Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 Escherichia coli WP2 (equivalent to OECD TG 471) (Mitsubishi, 2004a).
Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured human lymphocytes (OECD TG 473) (Mitsubishi, 2004b).
Mutagenicity to mammalian cells: negative with and without metabolic activation in mouse lymphoma L5178Y cells (OECD TG 476) (Flanders, L, 2012)

Link to relevant study records

Referenceopen allclose all

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.
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
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 / 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.
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.
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.

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)

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.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
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.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Remarks:
Evidence of GLP is not included in the study report.
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: CHL/IU cell line derived from lung of female Chinese hamster
Details on mammalian cell type (if applicable):
Cell line was purchased from Dainippon Pharma Co. Ltd., on July 17, 2001 (number of passage at purchasing: 14), and DMSO (lot number: 210G1441, Kanto Chemical Co., Inc.,) was added to the cell suspension to obtain the final concentration of 10 v/v%, and the solution was dispensed to 1 ml and stored frozen in liquid nitrogen (number of passage at freezing: 17). The frozen lot of cells was supplied for characteristic inspection. It was confirmed that they had chromosome number of 25, cell cycle of 12.5 hours and to negative in mycoplasma contamination. The frozen lot of cells was thawed, incubated and used for the test within 4 weeks after thawing (number of passage: 18 - 24). Incubation of cells was conducted on plastic plate (6 cm or 10 cm of diameter, Becton Dickinson and Company) in CO2 incubator (set to 5% of CO2 and 37°C of temperature, with humidification, Type 7300 or 6301C, NAPCO).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Test substance;
Short-term treatment (6-hrs treatment - 18-hrs recovery incubation); 257.5, 515 and 1030 µg/ml
24-hours continuous treatment; 257.5, 515 and 1030 µg/ml
Positive control;
Short-term treatment; without S9-mix: MMC; 0.1 µg/ml, with S9-mix: BP; 20 µg/ml
24-hours continuous treatment; MMC; 0.05 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Physiological saline (PS) (Otsuka Pharmaceutical Factory, Inc., Lot No.; K2A79)
- Justification for choice of solvent/vehicle: The test substance was soluble in PS at the concentration of 50 mg/ml in the test for selection of solvent. No exothermic, bubbling and discoloration was observed when PS was added. Based on these results, PS was used for solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix: abbreviated as MMC, Lot No. 342AJH, content; 103%, Kyowa Hakko Kogyo Co.
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9-mix: abbreviated as BP, Lot No. GG01, content; 95.6%, Tokyo Chemical Industry Co., Ltd.
Details on test system and experimental conditions:
(1) Cell line and selection rationale;
The CHL/IU cell line derived from lung of female Chinese hamster was used. The cell is widely used in chromosome aberration test for noted advantages, which include that the number of chromosomes is only 25, and that chromosomes are relatively large, contributing to easier specimen observation.
(2) Culture medium;
Approximately 8.3 g of Eagle MEM Medium “Nissui” (1) (lot number: 460201, Nihon Pharmaceutical Co., Ltd.,) was dissolved in 880 ml of purified water, autoclaved (121°C, 20 minutes) and 8.8 ml of 2.92 w/v% L-glutamine aqueous solution and 11.2 ml of 10 w/v% sodium bicarbonate which were sterilized separately were added, respectively in order to prepare a MEM solution. Then, 100 ml of inactivated (heated at 56°C for 30 minutes) bovine serum (Lot No. 296130, GIBCO BRL) was added to 900 ml of MEM solution. The obtained solution was used as MEM medium.
(3) Manufacture of S9-mix;
(a) S9;
S9 was purchased from Kikkoman Corporation on March 5, 2002. S9 was derived from liver of 7-week old male SD strain rat (body weight: 214 to 239 g) which was enzyme-induced with phenobarbital (single intraperitoneal administration at 30 mg/kg on Day 1, daily single intraperitoneal administration at 60 mg/kg for 3 days after Day 2) and 5,6-benzoflavone (single intraperitoneal administration at 80 mg/kg on Day 3). S9 of lot number RAA-459 (final protein concentration: 1.26 mg/ml, manufactured on February 22, 2002) was used for the test. Purchased S9 was stored frozen in Deep Freezer with controlled temperature of below -80°C (measured temperature: -84 to -82°C) until use.
(b) S9-mix;
Composition of S9-mix per 1 ml was shown in Table 3 and prepared when needed. S9-mix was stirred in ice until use.
(4) Preparation of test substance solution and positive control substance solution;
(a) Test substance solution;
Designated quantity of the test substance was weighed and dissolved in added PS by shaking stirring. This solution was used as the highest concentration solution.
The highest concentration solution was serially diluted with PS to prepare 10-fold concentration of the test substance solutions for each dose level. Test substance solution was prepared at use, and weighing, dilution, dispense and storage after preparation of test substance was done under yellow light. The storage period before treatment was 10 to 20 minutes in cell growth inhibition test, 5 to 15 minutes in chromosome aberration test (short term treatment) and 5 minutes in chromosome aberration test (continuous treatment).
(b) Positive control substance solution;
MMC in 2 mg-volume vial was dissolved in 5 ml of water for injection (Lot No.: K2A84, Otsuka Pharmaceutical Factory, Inc.,) at use. The solution was serially diluted with PS (Lot No.: K2A79, Otsuka Pharmaceutical Factory, Inc.,) to prepare solution at 10-fold concentration of treatment dose in each treatment condition (short term treatment: 1 µg/ml, continuous treatment: 0.5 µg/ml).
For BP, solution was prepared at 200-fold concentration of treatment dose by dissolving in DMSO (Lot No.: 207G1673, Kanto Chemical Co., Inc.,) at 4 mg/ml and was stored frozen until use.
(5) Cell growth inhibition test;
(a) Dose of test substance;
Prior to cell growth inhibition test, preliminary test was conducted by 24-hour treatment with and without S9 mix. Preliminary test (dose level: 10.3, 103, 1030 µg/ml [equivalent to 0.1, 1 and 10 mmol]) was conducted using 1 plate per each dose level. After the completion of the treatment, plates were observed by phase contrast inverted microscope and relative cell density was determined with the cell density in the negative control as 100%. As a result, the cell density in each dose level was showed 100%. According to above result, dose levels in cell growth inhibition test were set as follows:
-S9 mix: 64.4, 128.8, 257.5, 515, 1030 µg/ml
+S9 mix: 64.4, 128.8, 257.5, 515, 1030 µg/ml
24-hour treatment: 64.4, 128.8, 257.5, 515, 1030 µg/ml
(b) Treatment of cells;
Each 5 ml of cell suspension prepared at 4x10E3/ml was inoculated onto 6 cm plate contained MEM medium and incubated for 3 days.
After removal of MEM medium, solution for cell treatment of the following composition (Table 4) was added to 2 plates per 1 dose level, and cells were treated for 24 hours (continuous treatment) or 6 hours (short term treatment). In short term treatment, cell surface was washed with MEM after 6-hour treatment, 5 ml of fresh MEM medium was added and incubation was continued for further 18 hours. The solvent of the test substance was used as negative control material and similarly treated.
(c) Measurement of cell growth rate;
Surface of cell was washed with Dulbecco's phosphate buffered saline (referred as PBS(-) hereinafter, Nissui Pharmaceutical Co., Ltd.,) which was free of Ca2+ and Mg2+. Then cells were fixed with methanol for 10 minutes, stained with 3 v/v% Giemsa solution for 10 minutes and washed gently with water and dried. The cell growth rate was measured using mono layer culture cell density meter (Monocellater, Olympus Optical Co., Ltd.,) for each stained plate. As a result, 50% inhibitory concentration (IC50) of the test substance could not be calculated because cell growth inhibition greater than 50% was not observed at any treatment conditions.
(6) Chromosome aberration test;
Initially, short term treatment only was conducted for chromosome aberration test. The result of short term treatment was negative and continuous treatment was conducted consequently.
(a) Dose level for test substance and positive control substance;
Based on result of cell growth inhibition test, following dose levels were set in chromosome aberration test. No precipitation or deposition was observed at test substance treatment at any treatment condition and any test substance treatment group.
-S9 mix: 257.5, 515 and 1030 µg/ml
+S9 mix: 257.5, 515 and 1030 µg/ml
24-hour treatment: 257.5, 515 and 1030 µg/ml
For dose levels of positive control materials, MMC was set at 0.1 µg/ml in -S9 mix and BP was set at 0.05 µg/ml in 24-hour treatment. 20 µg/ml of BP was used in +S9 mix. These dose levels are known to induce clastogenicity.
(b)Treatment of cells;
Cells were treated similarly as in cell growth inhibition test.
For positive control, cells were treated similarly with cell treatment solution with the following composition (Table 5).
For negative control group and test substance treatment group, 4 plates were used per each treatment condition (2 plates for specimen preparation and 2 plates for measurement of cell growth rate). For positive control group, 2 plates were used per each treatment condition because the measurement of cell growth inhibition test was not conducted.
(c) Preparation of specimen;
Colcemid was added on each plate for specimen preparation at 2 hours prior to specimen preparation to make the final dose of 0.1 µg/ml and metaphase cells were accumulated. After treatment was completed, surface of cells was washed with PBS(-) and cells were separated by treatment of 0.25 w/v% trypsin. The solution was taken into centrifuge tube and cells were collected by centrifugation (1000 rpm, 5 minutes; following procedure was done under the same condition). After removal of supernatant, 4 ml of 0.075 mol/L potassium chloride solution was taken into each centrifuge tube and hypotonically treated (37°C, 15 minutes). 0.5 ml of cooled fixative (mixture of methanol and acetic acid [3:1, v/v]; following procedure was done under the same condition) was added and mixed, and then centrifuged to remove supernatant. 4 ml of fixative was added and the same procedure was repeated for 2 times. Then, cells were suspended in appropriate volume of fixative and the solution was dropped onto 2 positions of slide glass which was placed on wetted towel and the slide glass was dried. Specimen was stained with 3 v/v% Giemsa stain for 20 minutes, washed with water, dried and enclosed to obtain specimen of chromosome. 2 specimens were prepared per plate.
Evaluation criteria:
1. Structural and numerical aberration;
Observation was performed by blind test on 100 specimens per plate, i.e., 200 metaphases per 1 dose level. Cells with well-spread chromosome were observed in the metaphase observation.
Structural aberration was observed based on the following criteria:

Cells with centromere of 25 ± 2 or over 35 were excluded.

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange (dicentric chromosome, circular chromosome and etc)

Fragmentation

Gap was defined as a condition that width of achromatic region of chromatid is smaller than width of chromatid. Gap was recorded separately from other aberrations and not included in structural aberration.

Numerical aberration was defined as cells with centromeres over 35 and polyploid cells including endoreduplicated cells. Cells with numerical aberrations were counted.

2. Acceptance criteria for the test results:

Cells with 1 or more structural aberration were counted as structural chromosome aberration. Specimens with less than 50 observable metaphases per plate were excluded from counting.

3. Criteria for judgment of clastogenicity in each treatment condition are as follows:
- Negative (-): frequencies of both structural and numerical aberration are less than 5%
- Pseudo-Positive [equivocal] (+/-): frequencies of structural or numerical aberration or both are more than 5% and less than 10%.
- Positive (+):frequencies of structural or numerical aberration or both are more than 10%.
Statistics:
Statistical method was not used for evaluation of the results.
Key result
Species / strain:
other: CHL/IU cell line derived from lung of female Chinese hamster
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:
Chromosome aberration test using cultured mammalian cells was conducted to investigate clastogenicity of tetrahydrofurfuryl alcohol.
Frequencies of cells with structural and numerical aberration were less than 5% in both short term treatment and continuous treatment.
Frequencies of cells with structural and numerical aberration were less than 5% in all negative control groups and frequency of cells with structural aberration was more than 10% in all positive control groups. Therefore, it was confirmed that this study was valid technically.
Based on above, it is concluded that tetrahydrofurfuryl alcohol does not induce chromosomal aberration for CHL/IU cells under the conditions of this study.

Table 1.; Result of chromosome aberration test (short-term treatment)

Treatment-recovery period (h)

S9mix

Dose of test substance (µg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell proliferation rate (%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Fragment

Total number of aberrations (%)

Number of cells observed

Polyploid

endoreduplication

Total number of aberrant cells (%)

6-18

-

Negative control (NSS)

100

100

200

0

1

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

1

1 (0.5)

0

0

0

101

99

100

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

-

257.5

100

100

200

1

1

2 (1.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

1

1 (0.5)

1

0

1 (0.5)

2

2

4(2.0)

0

0

0

107

103

105

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

-

515

100

100

200

1

0

1(0.5)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

1

0

1 (0.5)

0

0

0

105

103

104

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

-

1030

100

100

200

0

1

1 (0.5)

1

0

1 (0.5)

1

0

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

1

1

2 (1.0)

0

0

0

105

107

106

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

-

Positive control (MMC 0.1)

100

100

200

15

19

34 (17.0)

23

28

51 (25.5)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

36

46

82 (41.0)

0

0

0

 

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

+

Negative control (NSS)

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

1

0

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

1

0

1 (0.5)

0

1

1

111

89

100

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

+

257.5

100

100

200

0

0

0 (0.0)

1

0

1 (0.5)

1

0

1 (0. 5)

0

0

0 (0.0)

0

0

0 (0.0)

2

0

2 (1.0)

0

0

0

111

105

108

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

+

515

100

100

200

0

0

0 (0.0)

1

0

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

1

0

1 (0.5)

0

0

0

106

108

107

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

+

1030

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0

109

114

112

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

6-18

+

Positive control (BP20)

100

100

200

15

9

24 (12.0)

78

79

157 (78.5)

1

1

2 (1.0)

0

0

0 (0.0)

0

0

0 (0.0)

81

82

163 (81.5)

0

1

1

 

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

NSS: Normal saline solution

DMSO: Dimethyl sulfoxide, MMC: mitomycin C, BP: benzo(a)pyrene

Table 2.; Result of chromosome aberration test (continuous treatment)

Treatment-recovery period (h)

Dose of test substance (µg/ml)

Number of cells showing structural chromosome aberration (incidence, %)

Number of gap appearances

Cell proliferation rate (%)

Number of cells showing numerical chromosome aberration (incidence, %)

Number of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Fragment

Total number of aberrations (%)

Number of cells observed

Polyploid

endoreduplication

Total number of aberrant cells (%)

24-0

Negative control (NSS)

100

100

200

0

1

1 (0.5)

0

0

0 (0.0)

0

1

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

0

2

2 (1.0)

1

0

1

106

94

100

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

24-0

257.5

100

100

200

0

0

0 (0.0)

1

0

1 (0.5)

1

0

1 (0.5)

1

0

1 (0.5)

0

0

0 (0.0)

3

0

3 (1.5)

0

0

0

104

102

103

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

24-0

515

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0

108

96

102

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

24-0

1030

100

100

200

0

0

0 (0.0)

2

0

2 (1.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

2

0

2 (1.0)

0

0

0

101

101

101

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

24-0

Positive control (MMC 0.05)

100

100

200

14

7

21 (10.5)

18

15

33 (16.5)

0

1

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

30

23

53 (26.5)

0

0

0

 

100

100

200

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0)

NSS: Normal saline solution

MMC: Mitomycin C

Conclusions:
Tetrahydrofurfuryl alcohol has been tested according to a Japanese national protocol that is equivalent to OECD 473. No substance-induced increase in the number of cells with aberrations was observed in CHL/IU cells when tested with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO 2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0, 63.75, 127.5, 255, 510, 765, 1020 μg/ml +/- MA, both experiments
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 medium
- Justification for choice of solvent/vehicle: following solubility checks
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (-MA, expt 1, +MA expts 1 and 2); 24 hours (-MA, expt 2)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 4 μg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures, experiment repeated. 96-well microtitre plates used for selection/viability assessment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other: numbers of small and large colonies were recorded.

Evaluation criteria:
A result is considered positive if the mutant frequency exceeds the solvent mutant frequency by the Global Evaluation Factor (GEF) 126 x 10E-06, and demonstrates a positive linear trend and is reproducible.
Statistics:
UKEMS statistical package
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none (see Table 1 below)
- Effects of osmolality: none (see Table 1 below)

RANGE-FINDING/SCREENING STUDIES: no toxicity observed up to the 10 mM limit dose of 1020 μg/ml.

COMPARISON WITH HISTORICAL CONTROL DATA: results were within the range of historical control

ADDITIONAL INFORMATION ON CYTOTOXICITY: see Table 2 below)

Table 1 pH and osmolality readings from the solubility test

μg/ml

0

3.99

7.97

15.94

31.88

63.75

127.5

255

510

1020

pH

7.31

7.31

7.31

7.33

7.33

7.33

7.33

7.33

7.33

7.34

mOsm

297

296

300

298

297

298

299

299

304

308

 

Table 2 Preliminary Toxicity Test

Dose

(μg/ml)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

3.99

89

104

95

7.97

87

103

94

15.94

96

100

79

31.88

88

100

93

63.75

85

100

90

127.5

94

109

72

255

85

110

86

510

87

109

80

1020

87

108

81

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

 

Table 3 Results experiment 1

Treatment

(μg/ml)

4 Hours -MA

Treatment

(μg/ml)

4 Hours +MA

%RSG

RTG

MF*

%RSG

RTG

MF*

0

100

1.00

145.64

0

100

1.00

177.18

63.75

98

1.03

185.92

63.75

109

1.09

206.57

127.5

98

1.00

197.08

127.5

101

1.06

205.50

255

101

1.05

180.91

255

108

1.10

190.59

510

99

1.10

154.04

510

110

1.02

200.68

765

106

1.07

181.93

765

106

1.02

191.57

1020

102

1.06

175.32

1020

105

1.08

177.59

Linear trend

NS

Linear trend

NS

EMS

400

58

0.35

1334.46

CP

2

58

0.33

1254.84

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

*Positive wells per tray, 96 wells plated

NS = not significant

 

Table 4 Results experiment 2

Treatment

(μg/ml)

24 Hours -MA

Treatment

(μg/ml)

4 Hours +MA

%RSG

RTG

MF*

%RSG

RTG

MF*

0

100

1.00

141.18

0

100

1.00

131.90

63.75

95

1.02

147.90

63.75

93

1.02

152.72

127.5

112

1.15

92.03

127.5

98

1.05

121.28

255

106

1.07

125.96

255

96

0.98

141.72

510

97

0.87

154.35

510

99

1.13

142.60

765

107

1.10

136.45

765

95

0.98

140.60

1020

105

0.92

148.69

1020

102

1.13

124.91

Linear trend

NS

Linear trend

NS

EMS

150

35

0.35

971.89

CP

2

75

0.53

1179.29

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

*Positive wells per tray, 96 wells plated

NS = not significant

Conclusions:
Tetrahydrofurfuryl alcohol has been tested for mutagenicity to mouse lymphoma L5178Y cells in a study conducted according to OECD 476 and in compliance with GLP. No increase mutant frequency or in the ratio of small to large colonies was observed in the presence or absence of metabolic activation in either the first experiment (4 hour exposure with and without metabolic activation) or the independent repeat experiment (24 hour exposure without metabolic activation, 4 h exposure with metabolic activation). No cytotoxicity was observed up to the limit concentration of 1020 μg/ml (10 mM). Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data are available from reliable studies on mutagenicity to bacterial cells and mammalian cells, and cytogenicity in mammalian cells.

Tetrahydrofurfuryl alcohol has been tested according to a Japanese national method that is equivalent to OECD TG 471 (Mitsubishi, 2004a). No evidence of test-substance induced increases in 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.

Tetrahydrofurfuryl alcohol has been tested according to a Japanese national protocol that is equivalent to OECD 473 (Mitsubishi, 2004b). No substance-induced increase in the number of cells with aberrations was observed in CHL/IU cells when tested with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

Tetrahydrofurfuryl alcohol has been tested for mutagenicity to mouse lymphoma L5178Y cells in a study conducted according to OECD 476 and in compliance with GLP (Flanders, L, 2012). No increase mutant frequency or in the ratio of small to large colonies was observed in the presence or absence of metabolic activation in either the first experiment (4 hour exposure with and without metabolic activation) or the independent repeat experiment (24 hour exposure without metabolic activation, 4 h exposure with metabolic activation). No cytotoxicity was observed up to the limit concentration of 1020 μg/ml (10 mM). Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

Proposal of in vivo testing is not required as the results of all the in vitro studies were negative.


Justification for classification or non-classification

Based on the available in vitro genotoxicity data, tetrahydrofurfuryl alcohol is not classified for mutagenicity according to Regulation (EC) No 1272/2008.