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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 January 25, 2008 to March 17, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

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:
rat or hamster liver S9-mix
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
congo red
methylmethanesulfonate
other: Sodium azide, 2-aminoanthracene

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Remarks:
hamster liver S9-mix.
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Experiment 1 was a direct plate assay with rat liver S9-mix.

Experiment 2 was a preincubation assay with hamster liver S9-mix.

Experiment 3 was performed with tester strain TA100 in the presence of hamster liver S9-mix.

Table 1.Experiment 1: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose

(μg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

881 ± 15

356 ± 97

1002 ± 45

1007 ± 16

1288 ± 76

Solvent control

15 ± 4

4 ± 1

14 ± 3

115 ± 4

24 ± 6

3

 

 

 

95 ± 9

25 ± 4

10

 

 

 

108 ± 19

25 ± 1

33

 

 

 

105 ± 6

20 ± 5

100

9 ± 1

5 ± 1

18 ± 5

111 ± 13

28 ± 11

333

13 ± 3

5 ± 1

20 ± 6

115 ± 11

25 ± 4

1000

11 ± 1

5 ± 2

15 ± 1

109 ± 5

25 ± 5

3330

10 ± 1

3 ± 1

18 ± 3

96 ± 6

35 ± 3

5000

10 ± 1

2 ± 1

18 ± 5

102 ± 7

32 ± 4

With S9-mix

Positive control

220 ± 17

431 ± 32

891 ± 58

886 ± 60

420 ± 36

Solvent control

9 ± 6

5 ± 1

16 ± 2

81 ± 3

30 ± 5

3

 

 

 

73 ± 6

32 ± 6

10

 

 

 

78 ± 10

22 ± 6

33

 

 

 

91 ± 6

34 ± 4

100

12 ± 6

4 ± 1

18 ± 3

102 ± 10

28 ± 8

333

11 ± 2

3 ± 1

15 ± 2

105 ± 16

34 ± 2

1000

13 ± 1

4 ± 2

17 ± 2

113 ± 11

33 ± 7

3330

12 ± 4

3 ± 1

21 ± 1

91 ± 6

35 ± 10

5000

13 ± 3

4 ± 2

18 ± 4

109 ± 7

36 ± 3

Table 2. Experiment 2: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose

(μg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

847 ± 31

185 ± 37

975 ± 41

675 ± 59

240 ± 24

Solvent control

10 ± 3

3 ± 1

17 ± 4

112 ± 6

25 ± 9

100

7 ± 3

3 ± 1

19 ± 2

114 ± 9

22 ± 5

333

19 ± 6

4 ± 1

18 ± 2

97 ± 2

25 ± 4

1000

11 ± 1

4 ± 1

20 ± 2

106 ± 20

20 ± 3

3330

9 ± 5

4 ± 2

21 ± 3

104 ± 11

20 ± 2

5000

12 ± 2

3 ± 1

20 ± 2

85 ± 2

27 ± 2

With S9-mix

Positive control

185 ± 37

187 ± 23

1468 ± 55

833 ± 314

323 ± 41

Positive control (CR)

 

 

381 ± 20

 

 

Solvent control

6 ± 2

5 ± 1

29 ± 5

71 ± 12

28 ± 2

100

5 ± 2

7 ± 3

33 ± 7

66 ± 5

30 ± 1

333

10 ± 2

10 ± 4

35 ± 5

68 ± 4

32 ± 2

1000

6 ± 3

8 ± 4

36 ± 4

80 ± 5

29 ± 4

3330

12 ± 2

7 ± 0

22 ± 7

153 ± 10

27 ± 4

5000

8 ± 3

7 ± 4

24 ± 2

173 ± 17

31 ± 6

CR: Congo red

 

Table 3. Experiment 3: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay

Dose

(μg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) one strains of Salmonella typhimurium

TA100

With S9-mix

Positive control

505 ± 20

Solvent control

80 ± 4

100

84 ± 10

333

90 ± 12

1000

95 ± 5

3330

161 ± 12

5000

194 ± 32

Applicant's summary and conclusion

Conclusions:
According to OECD 471 test method and EU Method B.13/14, Everzol Red CDN Crude is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix in tester stain TA100.
Executive summary:

This test using the procedures outlined in the NOTOX Study Plan for 487480, OECD 471 (OECD, 1997) and EU Method B.13/14 (2000). The results of this test for Everzol Red CDN Crude show that test validity criteria was met.

The test was performed in two independent experiments, at first a direct plate assay was performed with rat liver S9-mix and secondly a preincubation assay was performed with hamster liver S9-mix. To obtain more information about the possible mutagenicity of Everzol Red CDN Crude, an additional experiment was performed with tester strain TA100 in presence of hamster liver S9-mix. Based on thedirect plate assay of the strains TA100 and WP2uvrA, 5000μg/plate was set as the highest dose in this study. In all assay, five doses of Everzol Red CDN Crude at 100, 333, 1000, 3330 and 5000 μg/plate, solvent control and strain-specific positive controls were tested in tester strains TA98, TA100, TA1535, TA1537 and WP2uvrA in triplicate with or without S9 Mix activation (rat or hamster liver S9-mix). No toxicity was observed in all five tester strains up to 5000μg/plate in the absence and presence of metabolite activations. Results showed that Everzol Red CDN Crude induced an up to 2.4-fold, dose-related increase in the number of revertant colonies compared to the solvent control in tester strain TA100.

Based on the results of this study it is concluded that Everzol Red CDN Crude is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix in tester stain TA100.