Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2002 to 03 July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Name: CAS 577978-76-8
Chemical Name: Polyglycidyl ether of 1,6-naphthalenediol-pxylyleneglycol (or p-xylyleneglycol dimethylether) condensation polymer
CAS number: CAS 577978-76-8
Batch/Lot number: 0950985
Description: Brown solid
Purity: 100%
Expiry date: 08 February 2020 (as per updated CoA)
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity).
Safety precautions: Enhanced safety precautions above the routine safety precautions (lab coat, gloves, safety glasses, face mask) will be applied considering the supplied safety data sheet to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report

Method

Target gene:
Histidine and Tryprophan
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 applicable
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
1) Rat liver S9
An S9 (Lot No. 02050902, manufactured on May 9, 2002, Oriental Yeast Co., Ltd.) prepared from the liver of rats (body weight 206. 7±8.4 g) administered phenobarbital and 5,6-benzoflavone was used. The S9 was cryopreserved in an ultra-deep freezer (MDF-291, SANYO Electric Co., Inc.) until use.

2) Composition of S9 mix
A S9 mix was prepared using a cofactor for S9 mix (Lot No. 999202, Oriental Yeast Co., Ltd.) immediately before use.
One mL of the S9 mix consisted of 8 µmol MgC12, 33 µmol KCl, 5 µmol G-6-P, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium-phosphate buffer (pH 7.4) and 0.1 mL of S9.
Test concentrations with justification for top dose:
Dose-range-finding test-1
A total of 6 doses was set, 5,000 µg/plate was the highest dose and 5 lower doses diluted with a geometric progression of 4.
Dose-range-finding test-2
In the dose-range-finding test-1, growth inhibition were observed in all the tester strains. Four or more doses without growth inhibition (effective dose) were not obtained in all the tester strains without S9 mix and in TAlO0 and TA1535 with S9 mix. Therefore, dose-range-finding test-2 was carried out due to secure for four or more effective dose in a main test. Highest doses in the second test were selected at 78.1 µg/plate without S9 mix, at 313 µg/plate with S9 mix, and added at each 5 lower doses diluted with a geometric progression of 4.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino] acridine-2H Cl
Details on test system and experimental conditions:
The tests were performed using the pre-incubation method with and without S9 mix. Three plates were used for the negative control group and two plates for the test substance treatment and positive control groups. The test code number, name of tester strain, presence or absence of S9 mix and dose level were noted on the plate.

Procedures
fter 0. lmL of the test substance solution or positive control substance solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix and 0.1 mL of the bacterial culture were added to a test tube, and the mixture was shaken for 20 minutes at 37±0.5°C. Two mL of the soft agar was then added to the test tube, and the mixture was poured onto a minimal glucose agar plate. The number of reve1iant colonies was counted after incubation at 37±0.5°C for 48 hours.
Sterility
Each of the test substance solution of the highest dose and S9 mix was mixed with 2 mL of the soft agar and poured onto a minimal glucose agar plate. The plate was incubated at 37±0.5°C for 48 hours to investigate a bacterial contamination.

Main test
n the dose-range-finding tests, the growth inhibition and increase of the revertant colonies were observed, so highest doses were selected at 39.1 µg/plate in TAlO0 and TA1535, at 78.1 µg/plate in WP2 uvrA and TA98, at 19.5 µg/plate in TA1537 in the without S9 mix, and added at each 5 or 6 lower doses diluted with a geometric progression of 2. In the with S9 mix, highest doses were selected at 625 µg/plate in TAl00 and TA1535, at 1,250 µg/plate in WP2 uvrA, TA98 and TA 153 7, and added 6 lower doses diluted with a geometric progression of 2.


Rationale for test conditions:
Not specified
Evaluation criteria:
The test substance was judged positive when the number of revertant colonies was increased two times or more that of the negative control in a dose-dependent manner, and also when reproducibility of the test results was determined. In all the other cases, it was judged negative. Statistical treatment was not carried out.
In the case of a positive result, specific activities were calculated for dose levels of which the number of revertant colonies was twice or more than that in the negative
Statistics:
Statistical treatment was not carried out.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE-RANGE-FINDING TEST-1
Growth inhibition was observed in all the tester strains, so there were not four or more effective doses in all the tester strains without S9 mix and in TAlO0 and TA1535 with S9 mix. Correctly, evaluation of the test results were not completed. In WP2 uvrA and TA98 with S9 mix, the numbers of revertant colonies were increased two times or more that of the each negative control. And dose-related increase was showed in WP2 uvrA.

DOSE-RANGE-FINDING TEST-2
In TA1535, WP2 uvrA and TA98 without S9 mix and TA1535 with S9 mix, the numbers of revertant colonies were increased two times or more that of the negative control, and dose-related increase were shown in WP2 uvrA and TA98.
The numbers of revertant colonies were less than two times that of the negative control in the other test strains.
Growth inhibition was observed at 19.5 µg/plate or more in TAIO0, TA1535, TA98 and TA1537 and at 78.1 µg/plate in WP2 uvrA without S9 mix. Growth inhibition was observed at 313 µg/plate in TAl00 and TA1535 with S9 mix.
The precipitate of the test substance was not observed.

MAINTEST
The numbers of revertant colonies were increased in TA1535, WP2 uvrA and TA98 with and without S9 mix, and shown dose-related increased in WP2 uvrA and TA98. The numbers of revertant colonies were less than two times that of the negative control in TAIO0 and TA1537 with and without S9 mix.
Growth inhibition was observed at 19.5 µg/plate or more in TAIO0, TA1535 and TA98, at 39.1 µg/plate or more in WP2 uvrA and at 9.77 µg/plate or more in TA1537 without S9 mix. Growth inhibition was observed at 313 µg/plate or more in TAl00 and TA1535 and at 625 µg/plate or more in WP2 uvrA, TA98 and TA1537 with S9 mix. The precipitate of the test substance was observed at 625 µg/plate or more with S9 mix.

Mutagenicity of the test substance was judged positive because the numbers of revertant colonies were reproducible increased two times or more that of negative control in TA1535, WP2 uvrA and TA98 with and without S9 mix. And in WP2 uvrA and TA98 with and without S9 mix, the numbers of revertant colonies were increased two times or more that of negative control and dose-related increase was shown. The specific activity was 13,730 Revertants/mg. In the other test strains, the number of revertant colonies was not increased.
The numbers of revertant colonies of the positive and negative controls were within the range of the historical data in our laboratory. It was also confirmed that the test system was free from bacterial contamination, indicating that this study was appropriately performed.
Based on the above results, GK-5016-2 was considered to have mutagenic potential under the conditions in the present study.

Any other information on results incl. tables

Dose-range-finding test-I 

 

Test substance: GK-5016-2

Test dates

From June 7 2002 to June 10 2002

With (+) or without (-) S9 mix

Test substance dose (μg/plate)

Number of revertants per plate

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

 

 

 

-S9 mix

 

Negative control

97

84           (94)

102

7

10        (8)

8

29

25           (29)

34

20

28        (24)

24

8

4

13           (8)

4.88

147      

131        (139)

21

19     (20)

109

74           (92)

46

40        (43)  

7

5          (6)

19.5

137*

152*       (145)

9*

22*     (16)

131

128        (130)

86*

89*       (88)

2*

3*          (3) 

78.1

 36*

38*          (37)

6*

6*        (6)

77*

66*          (72) 

42*

42*       (42)

1*

0*          (1)

+313

0*

0*             (0)

0*

0*        (0)

28*

33*          (31)

0*

0*          (0)

0*

0*          (0)

+1250

0*

0*             (0)

0*

0* (0)

0*

0*              (0)

0*

0*         (0)  

0*

0*         (0)

+5000

0*

0*             (0)

0*

0*        (0)

0*

0*             (0)

0*

0*          (0)  

0*

0*          (0)

 

 

 

+S9 mix

 

Negative control

108

123        (120)

129

8

11

5         (9)

28

26

22           (25)

28

37

23        (29)

16

20

18        (18) 

4.88

110

93         (102)

21

8        (15)

43

42           (43)

33

26        (30)

22

21        (22)

19.5

130

130        (130)

11

5         (8)

31

27           (29)

33

51        (42)

23

12        (18)

78.1

127

137        (132)

18

16      (17)

72

49           (61)

37

34        (36)

16

23        (20)

313

107*

91*          (99)

12*

11*     (12)

165

114        (140)  

59

66        (63)

20

17        (19) 

+1250

0*

0*             (0)

0*

0*        (0)

14*

15*          (15)  

0*

1*         (1)

0*

0*          (0)     

+5000

0*

0*           (0)

0*

0*       (0)

14*

11*          (13)

0*

0*          (0)

0*

0*          (0)

 

 

Positive control -S9 mix

Chemical

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose(μg/plate)

 

0.01

 

05

 

0.01

 

01

 

05

Number of revertants/plate

236

223        (230)

433

409   (421)

187

159        (173)

414

424     (419) 

358

420     (389)

 

 

Positive control +S9 mix

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

 

1

 

2

 

10

 

0.5

 

2

Number of revertants/plate

838

805       (822)    

199

191    (195)

580

677        (629)

425

462    (444)  

416

457     (437)

[Notes] Parenthesis shows the mean of each plate.

+: Precipitate of test substance.

*: Observed bacterial growth inhibition.

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

• NaN3 : Sodium azide

· ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HCI

· 2AA: 2-Aminoanthracene

Dose-range-finding test-2

 

Test substance: GK-5016-2

Test dates

From June 7 2002 to June 10 2002

With (+) or without (-) S9 mix

Test substance dose (μg/plate)

Number of revertants per plate

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

 

 

 

-S9 mix

 

Negative control

97

87               (91)

91

10

7             (8)

8

32

32               (29)

24

18

29            (22)

20

9

5

9                (8)

0.0763

88      

87               (88)

11

9           (10)

39

36               (38)

29

15            (22)  

8

6                (7)

0.305

111

93             (102)

12

9           (11)

37

28               (33)

16

21            (19)

7

3                (5) 

1.22

122

115           (119)

7

8             (8)

44

64               (72) 

29

20            (25)

5

1                (3)

4.88

141

133           (137)

23

14         (19)

89

102             (96)

32

37            (35)

6

5                (6)

19.5

105*

111*          (108)

12*

11*       (12)

139

130           (135)

70*

83*           (77)  

3*

5*               (4)

7801

21*

18*              (20)

2*

2*           (2)

67*

57*              (62)

44*

38*           (41)  

2*

0*               (1)

 

 

 

+S9 mix

 

Negative control

105

111

119           (112)

5

12

5             (7)

 

--

 

--

 

 

--

0.305

97

122           (110)

9

6             (8)

 

--

 

--

 

--

1.22

92

100           (96)

7

7             (7)

 

--

 

--

 

--

4.88

118

94             (106)

3

6             (5)

 

--

 

--

 

--

19.5

106

132           (119)

8

12         (10)

 

--

 

--

 

--

78.1

147

153           (150)

17

13         (15)

 

--

 

--

 

--

313

85*

106*            (96)

23*

13*       (18)

 

--

 

--

 

--

 

 

Positive control -S9 mix

Chemical

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose(μg/plate)

 

0.01

 

05

 

0.01

 

01

 

05

Number of revertants/plate

284

263           (274)

407

448     (428)

183

181           (182)

315

350        (333) 

302

292        (297)

 

 

Positive control +S9 mix

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

 

1

 

2

 

10

 

0.5

 

2

Number of revertants/plate

973

824           (899)    

210

216     (213)

 

          --

 

         --

 

          --

[Notes] Parenthesis shows the mean of each plate.

*: Observed bacterial growth inhibition.

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

· NaN3 : Sodium azide

• ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HC1

· 2AA: 2-Aminoanthracene

Main test

Test substance: GK-5016-2

Test dates

From June 7 2002 to June 10 2002

With (+) or without (-) S9 mix

Test substance dose (μg/plate)

Number of revertants per plate

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

 

 

 

-S9 mix

 

Negative control

114

96             (111)

124

12

10         (10)

7

24

26               (29)

38

26

15            (20)

18

7

7

3                (6)

0.610

88      

87               (88)

7

12         (10)

41

23               (32)

 

--

9

4                (7)

1.22

117

113           (115)

11

12         (12)

30

36               (33)

19

30            (25)

8

2                (5) 

2.44

150

133           (142)

14

13         (14)

51

53               (52) 

28

30            (29)

8

6                (7)

4.88

145

121           (133)

23

12         (18)

66

71              (69)

41

36            (39)

5

6                (6)

9.77

156

158          (157)

19

24         (22)

95

88             (92)

71

54            (63)  

9*

4*               (7)

19.5

160

148           (154)

11*

6*           (9)

130

131           (131)

86*

92*           (89)  

3*

9*               (6)

39.1

53*

51*              (52)

5*

6*           (6)

123*

111*          (117)

82*

85*           (84)   

 

--

78.1

 

--

 

--

99*

98*              (99)

42*

50*           (46)

 

--

 

 

 

+S9 mix

 

Negative control

113

113

124           (117)

6

11

5             (7)

36

29

32               (32)

30

28

30            (29)

21

16

16            (18)

9.77

132

125           (129)

10

6             (8)

 

--

 

--

 

--

19.5

145

138           (142)

7

11           (9)

24

39               (32)

45

25            (35)

10

15            (13)

39.1

135

155           (145)

12

11         (12)

19

42               (31)

38

34            (36)

15

18            (17)

78.1

173

156           (165)

19

13         (16)

39

47               (43)

34

25            (30)

19

19            (19)

156

198

180           (189)

17

17         (17)

80

79               (80) 

47

45            (46)

18

21            (20)

313

85*

94*              (90)

14*

11*       (13)

159

146           (153) 

63

71            (67)

20

12            (16)

+625

54*

58*              (56)  

9*

7*           (8) 

41*

49*              (45)

14*

18*           (16)

7*

4*               (6)

+1250

 

--

 

--

21*

20*              (21)

8*

4*               (6)

3*

6*               (5)

 

 

Positive control -S9 mix

Chemical

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose(μg/plate)

 

0.01

 

05

 

0.01

 

01

 

05

Number of revertants/plate

320

313           (317)

400

367     (384)

190

164           (177)

436

425        (431) 

378

287        (333)

 

 

Positive control +S9 mix

Chemical

2AA

2AA

2AA

2AA

2AA

Dose(μg/plate)

 

1

 

2

 

10

 

0.5

 

2

Number of revertants/plate

1343

1287       (1315)    

215

206     (211)

523

604           (564) 

478

469        (474)

504

473        (489) 

[Notes] Parenthesis shows the mean of each plate.

+: Precipitate of test substance.

*: Observed bacterial growth inhibition.

· AF-2: 2-(2-Furyl)-3-(5-nitro-2-fmyl)acrylamide

· NaN3: Sodium azide

· ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HCI

· 2AA: 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:
GK-5016-2 was considered to have mutagenic potential under the conditions in the present study.
Executive summary:

The ability of GK-5016-2 to induce mutations was investigated in Salmonella typhimurium strains TAlO0, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA using a pre-incubation method with and without metabolic activation system (S9 mix).

As a result, the mutagenicity of the test substance was judged positive because the number of revertant colonies was increased two times or more that of negative control and the numbers increased with reproducibility in TA1535, WP2 uvrA and TA98 with and without S9 mix. Consequently, GK-5016-2 was considered to have mutagenic potential.