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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:
02-Jul-2001 to 26-Jul-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-AA used as positive control +S9)
Qualifier:
according to
Guideline:
other: Japanese Guidelines on Industrial Chemicals
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
brown liquid
Details on test material:
The tested salt was reported to be the 7-sodium salt.

Method

Target gene:
histidine (Salmonella typhimurium)
tryptophan (Escherichia coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and 5,6-benzoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Test 1-1: 78-5000 µg mixed sodium salts/plate -S9, 156-5000 µg/plate +S9
Test 1-2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium
Test 2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium, 78-5000 µg mixed sodium salts/plate -S9 for E coli, 156-5000 µg mixed sodium salts/plate +S9
20-5000ug/plate -S9; 156-5000ug/plate +S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9; TA98, 0.1 µg/plate; TA100, 0.01 µg/plate
Positive control substance:
furylfuramide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 ; TA1535, 0.5 µg/plate
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9; TA1537, 80 µg/plate
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9, WP2 uvrA, 2 µg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9; TA98, 0.5 µg/plate; TA100, 1 µg/plate; TA1535, TA1537, 2 µg/plate; WP2 uvrA, 10 µg/plate
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition
Evaluation criteria:
Posltive: mean number of revertant colonies more than double the negatlve control value and signlflcant concentration-dependent Increase
Statistics:
No statistical analysis performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 625 µg mixed sodium salts/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=1250 µg mixed sodium salts/plate -S9
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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations of 5-5000 µg/plate
- No growth inhibition in any strain
- Bacteriostatics observed at 1250 µg/plate -S9 in all strains
- No precipitation

COMPARISON WITH HISTORICAL CONTROL DATA:
- Negative/solvent control: within the range of historical data
- Positive controls: within the range of historical data

Any other information on results incl. tables

All treated cultures had less than 2-fold increase in mutant frequency and no evidence of a dose-response.  All positive controls gave a greater than 2-fold increase in mutant numbers.

Table 1 Experiment 1 Mean number of revertants (average of 3 plates)

 

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

102

10

23

14

7

78

-

102

8

21

16

9

156

-

88

7

18

16

6

313

-

71

5

18

8

1

625

-

0

0

7

0

0

1250

-

0

0

0

0

0

2500

-

0

0

0

0

0

5000

-

0

0

0

0

0

Positive control

-

528

422

631

559

194

Control

+

111

11

25

24

6

156

+

133

11

30

24

10

313

+

148

11

21

22

6

625

+

140

9

23

17

6

1250

+

122

6

21

11

3

2500

+

108

4

15

9

3

5000

+

98

5

9

8

2

Positive control

+

501

260

648

446

154

Mean number or revertants 16-19.07.2001

Table 2 Experiment 2 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

104

11

-

16

7

20

-

101

6

-

16

8

39

-

94

8

-

12

8

78

-

106

8

-

14

8

156

-

97

9

-

14

5

313

-

81

6

-

14

4

1250

-

0

0

-

0

0

5000

-

0

0

-

0

0

Positive control

-

522

411

-

604

211

 

Table 3 Experiment 3 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

99

8

20

21

7

20

-

105

10

-

18

7

39

-

107

11

-

19

6

78

-

110

10

22

19

6

156

-

103

11

14

17

7

313

-

86

8

16

15

4

625

-

-

-

13

-

-

1250

-

0

0

0

0

0

2500

-

-

-

0

-

-

5000

-

0

0

0

0

0

Positive control

-

510

419

655

601

183

Control

+

127

10

21

23

8

156

+

130

9

29

26

9

313

+

132

12

17

19

7

625

+

127

12

22

18

6

1250

+

114

8

15

14

7

2500

+

119

6

13

6

3

5000

+

103

6

12

3

2

Positive control

+

679

304

758

517

152

 

Applicant's summary and conclusion

Conclusions:
In a reliable study, conducted according to OECD guideline 471, no genotoxicity was seen in a bacterial mutagenicity assay. The study was performed in compliance with GLP.