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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
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:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Refer any other information on material and methods section
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
Refer any other information on material and methods section
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
yes
Remarks:
Refer any other information on material and methods section
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Deviations:
yes
Remarks:
Refer any other information on material and methods section
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Refer any other information on material and methods section
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trifluoro(pentafluoroethoxy)ethylene
EC Number:
234-018-7
EC Name:
Trifluoro(pentafluoroethoxy)ethylene
Cas Number:
10493-43-3
Molecular formula:
C4F8O
IUPAC Name:
1,1,2-trifluoro-2-(1,1,2,2,2-pentafluoroethoxy)ethene
Test material form:
gas
Details on test material:
- Purity: 99%

Method

Target gene:
Histidine biosynthetic enzymes
Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA100, TA1535, TA97a, and TA98
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 Liver homogenate activation system (S9 mix)
Test concentrations with justification for top dose:
Trial I (average measured concentrations): 0.49, 1.08, 1.27, 1.74, and 1.92% without S9; 0.50, 1.05, 1.29, 1.73, and 1.90% with S9
Trial II (average measured concentrations): 0.62, 0.92, 1.27, 1.86, and 2.08% without S9; 0.61, 0.87, 1.19, 1.86, and 2.10% with S9

The highest concentration was an atmosphere of 1.8%. This was approximately 60% of the lower explosive limit indicated by the sponsor.
Vehicle / solvent:
Dilution of the test substance with air was conducted immediately prior to treatment period. Gas chromatographic analysis was used to confirm the concentration of test atmospheres using the following conditions:
- Instrument: Hewlett-Packard 5890 Series II, gas chromatograph
- Column: 1/8" x 20"; 3% OV 17; 3% Chromosorb®; stainless steel
- Detector: Flame Ionization
- Carrier gas: Helium: Trial 1: 21.5 mL/min Trial 2: 21.0 mL/min;
- Detector gases: Hydrogen: Trial 1: 30.3 mL/min, Trial 2: 31.0 mL/min; Air: Trial 1: 330 mL/min, Trial 2: 299 mL/min
- Oven/Column temperature: 85°C, isothermal
- Injector port temperature: 68°C
- Detector temperature: 250°C
- Injection volume: 10 µL
Controls
Untreated negative controls:
yes
Remarks:
Air
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, ICR 191 acridine
Details on test system and experimental conditions:
This study consisted of two trials with and without activation. Three replicates were plated for each tester strain, test concentration, and condition. Positive controls, solvent controls, and air controls were included in all assays. Treatments with activation were conducted by adding 0.1 mL of solvent or positive control, 0.5 mL of S9 mix, and 0.1 mL of an overnight culture containing 1 x 10e8 bacteria to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCl (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium strains or 0.05 mM L-tryptophan for the E. coli strain. These components were mixed and poured onto a plate containing approximately 25 mL of Davis minimal agar with dextrose (minimal agar plates, purchased from MOLTOX™). Treatments in the absence of the metabolic activation system were identical to those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile phosphate buffered saline.

Plates were exposed to dilutions of the test gas after being placed on stainless steel racks specially designed to fit in 6-liter glass chambers. Chambers were equipped with Teflon® stopcocks and Viton® O-ring gaskets. As gases, the test substance and filtered air flows were regulated using individual rotameters, and mixed prior to entry into chambers. A flow rate of approximately 10 L/minute for approximately 5 minutes was used to create at least 5 volume changes to occur within the chambers to insure homogenous concentrations. Chambers were closed and 2-4 samplings of each chamber were taken and analyzed by gas chromatography to determine the initial concentration of the test substance. Chamber concentrations were determined by comparison of integrated peak areas to test substance standards by the analytical conditions previously described. Chambers were placed into an incubator at approximately 37°C for approximately 48 hours. Chambers were again sampled and analyzed to determine the ending test substance concentration. Chambers were flushed with at least 5-10 chamber volumes of filtered air and then refrigerated until counted. Plates were then removed for an evaluation of background lawns and colony formation.
Evaluation criteria:
A test substance was classified as positive when: (1) the average number of revertants in any strain at any test substance concentration studied was at least two times greater than the average number of revertants in the negative control; and (2) there was a positive dose-response relationship in that same strain.

A test substance was classified as negative when either: (1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control; or (2) there was no positive dose-response relationship.

Results not meeting these criteria for positive or negative assessments were evaluated using scientific judgment.
Statistics:
For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA100, TA1535, TA97a, and TA98
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
In the initial trial, end-treatment sampling for one concentration (1.73%) was not performed; therefore, the indicated exposure concentration represents the average of samples taken at the beginning of the exposure period. Analytical results indicated the test substance was stable under the conditions of the study, therefore, results in the one instance when post-treatment analytical was not performed were not rejected. All valid criteria were met.

Any other information on results incl. tables

Mutagenic Activity of the Test Substance in Strain TA100 Trial 1

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

80

81

78

80 (2)

T0,P0

0.49 ± 0.03

77

80

79

79 (2)

T0,P0

1.08 ± 0.03

82

80

80

81 (1)

T0,P0

1.27 ± 0.02

73

78

70

74 (4)

T0,P0

1.74 ± 0.03

77

72

79

76 (4)

T0,P0

1.92 ± 0.05

79

81

83

81 (2)

T0,P0

DMSO (Solvent) Control

0 µg/plate

66

71

72

70 (3)

T0,P0

NAAZ (positive control)

2 µg/plate

662

805

769

745 (74)

N

With Activation

Filtered Air

78

76

86

80 (5)

T0,P0

0.50 ± 0.04

78

85

76

80 (5)

T0,P0

1.05 ± 0.03

77

81

82

80 (3)

T0,P0

1.29 ± 0.07

74

79

81

78 (4)

T0,P0

1.73 ± 0.04*

70

85

83

79 (8)

T0,P0

1.90 ± 0.02

76

80

73

76 (4)

T0,P0

DMSO (Solvent) Control

0 µg/plate

82

79

75

79 (3)

T0,P0

2AA (positive control)

1 µg/plate

583

523

566

557 (31)

N

*Starting concentration – determination of the ending exposure concentration was not performed.

 

Mutagenic Activity of the Test Substance in Strain TA100 Trial 2

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

162

163

159

161 (2)

T0,P0

0.62 ± 0.08

159

161

175

165 (9)

T0,P0

0.92 ± 0.05

164

154

167

162 (7)

T0,P0

1.27 ± 0.03

154

168

163

162 (7)

T0,P0

1.86 ± 0.17

164

153

166

161 (7)

T0,P0

2.08 ± 0.28

150

163

161

158 (7)

T0,P0

DMSO (Solvent) Control

0 µg/plate

149

149

157

152 (5)

T0,P0

NAAZ (positive control)

2 µg/plate

853

854

885

864 (18)

N

With Activation

Filtered Air

148

160

164

157 (8)

T0,P0

0.61 ± 0.10

168

166

160

165 (4)

T0,P0

0.87 ± 0.03

163

154

170

162 (8)

T0,P0

1.19 ± 0.04

151

156

138

148 (9)

T0,P0

1.86 ± 0.10

166

184

177

176 (9)

T0,P0

2.10 ± 0.35

149

157

167

158 (9)

T0,P0

DMSO (Solvent) Control

0 µg/plate

157

172

164

164 (7)

T0,P0

2AA (positive control)

1 µg/plate

1593

1471

1571

1545 (65)

N

 

Mutagenic Activity of the Test Substance in Strain TA1535 Trial 1

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

11

11

12

11 (1)

T0,P0

0.49 ± 0.03

16

7

13

12 (5)

T0,P0

1.08 ± 0.03

11

16

12

13 (3)

T0,P0

1.27 ± 0.02

13

19

9

14 (5)

T0,P0

1.74 ± 0.03

11

10

11

11 (1)

T0,P0

1.92 ± 0.05

10

15

11

12 (3)

T0,P0

DMSO (Solvent) Control

0 µg/plate

12

11

15

13 (2)

T0,P0

NAAZ (positive control)

2 µg/plate

647

538

594

593 (55)

N

With Activation

Filtered Air

21

18

21

20 (2)

T0,P0

0.50 ± 0.04

21

19

22

21 (2)

T0,P0

1.05 ± 0.03

20

16

20

19 (2)

T0,P0

1.29 ± 0.07

15

9

12

12 (3)

T0,P0

1.73 ± 0.04*

14

15

9

13 (3)

T0,P0

1.90 ± 0.02

14

17

20

17 (3)

T0,P0

DMSO (Solvent) Control

0 µg/plate

9

11

14

11 (2)

T0,P0

2AA (positive control)

2 µg/plate

355

339

277

324 (41)

N

*Starting concentration – determination of the ending exposure concentration was not performed.

 

Mutagenic Activity of the Test Substance in Strain TA1535 Trial 2

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

8

14

12

11 (3)

T0,P0

0.62 ± 0.08

12

8

9

10 (2)

T0,P0

0.92 ± 0.05

13

9

15

12 (3)

T0,P0

1.27 ± 0.03

12

11

10

11 (1)

T0,P0

1.86 ± 0.17

11

12

10

11 (1)

T0,P0

2.08 ± 0.28

7

8

8

8 (1)

T0,P0

DMSO (Solvent) Control

0 µg/plate

10

9

8

9 (1)

T0,P0

NAAZ (positive control)

2 µg/plate

605

587

653

615 (34)

N

With Activation

Filtered Air

8

10

13

10 (3)

T0,P0

0.61 ± 0.10

9

12

8

10 (2)

T0,P0

0.87 ± 0.03

12

14

13

13 (1)

T0,P0

1.19 ± 0.04

12

8

7

9 (3)

T0,P0

1.86 ± 0.10

11

9

8

9 (2)

T0,P0

2.10 ± 0.35

8

10

10

9(1)

T0,P0

DMSO (Solvent) Control

0 µg/plate

10

12

12

11 (1)

T0,P0

2AA (positive control)

2 µg/plate

307

285

277

290 (16)

N

 

Mutagenic Activity of the Test Substance in Strain TA97a Trial 1

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

86

102

82

90 (11)

T0,P0

0.49 ± 0.03

83

87

83

84 (2)

T0,P0

1.08 ± 0.03

91

83

82

85 (5)

T0,P0

1.27 ± 0.02

77

70

69

72 (4)

T0,P0

1.74 ± 0.03

94

83

72

83 (11)

T0,P0

1.92 ± 0.05

81

81

81

81 (0)

T0,P0

DMSO (Solvent) Control

0 µg/plate

86

82

77

82 (4)

T0,P0

ICR 191 (positive control)

2 µg/plate

1253

1211

1236

1233 (21)

N

With Activation

Filtered Air

100

95

101

99 (3)

T0,P0

0.50 ± 0.04

86

92

85

88 (4)

T0,P0

1.05 ± 0.03

87

84

92

88 (4)

T0,P0

1.29 ± 0.07

83

90

87

87 (4)

T0,P0

1.73 ± 0.04*

82

85

83

83 (2)

T0,P0

1.90 ± 0.02

82

79

74

78 (4)

T0,P0

DMSO (Solvent) Control

0 µg/plate

89

107

94

97 (9)

T0,P0

2AA (positive control)

1 µg/plate

530

541

612

561 (45)

N

*Starting concentration – determination of the ending exposure concentration was not performed.

 

Mutagenic Activity of the Test Substance in Strain TA197a Trial 2

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

85

96

88

90 (6)

T0,P0

0.62 ± 0.08

77

87

85

83 (5)

T0,P0

0.92 ± 0.05

68

73

86

76 (9)

T0,P0

1.27 ± 0.03

73

79

81

78 (4)

T0,P0

1.86 ± 0.17

79

68

80

76 (7)

T0,P0

2.08 ± 0.28

62

81

72

72 (10)

T0,P0

DMSO (Solvent) Control

0 µg/plate

93

95

79

89 (9)

T0,P0

ICR 191 (positive control)

2 µg/plate

1247

1179

1050

1159 (100)

N

With Activation

Filtered Air

97

89

79

88 (9)

T0,P0

0.61 ± 0.10

77

80

75

77 (3)

T0,P0

0.87 ± 0.03

82

84

97

88 (8)

T0,P0

1.19 ± 0.04

81

70

73

75 (6)

T0,P0

1.86 ± 0.10

99

88

97

95 (6)

T0,P0

2.10 ± 0.35

64

74

70

69 (5)

T0,P0

DMSO (Solvent) Control

0 µg/plate

95

96

105

99 (5)

T0,P0

2AA (positive control)

1 µg/plate

764

720

737

740 (22)

N

 

Mutagenic Activity of the Test Substance in Strain TA98 Trial 1

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

11

17

9

12 (4)

T0,P0

0.49 ± 0.03

16

15

10

14 (3)

T0,P0

1.08 ± 0.03

17

15

13

15 (2)

T0,P0

1.27 ± 0.02

11

16

15

14 (3)

T0,P0

1.74 ± 0.03

14

20

11

15 (5)

T0,P0

1.92 ± 0.05

19

17

12

16 (4)

T0,P0

DMSO (Solvent) Control

0 µg/plate

13

10

9

11 (2)

T0,P0

2NF (positive control)

25 µg/plate

1374

1354

1235

1321 (75)

N

With Activation

Filtered Air

14

16

15

15 (1)

T0,P0

0.50 ± 0.04

14

15

17

15 (2)

T0,P0

1.05 ± 0.03

19

22

21

21 (2)

T0,P0

1.29 ± 0.07

20

22

17

20 (3)

T0,P0

1.73 ± 0.04*

17

16

15

16 (1)

T0,P0

1.90 ± 0.02

15

14

13

14 (1)

T0,P0

DMSO (Solvent) Control

0 µg/plate

12

17

18

16 (3)

T0,P0

2AA (positive control)

2 µg/plate

1214

1174

1346

1245 (90)

N

*Starting concentration – determination of the ending exposure concentration was not performed.

 

Mutagenic Activity of the Test Substance in Strain TA198 Trial 2

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

20

17

11

16 (5)

T0,P0

0.62 ± 0.08

19

15

21

18 (3)

T0,P0

0.92 ± 0.05

16

20

10

15 (5)

T0,P0

1.27 ± 0.03

9

11

15

12 (3)

T0,P0

1.86 ± 0.17

11

12

22

15 (6)

T0,P0

2.08 ± 0.28

13

13

17

14 (2)

T0,P0

DMSO (Solvent) Control

0 µg/plate

15

19

21

18 (3)

T0,P0

2 NF (positive control)

25 µg/plate

1312

1363

1437

1371 (63)

N

With Activation

Filtered Air

26

30

33

30 (4)

T0,P0

0.61 ± 0.10

21

21

25

22 (2)

T0,P0

0.87 ± 0.03

19

19

16

18 (2)

T0,P0

1.19 ± 0.04

13

14

16

14 (2)

T0,P0

1.86 ± 0.10

19

18

19

19 (1)

T0,P0

2.10 ± 0.35

20

22

21

21 (1)

T0,P0

DMSO (Solvent) Control

0 µg/plate

22

23

20

22 (2)

T0,P0

2AA (positive control)

2 µg/plate

1802

2153

1921

1959 (179)

N

 

Mutagenic Activity of the Test Substance in Strain WP2 uvrA (pKM101) Trial 1

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

125

155

161

147 (19)

T0,P0

0.49 ± 0.03

153

151

155

153 (2)

T0,P0

1.08 ± 0.03

151

148

153

151 (3)

T0,P0

1.27 ± 0.02

128

125

138

130 (7)

T0,P0

1.74 ± 0.03

145

128

143

139 (9)

T0,P0

1.92 ± 0.05

128

142

136

135 (7)

T0,P0

DMSO (Solvent) Control

0 µg/plate

129

127

124

127 (3)

T0,P0

MMS (positive control)

1000 µg/plate

1968

1947

1851

1922 (62)

N

With Activation

Filtered Air

134

135

141

137 (4)

T0,P0

0.50 ± 0.04

132

127

127

129 (3)

T0,P0

1.05 ± 0.03

124

148

127

133 (13)

T0,P0

1.29 ± 0.07

128

126

131

128 (3)

T0,P0

1.73 ± 0.04*

155

157

134

149 (13)

T0,P0

1.90 ± 0.02

128

126

118

124 (5)

T0,P0

DMSO (Solvent) Control

0 µg/plate

162

175

151

163 (12)

T0,P0

2AA (positive control)

25 µg/plate

2082

1894

1939

1972 (98)

N

*Starting concentration – determination of the ending exposure concentration was not performed.

 

Mutagenic Activity of the Test Substance in Strain WP2 uvrA (pKM101) Trial 2

Average Concentration (%)

Mean ± S.D.

Revertants

 

 

Average

(S.D.)

Observations

Plate 1

Plate 2

Plate 3

Without Activation

Filtered Air

174

146

150

157 (15)

T0,P0

0.62 ± 0.08

143

163

143

150 (12)

T0,P0

0.92 ± 0.05

133

145

146

141 (7)

T0,P0

1.27 ± 0.03

147

158

139

148 (10)

T0,P0

1.86 ± 0.17

151

129

152

144 (13)

T0,P0

2.08 ± 0.28

134

141

129

135 (6)

T0,P0

DMSO (Solvent) Control

0 µg/plate

141

149

145

145 (4)

T0,P0

MMS (positive control)

25 µg/plate

2114

1783

2199

2032 (220)

N

With Activation

Filtered Air

133

145

152

143 (10)

T0,P0

0.61 ± 0.10

135

138

144

139 (5)

T0,P0

0.87 ± 0.03

126

129

137

131 (6)

T0,P0

1.19 ± 0.04

126

138

129

131 (6)

T0,P0

1.86 ± 0.10

142

138

140

140 (2)

T0,P0

2.10 ± 0.35

143

144

147

145 (2)

T0,P0

DMSO (Solvent) Control

0 µg/plate

181

174

177

177 (4)

T0,P0

2AA (positive control)

25 µg/plate

1805

1645

2057

1836 (208)

N

 

Applicant's summary and conclusion

Conclusions:
No evidence of mutagenic activity was detected in either of the two independent trials.
Executive summary:

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9) in accordance with OECD Guideline 471 and 472.

In comparison to negative air and dimethyl sulfoxide (DMSO) controls, tester strains were exposed to the test substance in top agar overlays at average concentrations of 0.49, 1.08, 1.27, 1.74, and 1.92% in the absence of S9, and 0.50, 1.05, 1.29, 1.73, and 1.90% in the presence of S9 for approximately 48 hours. No evidence of mutagenicity was detected in the first trial.

In a second confirmatory trial, plates were exposed to average concentrations of 0.62, 0.92, 1.27, 1.86 and 2.08% in the absence of S9 and 0.61, 0.87, 1.19, 1.86 and 2.10% were tested in the presence of S9 in comparison negative (air) and DMSO controls. No evidence of mutagenicity was detected in the second trial.

Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. In this study, the test substance was negative.