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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
Study period:
not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The Japanese relevant authority has stated clearly in secondary summaries that this study was conducted in compliance with GLP. However, some pages, which may contain the confirmation of GLP compliance, are missing from the published study report. Overall, the studies are the part of safety evaluation by the national authority of Japan (from Japan Existing Chemical Data Base (JECDB)). The data are collected under the control of the authority and approved by the GLP authority, which are under national control. Therefore we consider that the studies are in compliance with GLP although GLP or QA statement is not included in the available study reports.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
2AA- only control with metabolic activation
GLP compliance:
yes
Remarks:
evidence of GLP is not included in the report
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydrofurfuryl alcohol
EC Number:
202-625-6
EC Name:
Tetrahydrofurfuryl alcohol
Cas Number:
97-99-4
Molecular formula:
C5H10O2
IUPAC Name:
tetrahydrofuran-2-ylmethanol
Details on test material:
Name of test substance; Tetrahydrofurfuryl alcohol
Structural formula; see attachment
Molecular weight; 102.13
Purity; 99.5%
Impurity; 5-Methyltetrahydrofurfuryl alcohol (GC): 0.34%
Appearance under ambient temperature; transparent liquid
Substance type; Pure active substance
Lot. No.; not reported

Method

Target gene:
S. typhimurium TA100 and TA1535; his G (base-pair substitution)
S. typhimurium TA98; his D (frameshift)
S. typhimurium TA 1537; his C (frameshift)
E. coli WP2uvrA/pK101; trp E (base-pair substitute)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and tetrahydrofurfuryl alcohol induced rat liver S9
Test concentrations with justification for top dose:
Tester strains; TA 100, TA1535, WP2uvrA/pK101, TA 98 and TA 1537: 5000, 2500, 1250, 625 and 313 µg/plate (with or without S9 mix)
Positive substance;
with S9 mix: TA 100 (2-aminoanthracene (2-AA); 1 µg/plate), TA1535 (2-AA; 2 µg/plate), WP2uvrA/pK101 (2-AA; 2 µg/plate), TA 98 (2-AA; 0.5 µg/plate) and TA 1537 (2-AA; 2 µg/plate)
without S9 mix: TA 100 (furfurylamide (AF-2); 0.01 µg/plate), TA1535 (sodium azide (NaN3); 0.5 µg/plate), WP2uvrA/pK101 (ENNG; 2 µg/plate), TA 98 (AF-2; 0.5 µg/plate) and TA 1537 (9-AA; 2 µg/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water for injection (Otsuka Pharmaceutical Factory, Inc., Lot No.; K0C78)
- Justification for choice of solvent/vehicle: The test substance was soluble in water for injection (DW) at the concentration of 50 mg/ml in the preliminary test for selection of solvent. No exothermic, bubbling and discoloration was observed when DW was added. Based on these results, DW was used for solvent.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
AF-2 (Lot No.; CAP0185, content; 98.9%, Wako Pure Chemical Industries, Ltd.), Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
NaN3 (Lot No.; KWE6685, content; 96.5%, Wako Pure Chemical Industries, Ltd.), Solvent: DW
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG (Lot No. 56F-3651, content; 99.0%, Sigma Chemical Company), Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DW
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-AA (Lot No., 80F-0186, content; >99%, Sigma Chemical Company), Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2-AA (Lot No. TWH2355, content; 98.0%, Wako Pure Chemical Industries, Ltd.), Solvent: DMSO
Details on test system and experimental conditions:
1. Tester strain;
Five tester strains, Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 which were obtained from the University of California on May 27, 1983 and Escherichia coli strain WP2uvrA/pKM101 which was obtained from Japan Bioassay Research Center were used. These strains are widely used for bacterial reverse mutation test, and are recommended by OECD Guidelines and the guideline in Japanese Chemical Substances Control Law.
2. Bacterial suspension;
Each frozen tester strains were thawed at use, and aliquot of 20 µl was taken and inoculated to 10 µl of liquid complete medium. They were incubation under shaking culture for 8 hours at 37°C (90 agitation/min.). L-shaped tube (capacity; 22 ml) was used for culturing vessel. After incubation period was finished, turbidity of bacterial suspension was measured with turbidity meter. Viable bacteria count was calculated by conversion from turbidity. The appropriate bacterial concentration (10 E+09/ml or more) was confirmed in the suspension and then used for the test (Note 2).
3. Reverse mutation test;
The test was conducted by pre-incubation method with and without S9 mix. For each dosage, 0.1 ml of test substance solution or negative (solvent) control material was added into sterilized tube, and then 0.5 ml of 0.1 mol/l sodium-phosphate buffer (pH 7.4) (without S9 mix) or 0.5 ml of S9 mix (with S9 mix) was added. Then 0.1 ml of bacterial suspension was added and was shaking-incubated at 37°C for 20 minutes. After pre-incubation, 2 ml of top agar was added to the mixture and was multilayered onto minimum glucose agar plate medium. After solidification of multilayered top agar, it was incubated at 37°C for 48 hours. Growth of bacterial flora was observed with stereoscopic microscope to investigate degree of bacterial growth inhibition by test material and then the presence of precipitation was investigated by visual inspection. Number of revertant colonies on the plate was counted with automatic colony counter (CA-11, System Science Co., Ltd.; correction method: area and miscount correction). Tests for the positive control materials were conducted similarly.
Evaluation criteria:
If more than 2-fold increase is noted in the number of revertant colonies (mean value) compared with negative (solvent) control with our without S9 mix in any tester strain and also if reproducibility is noted in such increase, test substance is judged to have mutagenicity (positive). For other case, it is judged as negative.
Statistics:
No statistical method was used for judgment of test results.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The numbers of revertant colonies were below 2 fold of negative (solvent) control value at the dosages of 5000, 1250, 313, 78.1, 19.5, 4.88 and 1.22 µg/plate for all tester strains with or without S9 mix as the result of the preliminary test. No growth inhibition and no precipitation were observed for any tester strain with or without S9 mix.
Based on above results, main tests were conducted at 5 dosages of 5000, 2500, 1250, 625 and 313 µg/plate for all tester strains.
As the result of 2 main tests, the numbers of revertant colonies for all tester strains were below 2 folds of negative (solvent) control value with or without S9 mix. No growth inhibition and no precipitation were observed for any tester strain with or without S9 mix.
From sterility tests for the test substance solution at the highest concentration and S9 mix, no contamination by bacteria or mould which affected the results of the tests was observed.
Based on the results of preliminary test, main tests were conducted with the highest concentration of 5000 µg/plate. 
Number of revertant colonies was below 2 folds of negative (solvent) control value for all tester strains with
or without S9 mix.
It was confirmed that the test was appropriately conducted because negative (solvent) control value and positive
control value in this test are within the normal range which calculated from historical background data, and because
positive control material showed positive result with significant increase of over 2 fold in the number of revertant
colonies which was induced by the positive control material in each tester strains with or without S9 mix compared
with the number of negative (solvent) control in each tester strains.
Based on above results, it was concluded that tetrahydrofurfuryl alcohol has no mutagenicity (negative) for
bacterial reverse mutation test.

Any other information on results incl. tables

Pre-experiment for toxicity

Whether or not there is metabolic activation

Dosage level of the test substance

(µg/plate)

Number of reverse mutations (number of colonies/plate)

Base pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA/pKM101

TA98

TA1537

－S9 mix

Negative control

114

9

81

16

17

1.22

130

10

87

23

23

4.88

109

8

84

24

24

19.5

123

13

67

26

29

78.1

116

14

71

24

24

313

126

10

83

18

22

1250

118

8

65

27

24

5000

105

13

73

26

25

+S9 mix

Negative control

113

8

78

32

24

1.22

117

10

86

26

31

4.88

126

14

114

35

20

19.5

120

11

112

25

24

78.1

128

8

89

34

30

313

123

9

125

39

22

1250

114

8

104

32

27

5000

108

11

88

33

25

Positive control

Positive control groups for which  S9 mix is not necessary

Name

AF-2

NaN3

ENNG

AF-2

9-AA

Dosage (µg/plate)

0.01

0.5

2

0.1

80

(Number of colonies/plate)

551

467

3659

697

263

Positive control groups for whichS9mix is necessary

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Dosage (µg/plate)

1

2

2

0.5

2

(Number of colonies/plate)

1298

182

625

400

184

Results of main experiment 1, preincubation; revertants per plate (mean and s.d.)

Whether or not there is metabolic activation

Dosage level of test substance

(µg/plate)

Number of reverse mutations (number of colonies/plate)

Base pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA/ pKM101

TA98

TA1537

－S9 mix

Negative control

103

123

113

(113)

(± 10)

14

11

11

(12)

(± 2)

82

75

87

(81)

(± 6)

22

20

22

(21)

(± 1)

17

16

17

(17)

(± 1)

313

97

103

122

(107)

(± 13)

11

8

10

(10)

(± 2)

93

73

92

(86)

(± 11)

17

23

21

(20)

(± 3)

16

21

15

(17)

(± 3)

625

112

122

108

(114)

(± 7)

10

8

12

(10)

(± 2)

76

76

98

(83)

(± 13)

21

20

19

(20)

(± 1)

15

18

14

(16)

(± 2)

1250

110

134

96

(113)

(± 19)

14

11

10

(12)

(± 2)

63

77

95

(78)

(± 16)

31

18

25

(25)

(± 7)

18

20

15

(18)

(± 3)

2500

116

109

111

(112)

(± 4)

11

9

9

(10)

(± 1)

93

81

92

(89)

(± 7)

20

17

24

(20)

(± 4)

17

11

17

(15)

(± 3)

5000

118

119

102

(113)

(± 10)

10

16

11

(12)

(± 3)

87

70

91

(83)

(± 11)

17

21

17

(18)

(± 2)

17

23

15

(18)

(± 4)

+S9 mix

Negative control

128

127

102

(119)

(± 15)

13

8

15

(12)

(± 4)

99

106

99

(101)

(± 4)

33

24

23

(27)

(± 6)

25

24

22

(24)

(± 2)

313

114

120

119

(118)

(± 3)

9

11

11

(10)

(± 1)

87

111

92

(97)

(± 13)

40

23

29

(31)

(± 9)

26

27

21

(25)

(± 3)

625

137

98

125

(120)

(± 20

13

10

12

(12)

(± 2)

92

102

77

(90)

(± 13)

33

30

23

(29)

(± 5)

27

23

22

(24)

(± 3)

1250

108

126

106

(113)

(± 11)

12

11

17

(13)

(± 3)

104

111

112

(109)

(± 4)

30

31

37

(33)

(± 4)

22

27

24

(24)

(± 3)

2500

113

120

123

(119)

(± 5)

10

16

15

(14)

(± 3)

109

95

112

(105)

(± 9)

29

29

33

(30)

(± 2)

26

24

18

(23)

(± 4)

5000

101

120

113

(111)

(± 10)

10

18

13

(14)

(± 4)

86

113

116

(105)

(± 17)

23

33

23

(26)

(± 6)

25

30

29

(28)

(± 3)

Positive control

Positive control groups for which  S9 mix is not necessary

Name

AF-2

NaN3

ENNG

AF-2

9-AA

Dosage (µg/plate)

0.01

0.5

2

0.1

80

(Number of colonies/plate)

564

555

533

 

(551)

(± 16)

 

520

476

468

(488)

(± 28)

4412

4259

3992

(4221)

(± 213)

632

644

625

(634)

(± 10)

292

237

346

(292)

(± 55)

Positive control groups for whichS9mix is necessary

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Dosage (µg/plate)

1

2

2

0.5

2

(Number of colonies/plate)

1381

1446

1443

(1423)

(± 37)

169

149

171

(163)

(± 12)

935

955

925

(938)

(± 15)

436

437

451

(441)

(± 8)

171

167

175

(171)

(± 4)

AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

ENNG:N-ethyl-N’-nitro-N-nitrosoguanidine,

9-AA:9-Aminoacridine hydrochloride,

2-AA:2-Aminoanthracene

Results of main experiment 2, preincubation; revertants per plate (mean and s.d.)

Whether or not there is metabolic activation

Dosage level of test substance

(µg/plate)

Number of reverse mutations (number of colonies/plate)

Base pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA/ pKM101

TA98

TA1537

－S9 mix

Negative control

121

118

102

(114)

(± 10)

16

15

14

(15)

(± 1)

82

85

88

(85)

(± 3)

19

22

22

(21)

(± 2)

18

14

11

(14)

(± 4)

313

127

111

108

(115)

(± 10)

14

11

12

(12)

(± 2)

96

94

86

(92)

(± 5)

24

24

26

(25)

(± 1)

17

16

15

(16)

(± 1)

625

95

106

107

(103)

(± 7)

9

10

10

(10)

(± 1)

95

86

89

(90)

(± 5)

16

24

25

(22)

(± 5)

16

16

15

(16)

(± 1)

1250

110

112

117

(113)

(± 4)

10

11

12

(11)

(± 1)

76

90

88

(85)

(± 8)

17

25

21

(21)

(± 4)

14

18

17

(16)

(± 2)

2500

101

105

105

(104)

(± 2)

13

9

15

(12)

(± 3)

86

90

91

(89)

(± 3)

16

17

20

(18)

(± 2)

15

13

17

(15)

(± 2)

5000

121

98

130

(116)

(± 17)

8

11

14

(11)

(± 3)

93

87

81

(87)

(± 6)

26

18

23

(22)

(± 4)

12

10

12

(11)

(± 1)

+S9 mix

Negative control

110

127

108

(115)

(± 10)

11

11

16

(13)

(± 3)

93

95

87

(92)

(± 4)

32

29

35

(32)

(± 3)

23

23

15

(20)

(± 5)

313

123

114

126

(121)

(± 6)

12

13

13

(13)

(± 1)

88

111

91

(97)

(± 13)

24

31

27

(27)

(± 4)

20

17

22

(20)

(± 3)

625

124

119

110

(118)

(± 7)

14

16

14

(15)

(± 1)

90

87

103

(93)

(± 9)

39

21

24

(28)

(± 10)

18

22

29

(23)

(± 6)

1250

121

115

112

(116)

(± 5)

10

13

10

(11)

(± 2)

87

95

102

(95)

(± 8)

25

28

33

(29)

(± 4)

22

25

22

(23)

(± 2)

2500

108

99

116

(108)

(± 9)

14

10

13

(12)

(± 2)

90

96

108

(98)

(± 9)

24

27

30

(27)

(± 3)

22

16

20

(19)

(± 3)

5000

108

114

102

(108)

(± 6)

9

13

10

(11)

(± 2)

101

87

108

(99)

(± 11)

25

25

28

(26)

(± 2)

22

19

19

(20)

(± 2)

Positive control

Positive control groups for which  S9 mix is not necessary

Name

AF-2

NaN3

ENNG

AF-2

9-AA

Dosage (µg/plate)

0.01

0.5

2

0.1

80

(Number of colonies/plate)

563

520

482

 

(552)

(± 41)

 

369

387

440

(399)

(± 37)

4358

4439

4275

(4357)

(± 82)

632

618

590

(613)

(± 21)

242

209

209

(220)

(± 19)

Positive control groups for whichS9mix is necessary

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Dosage (µg/plate)

1

2

2

0.5

2

(Number of colonies/plate)

1398

1398

1301

(1366)

(± 56)

150

147

138

(145)

(± 6)

1347

1373

1072

(1264)

(± 167)

432

428

423

(428)

(± 5)

200

182

194

(192)

(± 9)

Applicant's summary and conclusion

Conclusions:
Tetrahydrofurfuryl alcohol has been tested according to a Japanese national method that is equivalent to OECD TG 471. No evidence of test-substance induced increases in the number of revertants was obtained using S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without metabolic activation, in either the initial or the repeat experiment, both of which used the pre-incubation method. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.