mean micronucleated PCE/1000 PCE were also calculated. The PCE/NCE ratios and frequencies of micronucleated PCE in vehicle control animals were compared with historical control ranges.

Statistics:
For each animal scored, at least 1000 polychromatic (PCE) and normochromatic (NCE) erythrocytes were analyzed to obtain a PCE/NCE ratio. At least 2000 PCE per animal were scored for micronuclei. The PCE/NCE ratio for each sex and the mean per treatment group were calculated as a potential indicator of bone marrow toxicity. The sex and group mean micronucleated PCE/1000 PCE were also calculated. The PCE/NCE ratios and frequencies of micronucleated PCE in vehicle control animals were compared with historical control ranges. For each group, inter individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity chi square test. The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups using a 2 x 2 contingency table to determine X2. Probability values of p <= 0.05 were accepted as significant. The statistical methods were according to Lovell et al. (1989).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 19124 ppm (mean of 3 days exposure)
- Clinical signs of toxicity in test animals: no deaths were observed
- Evidence of cytotoxicity in tissue analyzed: no decrease in group mean PCE/NCE ratio (this was 0.99 and 1.31 for control and 1.10 and 1.14 for chloroethane treated male and female B6C3F1 mice, respectively).


Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Under normal conditions chloroethane exists as a gas. In vitro tests referred to below, were conducted in closed environment of exposure chambers and in vivo studies animals were exposed by inhalation to chloroethane.

 

In vitro:

Mutagenicity in bacteria:

In bacterial reverse gene mutation assays equivalent to OECD guideline 471, chloroethane was shown to induce gene mutations in the Salmonella typhimurium test strains detecting base pair substitution mutations, namely TA 100 (Araki et al., 1994; Haskell Laboratories, 1978) and TA 1535 (Araki et al., 1994; NTP, 1986; Haskell Laboratories, 1978) with and without metabolic activation system. However, NTP (1989) reported, that chloroethane was mutagenic in the strain TA 100 in the presence of S9 but negative results were achieved in the absence of S9.

Positive mutagenic results in a bacteria strain with a A/T base pair as primary mutation site to detect crosslinking mutagens (E. coli WP2 uvr A) were also observed with and without S9-mix (Araki et al., 1994).

In contrast, no frameshift mutation induction was observed using the Salmonella typhimurium strains TA 98 and TA 1537 both in the presence and absence of metabolic activation system (Araki et al., 1994; NTP, 1986; Haskell Laboratories, 1978).

NTP (1989) examined cytotoxicty at approx. 42 µg/plate to all three tested strains TA 98, TA 100 and TA 1535. An inhibition of growth in the strain TA 1535 was found at a concentration of about 24% in the presence and absence of an activation system.

 

Mutagenicity in mammalian cells:

A HPRT assay was conducted in Chinese hamster ovary cells (Ebert et al., 1994). Chloroethane concentrations ranges of 0.09 – 1.89 mg/mL and 0.65 – 2.03 mg/mL were tested without exogenous metabolic activation in gas-tight glass culture containers and ranges of 0.10 – 2.34 mg/mL and 1.02 – 2.48 mg/mL were tested with metabolic activation system. An ambigous mutagenic potential of the test substance was observed at the respective highest concentrations in the absence and in the presence of a metabolic activation system.Cytotoxicity was observed at the highest test concentrations, either with or without addition of metabolic activation.At first glance, the mutation rate was increased. However, the supposed positive result was most likely due to cytotoxicity and not related to genotoxicity. Thus, under the experimental conditions chosen, the mouse lymphoma assay provided ambiguous results for genotoxicity in mammalian cells.

 

In a BALB/c-3T3 cell transformation assay, chloroethane induced a dose-dependent cytotoxiciy but no consistent transformation response at concentrations up to 467 mg/mL (Milman et al., 1988; Tu et al., 1985).

Chloroethane did not exert any genotoxic potential to mouse hepatocytes (B6C3F1) in the DNA repair test at the highest non-cytotoxic concentration of 5% (Milman et al., 1988).

 

In vivo:

In a GLP-guideline study according to guideline 474 B6C3F1 mice were exposed to 0 or 25000 ppm chloroethane for 3 consecutive days (6 h/day) (Ebert et al., 1994). Samples of the bone marrow were taken from 5 males and 5 females each. Results from the data analysis show, that there was no significant difference in the number of micronucleated polychromatic erythrocytes (PCE) (per 1000 polychromatic erythrocytes) compared to the control value. The ratio of polychromatic to normochromatic erythrocytes (NCE) was not significantly different to the controls. Thus chloroethane did not reveal any clastogenic activity in this in vivo micronucleus assay in the mouse.

 

In another in vivo experiment equivalent to OECD Guideline 486, chloroethane was studied for its capability of inducing unscheduled DNA-synthesis in B6C3F1 mice (Ebert et al., 1994). The animals were exposed to 25000 ppm chloroethane via nose only inhalation, 6 hours/day for 3 consecutive days. The hepatocytes were isolated correspondingly 12-14 hours and 2-4 hours after the last dosing. Chloroethane caused no consistent increase in S-phase DNA-synthesis.

 

In summary, chloroethane did induce gene mutations in bacteria (with and without metabolic activation); gene mutations in mammalian cells were equivocal. However, in in vivo studies (micronucleus test and UDS assay) chloroethane did not induce chromosal damage and no consistent increase in S-phase DNA-synthesis.

Justification for classification or non-classification

The data is conclusive but not sufficient for classification according to CLP (1272/2008/EC).