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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:
From February 28, 2000 to March 25, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: paste
Details on test material:
- Purity: approximately 79%
- Lot/batch No.: 876 TK
- Physical state: off white paste
- Storage condition of test material: room temperature, in the dark
Specific details on test material used for the study:
-an allowance for test material purity was made prior to each days formulation.

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
Experiment 1:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
15, 50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A

Experiment 2:
5, 15, 50, 150, 500 and 1500 µg/plate for the strains S. typhimurium TA 1535, TA 98 and TA 100
50, 150, 500, 1500 and 5000 µg/plate for E. coli WP2 uvr A
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537

Experiment 3:
15, 50, 100, 150, 200 and 500 µg/plate for the strain S. typhimurium TA 1537
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl formamide

Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(Dimethyl formamide)
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Positive controls:
yes
Remarks:
(without metabolic activation)
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
other: 2-aminoanthracene
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP uvrA
Positive controls:
yes
Remarks:
(with metabolic activation)
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Evaluation criteria:
The test material was considered positive in this test system if there is a reproducible dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression

Results and discussion

Test resultsopen allclose all
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 500 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: E. coli WP2 uvr A, TA1535, TA1537, TA98, TA100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at and above 500 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation of the test substance was noted at the dose levels tested.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity study was performed with strains TA100 and WPuvrA with and without metabolic activation. Ten concentrations of the test material and a vehicle control (dimethyl formamide) were tested. The dose range of the test material was: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number, within the range of historical data, of spontaneous revertants per plate in the vehicle and untreated controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1

 

Table 1: without metabolic activation

With or without S9-Mix

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  

TA100             TA1535        WP2uvrA

Frameshift type

   

    TA98         TA1537

-

0

146

10.1 #

22

2.9

19

3.1

20

6.1

9

0.6

-

5

144

12.5

20

3.1

 

N/T

20

3.8

10

1.5

-

15

145

11.4

14

4.2

23

3.8

25

2.3

14

4.4

-

50

143

7.6

12

2.1

16

7.0

28

8.4

16*

3.8

-

150

112

4.0

13

2.3

24

3.1

25

4.0

18$$$

2.3

-

500

23

3.2

3

2.0

22

9.3

6

2.0

3V

1.7

-

1500

0V

0.0

0V

0.0

20

3.5

0V

0.0

0T

0.0

-

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

Name

ENNG

ENNG

ENNG

4NQO

9AA

S9-Mix

Concentration

(µg/plate)

3

5

2

0.2

80

 

Nº Colonies per plate

425

56.4

372

1.5

615

77.6

111

12.3

807

226.2

 

ENNG  N-ethyl-N'-nitro-N-nilrosoguanidine N/T  Not tested at this dose level

4NQO  4-Nitroquinoline-l-oxide                                   S  Sparse bacterial background lawn

9AA    9-Aminoacridine                                           T  Toxic, no bacterial background lawn

V        Very weak bacterial background lawn            $$$ p¿0.005

#         Standard deviation                                         *    p¿ 0,05         

 

 Table 2: with metabolic activation 

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

TA100           TA1535      WP2uvrA

Frameshift type

 

  TA98               TA1537

+

0

151

18.6#

13

2.1

22

0.0

24

3.6

7

1.0

+

5

174

51.0

14

1.5

N/T

21

3.2

12

2.0

+

15

132

13

11

5.1

22

1.2

29

2.5

9

3.1

+

50

144

11

12

2.1

19

4.5

30

8.3

13

5.3

+

150

154

14.6

14

1.7

23

1.0

27

3.6

13

4.2

+

500

38

5.3

5

4.0

27

6.1

17

7.2

4

1.7

+

1500

0V

0.0

0V

0.0

20

5.5

0V

0.0

0V

0.0

+

5000

N/T

N/T

0V

0.0

N/T

N/T

Positive controls

S9-Mix

+

 

 

 

Name

Concentration µg/plate)

Nº colonies per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

2122

505.5

212

41.0

479

7.6

231

25.4

546

60.5

 BP        Benzo(a)pyrene                                        N/T   Not testedat this dose level

2AA     2-Aminoanthracene                                      S      Sparse bacterial background lawn

V          Very weak bacterial background lawn           #      Standard deviation

*           p¿ 0,05

 

 Experiment 2

 

Table 3: without metabolic activation

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

 

 TA100           TA1535     WP2uvrA

Frameshift type

    

  TA98           TA1537

-

0

81

12.3#

19

1.7

18

6.1

15

2.3

9

5.3

-

5

89

1.0

16

3.2

N/T

14

2.1

N/T

-

15

79

15.0

15

6.2

N/T

14

1.5

9

1.2

-

50

85

3.2

22

7.0

20

9.8

16

2.3

10

3.8

-

100

N/T

N/T

N/T

N/T

13

3.2

-

150

76

9.6

15

1.2

17

3.8

15

3.1

9

4.4

-

200

N/T

N/T

N/T

N/T

6

1.5

-

500

10S

6.0

3S

1.0

16

3.8

6S

3.1

0V

0.0

-

1500

0V

0.0

0V

0.0

14

3.6

0V

0.0

N/T

-

5000

N/T

N/T

2

1.7

N/T

N/T

Positive controls

S9-Mix

-

 

 

Name

 

ENNG

ENNG

ENNG

4NQO

9AA

Concentration

(µg/plate)

3

5

2

0.2

80

No. colonies per plate

 

359

12.2

269

35.5

817

40.6

89

8.4

940

164.2

 

ENNG   N-ethyl-N'-nitro-N-nitrosoguanidine                  N/T   Not tested at this dose level

4NQO   4-Nttroquinoline-l-oxide                                   S      Sparse bacterial background lawn

9AA      9-Aminoacridine                                            V      Very weak bacterial background lawn

#          Standard deviation

 

Table 4: with metabolic activation

With or without

S9-Mix

 

Test substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

  TA100        TA1535       WP2uvrA

Frameshift type

TA98        TA1537

+

0

93

4.0#

12

4.5

21

2.6

22

3.1

10

4.2

+

5

97

8.2

13

2.3

N/T

23

4.6

N/T

+

15

83

11.8

12

1.7

N/T

17

3.5

11

3.1

+

50

91

12.1

12

1.0

20

2.5

29

9.2

10

2.0

 

100

N/T

N/T

N/T

N/T

13

1.7

+

150

82

12.7

13

2.1

20

3.5

31

12.4

17

6.6

+

200

N/T

N/T

N/T

N/T

11

4.6 

+

500

30

10.8

4S

1.7

19

5.3

11S

4.2

5S

2.5

+

1500

0V

0.0

0V

0.0

13

3.1

0V

0.0

N/T

+

5000

N/T

N/T

2S

2.1

N/T

N/T

Positive controls

S9-Mix

+

 

 

Name

 

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

929

68.6

128

36.0

708

24.3

174

36.3

435

56.5

 

BP       Benzo(a)pyrene                      N/T   Not tested at this dose level

2AA     2-Aminoanthracene                  S      Sparse bacterial background lawn

#         Standard deviation                    V      Very weak bacterial background lawn

 

 

Experiment 3

Table 5: with and without metabolic activation

Test substance concentration µg/plate)

Number of revertants (mean number of colonies per plate)

Frameshift type

(Without metabolic activation)

TA1537

Frameshift type

(With metabolic activation)

TA1537

0

8

3.2#

12

3.1

15

9

2.0

11

4.2

50

10

1.5

13

2.6

100

9

0.7

13

4.5

150

11

3.2

13

3.5

200

2S

1.7

14

4.0

500

0V

0.0

10S

7.8

Name

9AA

2AA

Concentration (µg/plate)

80

2

No. colonies per plate

940

87.0

399

137.2

9AA     9-Aminoacridine

2AA     2-Arninoanthracene

S         Sparse bacterial background lawn

V         Very weak bacterial background lawn

#          Standard deviation

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test and therefore is not to be classified according to CLP Regulation.
Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance was performed with five histidine dependents bacteria, four strains of Salmonella typhimurium T1535, TA98, TA100, and TA1537 and E.Coli WP2 uvrA. Plate Incorporation Method was carried out at the concentrations of 5000, 1500, 500, 150, 50 and 15 µg/plate for the strains of Salmonella typhimurium in the presence and absence of metabolic activation and at the concentrations of 1500, 500, 150, 50, 15 and 5 µg/plate for the E.coli WP2 uvrA in the presence and absence of metabolic activation.The dose range for TA1537 (with/without S9) was modified slightly, based on results observed in the Experiment 1 and was 500, 200, 100, 50 and 15 µg/plate. Intermediate doses were included to allow for small but statistically significant increases observed in the first experiment. The test material caused a visible reduction in the growth of the bacterial lawn to all of the bacterial tester strains both with and without metabolic activation. The toxicity of the test material to the bacterial tester strains varied both between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Very small, statistically significant increases were observed to TA1537 in Experiment 1 only both with and without S9. However, these increases were not considered to be biologically significant as they were within the strains historical range and were non-reproducible over three separate experiments. According to the obtained results, the test substance was, therefore, considered to be non-mutagenic under the conditions of this test.