Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-06-30 to 2004-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
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: particulate/powder

Method

Target gene:
his-; uvrB-; WP2uvrA-
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:
S9 Mix
Test concentrations with justification for top dose:
Preliminary Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Main Test:
Salmonella without S9-mix: 1,5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Salmonella with S9-mix (except TA1535), E. coli, with and without S9-mix: 5, 15, 50, 150, 500 and 1500 µg/plate.
TA1535 (with S9-mix only): 15, 50, 1513, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to insolubility of the test substance in water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): 1 to 9.9E+006 bacteria per mL

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3 (test concentrations, vehicle control and positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate and/or thinning or disappearance of the background bacterial lawn, or appearance of pinhead colonies in treated cultures.
- Any supplementary information relevant to cytotoxicity: The test material caused a visible reduction in the growth ef the bacterial background lawn to all of the strains, initially at 500 and 150 µg/plate, with and without S9-mix, respectively.
Rationale for test conditions:
Tested up to the toxic limit or, in case of TA1535, experiment 1 only the maximum recommended dose level of 5000 µg/plate (in the presence of S9 only).
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett´s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results
Key result
Species / strain:
other: TA98, TA100, TA1535, TA1537; E.coli WP2uvrA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of 3 plates)

Experiment 1:

 

[TA100]

[TA1535]

[WP2uvrA-]

[TA98]

[TA1537]

Conc.
[unit]

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

 87

 83

 no

 30

14 

 no

19

26

 no

15

23

 no

 15

 19

 no

1.5

 77

 75

 no

 39

 N/T

 no

N/T

28

 no

14

22

 no

 12

 21

 no

5

 71

 86

 no

 44

 9

 no

19

27

 no

17

27

 no

 9

 18

 no

15

 79

 78

 no

 44

 14

 no

21

25

 no

14

26

 no

 11

 18

 no

50

 65

 88

 no

 29

 10

 no

20

28

 no

13

31

 no

 6

 21

 no

150

 42

 92

 yes

 23

 17

 no

21

19

 no

16

27

 yes

 9

 15

 no

500

0

0

yes

0

0

yes

16

0

yes

0

0

yes

0

0

yes

1500

0

N/T

yes

0

0

yes

0

N/T

yes

0

N/T

yes

0

N/T

yes

Positive control

 465

 948

 

 441

294 

 

716

238

 

158

181

 

 979

193

 

Experiment 2:

 

[TA100]

[TA1535]

[WP2uvrA-]

[TA98]

[TA1537]

Conc.
[µg/plate]

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

101

 93

 no

 31

15

 no

27

35

 no

18

28

 no

 11

22

 no

0.5

102

 

no

35

 

no

N/T

 

no

25

 

no

11

 

no

1.5

 100

 

 no

 32

 

 no

N/T

 

 no

24

 

 no

 15

 

 no

5

 113

94

no

 34

 15

no

21

35

no

16

27

no

 18

 20

no

15

 103

 85

no

 29

 16

no

21

27

no

18

32

no

 15

 19

no

50

 93

 94

 no

29

 10

 no

24

26

 no

18

34

 no

 15

 23

 no

150

 94

 87

 no

32

 17

 no

22

33

 no

18

31

 no

 15

 20

 no

500

0

81

yes

0

22

yes

27

23

no

0

29

yes

0

20

yes

1500

N/T

 0

yes

N/T

0

yes

0

0

yes

N/T

3

yes

N/T

0

yes

Positive control

 385

826

 

 211

 248

 

789

332

 

177

228

 

 1020

308

 

Applicant's summary and conclusion

Conclusions:
In the present study according to OECD guideline 471 (adopted 21 July 1997) four Salmonella typhimurium strains and one Escherichia coli strain were incubated with the test substance at the following concentrations: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate. Since the validity criteria of the test were fulfilled and there were no increases in the revertant count up to cytotoxic concentrations, the substance is considered to be non-mutagenic under the described test conditions.Thus, the test substance does not nedd to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) with respect to mutagenicity.