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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-01-09 to 2001-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Method

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 (liver post mitochondrial fraction of rats induced with phenobarbitone/ beta-naphtoflavone)
Test concentrations with justification for top dose:
- First mutagenicity experiment with and without S9 mix: 0, 50, 150, 500, 1500 and 5000 µg/plate
- Second mutagenicity experiment with and without S9 mix: 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
vehicle used: Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity and second mutagenicity tests with or without S9 mix
- Exposure duration: 48h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C for Salmonella strains.
agar containing traces of tryptophan and biotin, maintained at 45°C for Escherichia coli.

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.
Evaluation criteria:
After incubation for 48 hours at 37 °C in the dark, colonies were counted
Statistics:
No statistical analysis performed

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity
Remarks:
Tested up to limit concentrations recommended by the test guideline
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Tested up to limit concentrations recommended by the test guideline
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 - Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

- A white precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies

- No toxicity was noted at any dose-level in any strain.

- The test item did not induce any noteworthy increase in the number of revertants, in any of the strains, either with or without S9 mix.

Table 7.6.1/1 Experiment 1- Without metabolic activation

 

With or Without S9-Mix

 

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

  TA 100  TA 1535

WP2uvrA-

 TA 98

TA 1537

 

-

 

0

102      (91) 

86 9.2

86 

12      (12)12 0.6

13

39       (28)

25 9.8

20

40       (28)

23 10.8

20

7         (6)

9 3.6

2         

 

-

 

50

95       (94)

87 6.6

100      

13       (13)

13 0.0

I3       

25       (29)

30 3.2

31   

18       (21)

22 2.6

23 

7         (6)

4 2.1

8        

 

-

 

150

85       (86)

84 2.1

88        

13       (13)

12 1.0

14        

22       (26)

34 6.9

22       

22       (18)

18 4.5

13       

3         (3)

3 0.0

3       

 

-

 

500

92       (93)

97 3.2

91       

14       (13)

12 1.2

12       

20       (23)

25 2.5

23       

18       (19)

19 0.6

19       

4         (6)

6 1.5

7        

-

 

1500

92       (87)

79 7.0

90       

15      (13)

12 2.1

11

22        (25)

31 4.9

23

16       (21)

24 4.6

24

6         (7)

5         2.1

9

 

-

 

5000

88P (95)

89P 11.8

109P      

llP (11)

11P 0.0 11P       

23P (26)

25P 4.2

31P      

24P (33) 38P      

38P       8.1

9P       (9)

9P 0.0

9P       

 

Positive controls

 

S9-Mix

-

 

Name

 

Concentration

(µg/plate)

 

No.colonies per plate

 

ENNG

 

ENNG

 

ENNG

 

4NQO

 

9AA

 

3

 

5

 

2

 

0.2

 

80

467      (479)

498       16.4

473

249     (240)

213      24.2

259

811     (802)

769      29.5

826

107     (107)

106      0.6

107

1801   (1826)

1649    190.2

2027

Table 7.6.1/2 Experiment 1- With metabolic activation

 

With or without S9-Mix

 

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

TA 100 

TA 1535  WP2uvrA-

   TA98

 

+

 

0

147     (131)

124

123     13.6

17       (16)

14

17       1.7

22       (23)

22

25        1.7

43       (31)

26

25       10.1

6         (7)

6

10       2.3

 

+

 

50

125     (123)

120

125      2.9

17       (16)

16

15       1.0

31       (26)

23

25        4.2

35      (29)

30

21        7.1

11       (9)

8

7         2.1

 

+

 

150

85       (92)

102

90       8.7

17       (17)

18

17       0.6

23      (27)

33

26        5.1

31      (33)

37

31        3.5

8         (6)

2

9         3.8

 

+

 

500

.134     (115)

109

103      16.4

10      (12)

13

12        1.5

30       (29)

31

27        2.1

24       (29)

28

34        5.0

7         (9)

10

Il       2.1

 

+

 

1500

112     (89)

96

60      26.6

10       (12)

16

11       3.2

24       (23)

21

25       2.1

27       (29)

29

31       2.0

7         (6)

5

7         1.2

 

+

 

5000

l01P     (96)

l24P

64P      30.3

16P     (16)

16P

16P     0.0

28P      (26)

22P

27P       3.2

26P      (32)

34P

37P       5.7

5P        (6)

5P

8P        1.7

 

Positive controls

 

S9-Mix

 

+

 

Narne

 

Concentration

(µg/plate)

 

No.colonies per plate

 

2AA

 

2AA

 

2AA

 

BP

 

2AA

 

1

 

2

 

10

 

5

 

2

1855   (1740)

1809    160.4

1557

472     (388)

357      73.6

335

965    (1024)

1029    56.2

1077

277     (240)

216      32.5

227

346    (467)

537     105.5

519

Table 7.6.1/3 Experiment 2- Without metabolic activation

 

With or without S9-Mix

 

Test substance

concentration (µg/plate)

Number of revertants (mean number of colonies per plate)

 TA 100  TA 1535

WP2uvrA-

TA 98

TA 1537  

 

-

 

0

157     (146)

133

149     12.2

16      (19)

15

26 6.1

45       (36)

35

28 8.5

45       (41)

39

40 3.5

20       (15)

14       4.2

12

-

 

50

150    (140)

135

134      9.0

20       (19)

17

20        1.7

48      {37)

33

31       9.3

29      (36)

45

33       8.3

9        (15)

18

17       4.9

-

 

150

160     (147)

128

154     17.0

24       (21)

15

25        5.5

.31        (32)

29

37        4.2

45       (48)

42

57       7.9

7        (13)

17

16       5.5

-

 

500

158     (146)

141

140      10.1

16      (18)

14

25       5.9

28      (35)

38

40        6.4

46      (53)

54

58       6.1

17      (19)

20

19        1.5

-

 

1500

122     (124)

129

122      4.0

30       (24)

15 7.8

26

39    (37)

38 3.2

33

39      (40)

40

40 0.6

20       (19)

18

19 1.0

 

-

 

5000

121P  (128)

114P    18.0 148P

19P      (17)

15P 2.1

16P

32P      {36)

41P       4.6

35P

44P      (35)

32P 7.9

29P

12P     (12)

14P 2.5 9P

 

Positive controls

 

S9-Mix

 

-

 

Name

 

Concentration

g/p1ate)

 

No.colonies per plate

 

ENNG

 

ENNG

 

ENNG

 

4NQO

 

9AA

 

3

 

5

 

2

 

0.2

 

80

663     (680)

697      17.0

681

279     (255)

266      30.4

221

639     (596)

606     49.3

542

109    (108)

113      5.0

103

946     (881)

829     59.7

867

Table 7.6.1/4 Experiment 2- With metabolic activation

 

With or without S9-Mix

 

Test substance concentration g/plate)

Number of revertants (mean number of colonies per plate)

 TA 100  TA 1535

WP2uvrA-

 TA 98

TA 1537

 

+

 

0

137     (146)

154

148     8.6

22       (19)

19

17       2.5

42       (44)

43

47        2.6

54       (53)

52        1.4

C

15       (16)

14

20        3.2

 

+

 

50

151     (143)

152

127     14.2

17      (26)

31

29        7.6

39       (41)

49

35       7.2

58       (53)

53

49       4.5

17       (15)

10

19       4.7

 

+

  150

148     (144)

148

137      6.4

19       (24)

24

30       5.5

45       (44)

47

39        4.2

57       (57)

57

57       0.0

13       (19)

21

22        4.9

 

+

 

500

125     (134)

128

149      13.1

24       (22)

21

21        1.7

52  (38)

34

28       12.5

64      (61)

58

61        3.0

19       (l5)

14

13        3.2

 

+

 

1500

119     (130)

132

138      9.7

26       (27)

21

33        6.0

38       (41)

43

41       2.5

55

43 (54)

63       10.1

10      (14)

20

13        .5.1

 

+

 

5000

130P    (136)

155P

124P     16.4

24P      (23)

25P      2.1

21P

26P      (32)

30P

41p      7.8

57P      (59)

64P      4.4

56P

llP       (11)

l2P

llP        0.6

 

Positive controls

 

S9-Mix

 

+

 

Name

 

Concentration

g/plate)

 

No.colonies per plate

 

2AA

 

2AA

 

2AA

 

BP

 

lAA

 

1

 

2

 

10

 

5

 

2

2202   (2240)

2286     42.6

2232

308     (295)

286      11.4

292

991     (985)

940      42.3

1024

240     (236)

220      14.0

247

570     (560)

552       9.1

559

2AA: 2 -Aminoanthracene

BP: Benzo(a)pyrene

P: Precipitate

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

4NQO: 4 -Nitroquinoline-1 -oxide

9AA: 9 -Aminoacridine

C: Contaminated

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The potential of the substance to induce reverse mutation in bacteria was assessed using four strains of Salmonella typhimurium and Escherichia Coli WP2 uvrA according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study (all doses were expressed in pure substance). The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with phenobarbitone and beta-naphtoflavone.

Both experiments were performed according to the direct plate incorporation. After 48 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The preliminary assays with and without metabolic activation showed that the test substance was freely soluble and non-toxic in all tested strains. The highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines with a dose range of 50 to 5000 µg/plate.

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

A white precipitate was observed in the petri plates at 5000 µg/plate, this did not prevent the scoring of the colonies. No reduction in the growth of the bacterial background lawn was observed.

In the two independent assays, no significant increase in the mean number of revertants was noted in the bacterial strains tested in the presence of the test substance neither with nor without metabolic activation.

Under these experimental conditions, the substance did not show any mutagenic activity in the bacterial reverse mutation test.