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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
Study period:
1988-02-12 to 1988-02-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Only 4 tester strains were used.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one
EC Number:
403-830-5
EC Name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one
Cas Number:
89331-94-2
Molecular formula:
C35H36N2O3
IUPAC Name:
6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 98, 100, 1535, 1537
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver (S9 mix)
Test concentrations with justification for top dose:
DRF: 5, 50, 500, 5000 µg/plate with or without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with or without metabolic activation
Vehicle / solvent:
- Vehicle used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 2-AA
Remarks:
with S9 mix; 2 µg/plate for strains TA 1535 and TA 1537; 0.5 µ/plate for strain TA 98; 1 µg/plate for strain TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1 µg/plate for strain TA 98, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for strain TA 1537, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix; 5 µg/plate for strain TA 1535; 3 µg/plate for strain TA 100.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: approximately 2 x 10^9 organisms per mL which was assessed photometrically.

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or by the absence of a complete background bacterial lawn.
Rationale for test conditions:
According to the guideline
Evaluation criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A Compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1538
Remarks:
Please refer to study entry of YKG26 for further information
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:
S. typhimurium TA 98
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:
S. typhimurium TA 1535
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:
S. typhimurium TA 100
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:
S. typhimurium TA 1537
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:
Concentration of the test substance resulting in precipitation: 1500 μg/plate

Any other information on results incl. tables

Table 1: Summary of the Results, Test 1

Strain

Dose Level (µg/plate)

S9 mix

Mean revertant colony count

SD

Individual revertant colony count

TA 1535

5000

-

7

0.6

7P,8P,7P

1500

-

10

1.2

11P,9P,11P

500

-

13

2.9

15,15,10

150

-

10

1.0

11,9,10

50

-

16

1.5

14,16,17

0

-

12

2.1

10,11,14

Solvent

-

14

2.3

15,11,15

5000

+

7

1.5

6P,7P,9P

1500

+

9

3.1

12P,10P,6P

500

+

10

1.2

11,11,9

150

+

12

6.1

9,19,8

50

+

10

4.0

6,14,11

0

+

10

1.5

12,9,10

Solvent

+

12

4.2

9,11,17

TA 1537

5000

-

7

2.6

6P,5P,10P

1500

-

8

1.2

7P,9P,9P

500

-

14

2.1

16,12,15

150

-

8

1.5

10,7,8

50

-

8

2.1

6,9,10

0

-

14

1.5

16,14,13

Solvent

-

16

3.5

18,12,18

5000

+

8

1.5

6P,9P,8P

1500

+

9

2.0

9P,11P,7P

500

+

11

1.5

9,11,12

150

+

14

5.1

8,18,15

50

+

11

3.1

8,12,14

0

+

10

1.2

11,11,9

Solvent

+

11

1.5

11,10,13

TA 98

5000

-

20

2.0

20P,22P,18P

1500

-

30

5.7

35P,32P,24P

500

-

27

12.5

23,41,17

150

-

28

3.1

25,31,29

50

-

32

7.5

33,39,24

0

-

35

5.8

38,28,38

Solvent

-

30

4.0

34,31,26

5000

+

11

1.0

12P,10P,11P

1500

+

19

5.3

13P,21P,23P

500

+

21

6.1

22,14,26

150

+

21

5.3

25,23,15

50

+

18

2.6

20,19,15

0

+

19

1.0

18,20,19

Solvent

+

19

1.2

20,20,18

TA 100

5000

-

81

2.5

81P,78P,83P

1500

-

104

15.7

97P,122P,93P

500

-

117

13.1

103,129,119

150

-

122

16.5

141,113,112

50

-

119

8.6

111,128,117

0

-

120

4.5

120,116,125

Solvent

-

113

10.0

103,114,123

5000

+

91

11.5

79P,92P,102P

1500

+

103

4.6

98P,104P,107P

500

+

135

6.1

140,128,136

150

+

129

8.5

132,119,135

50

+

125

15.8

139,129,108

0

+

132

9.1

128,142,125

Solvent

+

121

3.1

122,124,118

Positive Control

TA 1535

ENNG

5

-

771

38.4

740,759,814

TA 1537

9-AA

80

-

X

-

X, X, X,

TA 98

NF

1

-

107

11.4

110,116,94

TA 100

ENNG

3

-

378

25.0

361,367,407

TA 1535

2-AA

2

+

100

10.6

96,92,112

TA 1537

2-AA

2

+

74

9.1

81,64,78

TA 98

2-AA

0.5

+

135

17.1

133,119,153

TA 100

2-AA

1

+

348

32.1

351,378,314

P Precipitation

SD Standard deviation

X Too many colonies for accurate counting

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

9 AC 9-aminoacridine

2-AA 2-aminoanthracene

NF 2-nitrofluorene

Table 2: Summary of the Results, Test 2

Strain

Dose Level (µg/plate)

S9 mix

Mean revertant colony count

SD

Individual revertant colony count

TA 1535

5000

_

7

0.6

7P,8P,7P

1500

-

10

3.0

10P,7P,13P

500

-

11

1.0

12,11,10

150

-

9

3.5

13,7,7

50

-

13

2.6

10,15,14

0

-

15

1.5

16,15,13

Solvent

-

14

2.5

17,14,12

5000

+

5

1.5

5P,7P,4P

1500

+

11

2.0

9P,11P,13P

500

10

7

2.1

5,8,9

150

+

14

4.2

15,9,17

50

+

7

2.5

7,4,9

0

+

13

3.6

9,16,14

Solvent

+

14

4.6

18,15,9

TA 1537

5000

-

6

2.1

5P,4P,8P

1500

-

9

1.5

9P,10P,7P

500

-

12

1.5

14,11,12

150

-

9

2.5

7,12,9

50

-

9

3.2

8,13,7

0

-

12

3.6

16,11,9

Solvent

-

14

0.6

14,13,14

5000

+

6

1.0

7P,6P,5P

1500

+

10

0.0

10P,10P,10P

500

+

11

2.6

16,11,9

150

+

10

2.1

12,8,9

50

+

11

0.6

11,11,10

0

+

13

2.0

11,13,15

Solvent

+

15

1.5

13,15,16

TA 98

5000

-

24

6.0

30P,25P,18P

1500

-

23

1.7

24P,24P,21P

500

-

34

4.0

34,30,38

150

-

29

8.3

36,20,32

50

-

31

7.0

32,24,38

0

-

37

4.4

32,39,40

Solvent

-

36

8.0

28,37,44

5000

+

13

1.5

15P,13P,12P

1500

+

17

3.1

20P,16P,14P

500

+

22

6.1

15,23,27

150

+

20

5.3

26,18,16

50

+

17

2.5

17,20,15

0

+

19

2.1

17,21,18

Solvent

+

20

1.5

19,20,22

TA 100

5000

-

89

13.9

85P,77P,104P

1500

-

99

6.7

95P,107P,96P

500

-

135

17.0

118,134,152

150

-

133

13.5

133,120,147

50

-

138

2.1

137,140,136

0

-

142

9.1

146,132,149

Solvent

-

127

18.8

117,149,116

5000

+

107

9.0

116P,98P,108P

1500

+

109

19.1

102P,95P,131P

500

+

139

7.8

141,130,145

150

+

137

3.5

139,133,139

50

+

133

7.8

137,138,124

0

+

148

10.1

153,136,154

Solvent

+

142

13.3

157,134,134

Positive Control

TA 1535

ENNG

5

-

231

70.6

158,235,299

TA 1537

9-AA

80

-

2348

55.5

2292,2403,2350

TA 98

NF

1

-

127

4.7

131,129,122

TA 100

ENNG

3

-

357

12.1

368,344,358

TA 1535

2-AA

2

+

124

9.5

131,127,113

TA 1537

2-AA

2

+

76

7.1

77,68,82

TA 98

2-AA

0.5

+

129

7.0

122,136,129

TA 100

2-AA

1

+

432

48.6

377,450,469

P Precipitation

SD Standard deviation

X Too many colonies for accurate counting

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

9 AC 9-aminoacridine

2-AA 2-aminoanthracene

NF 2-nitrofluorene

Applicant's summary and conclusion

Conclusions:
The test substance did not show any mutagenic effects in any of the tester strains.
Executive summary:

In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), four strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. Precipitation was observed at concentrations of 1500 µg/plate and above. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.

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