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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:
2003-03-19 to 2003-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine operon
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 Beta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3 µg/plate - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone

- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 (without activation) 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine 10 µg/plate in TA 98, 50 µg/plate in TA 1537
Remarks:
TA 1537, TA 98 (without activation)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (without activation) 4 µl/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 µg/plate (WP2 uvrA)
Remarks:
All strains (with activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION

- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY

- Method: Background lawn assessment, revertant colony counts
Evaluation criteria:
A result is considered positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA and 3-fold of the solvent control for TA 1535 and TA 1537 in both experiments.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.





Statistics:
No statistical evaluation of the data is required

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
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:
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:
cytotoxicity
Remarks:
5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: With metabolic activation, the number of colonies did not quite reach the lower limit of historical control data in strain TA 1537, (negative and solvent control, exp 1), and in strain WP2 uvrA (negative control exp 1and 2, solvent control exp 1). Since these deviations are rather small, these effects are considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Any other information on results incl. tables

Table 2:  Pre-toxicity test

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

Negative control

23

29

No

108

107

No

10

12

No

0*

26

29

No

134

113

No

9

13

No

3

16

30

No

101

106

No

13

9

No

10

19

30

No

114

127

No

14

9

No

33

21

26

No

115

104

No

12

13

No

100

19

28

No

88

121

No

12

10

No

333

20

27

No

68

81

No

9

10

No

1000

16

26

No

76

84

No

9

10

No

2500

18

23

No

73

67

No

8

7

No

5000

17

20

No

52

58

Yes

5

6

Yes

Positive control

228

1495

No

1068

1629

No

697

211

No

*solvent control with acetone

Table 2:  Pre-toxicity test

 

TA1537

E. coli WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

Negative control

7

6

No

31

32

No

0*

4

5

No

29

31

No

3

3

5

No

28

25

No

10

5

5

No

28

34

No

33

5

5

No

27

33

No

100

4

7

No

32

34

No

333

3

4

No

30

27

No

1000

2

3

No

30

35

No

2500

3

3

No

31

32

No

5000

1

2

Yes

18

35

No

Positive control

50

226

No

804

215

No

*solvent control with acetone

Table 3: Experiment 1 Plate incorporation number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

Negative control

23

29

No

108

107

No

10

12

No

0*

26

29

No

134

113

No

9

13

No

33

21

26

No

115

104

No

12

13

No

100

19

28

No

88

121

No

12

10

No

333

20

27

No

68

81

No

9

10

No

1000

16

26

No

76

84

No

9

10

No

2500

18

23

No

73

67

No

8

7

No

5000

17

20

No

52

58

Yes

5

6

Yes

Positive control

228

1495

No

1068

1629

No

697

211

No

*solvent control with acetone

Table 3: Experiment 1 Plate incorporation - number of revertants per plate (mean of 3 plates)

 

TA1537

E. coli WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

Negative control

7

6

No

31

32

No

0*

4

5

No

29

31

No

33

5

5

No

27

33

No

100

4

7

No

32

34

No

333

3

4

No

30

27

No

1000

2

3

No

30

35

No

2500

3

3

No

31

32

No

5000

1

2

Yes

18

35

No

Positive control

50

226

No

804

215

No

*solvent control with acetone

Table 4: Experiment 2 Pre-incubation number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

Negative control

30

38

No

148

184

No

20

18

No

0*

28

35

No

154

187

No

17

17

No

33

30

40

No

150

199

No

17

19

No

100

28

40

No

146

214

No

17

16

No

333

23

32

No

177

198

No

18

16

No

1000

24

37

No

195

216

No

17

17

No

2500

28

35

No

188

242

No

21

15

No

5000

31

44

No

207

215

No

16

17

No

Positive control

239

1465

No

1536

1207

No

1479

299

No

*solvent control with acetone

Table 4: Experiment 2 Pre-incubation number of revertants per plate (mean of 3 plates)

 

TA1537

E. coli WP2 uvrA

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

Negative control**

9

11

No

38

33

No

0*

9

11

No

36

38

No

33

10

13

No

28

35

No

100

7

11

No

39

36

No

333

5

14

No

42

46

No

1000

6

10

No

31

46

No

2500

6

11

No

41

46

No

5000

5

12

No

40

41

No

Positive control

77

227

No

444

346

No

*solvent control with acetone

Applicant's summary and conclusion

Conclusions:
The substance RMS 808 MC has been tested according to OECD 471 and under GLP. No increase in the number of revertants was observed for the test substance tested up to cytotoxic or limit concentrations in any of the test strains either with or without metabolic activation in either the initial plate incorporation assay or the independent pre-incubation experiment . It is concluded that the test substance is not mutagenic to bacteria under the conditions of the test.