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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
Reliability:
1 (reliable without restriction)

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: "Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test material (as cited in the study report: FAT 40842/A TE
- Substance type: coloring dye
- Physical state: solid, dark bluish green powder
- Analytical purity: 96%
- Lot/batch No.: Blau DRI 2098 Op 1/07
- Expiration date of the lot/batch: June 30, 2014
- Storage condition of test material: at room temperature at about 20 ºC

Method

Target gene:
see Table 1 "any other information on materials and methods incl. tables"
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
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: NaN3 = sodium azide 4-NOPD = 4-Nitro-o-phenylene-diamine MMS = methyl methane sulfonate 2-AA = 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
For each strain and dose level, including the controls three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates (first experiment):
- 100 μl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μl Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000μl Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.


Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2).
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2) and Prival and Mitchell (1).
1. Prival. M.J. and V.D. Mitchell (1982)
Analysis of a method for testing azo dyes for mutagenic activity in Salmonella
typhimurium in the presence of flavin mononucleotide and hamster liver S9
Mutation Res. 97, 103-116
2. Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973)
Carcinogens are mutagens: a simple test system combining liver homogenates for
activation and bacteria for detection
Proc. Natl. Acad. Sci. (USA) 70, 2281-2285
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant effects by test item
- Effects of osmolality: no significant effects by test item
- Evaporation from medium: no
- Water solubility: low, suspended homogeniously in DMSO
- Precipitation: Precipitation of the test item was observed either in the overlay agar in the test tubes at 5000 Lg/plate and on the incubated agar plates from 333 Lg/plate up to 5000 Lg/plates.
- Other confounding effects: no


COMPARISON WITH HISTORICAL CONTROL DATA:
attached to the report


ADDITIONAL INFORMATION ON CYTOTOXICITY: no
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: reverse mutation assay

Any other information on results incl. tables

- Pre-Experiment/Experiment I

Metabolic

Activation

Test

Group

Dose Level

(µg/plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

23 ± 10

16 ± 5

33 ± 10

137 ± 17

49 ± 4

Untreated

 

 

25 ± 5

13 ± 4

35 ± 8

148 ± 10

57 ± 15

FAT 40842/ATE

3 µg

 

18 ± 5

14 ± 3

35 ± 13

127 ± 7

47 ± 6

 

10 µg

 

16 ± 4

16 ± 1

35 ± 11

127 ± 2

46 ± 3

 

33 µg

 

21 ± 7

18 ± 10

37 ± 8

152 ± 19

47 ± 3

 

100 µg

 

23 ± 8

17 ± 2

45 ± 3

143 ± 12

52 ± 10

 

333 µg

 

23 ± 3P

16 ± 2P

39 ± 7P

146 ± 6P

51 ± 17P

 

1000 µg

 

20 ± 7P

19 ± 4P

48 ± 2P

154 ± 10P

44 ± 4P

 

2500 µg

 

16 ± 1P

20 ± 8P

61 ± 9P

146 ± 24P

51 ± 14P

 

5000 µg

 

18 ± 3P

17 ± 2P

52 ± 13P

158 ± 4P

45 ± 4P

NaN3

10 µg

 

2313 ± 42

 

 

2080 ± 71

 

4-NOPD

10 µg

 

 

 

405 ± 17

 

 

4-NOPD

50 µg

 

 

110 ± 10

 

 

 

MMS

3.0 µL

 

 

 

 

 

1226 ± 92

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

23 ± 4

23 ± 10

48 ± 13

142 ± 13

67 ± 3

Untreated

 

 

22 ± 5

15 ± 2

40 ± 2

149 ± 19

71 ± 10

FAT 40842/ATE

3 µg

 

33 ± 10

31 ± 6

166 ± 21

178 ± 5

70 ± 6

 

10 µg

 

25 ± 8

37 ± 3

173 ± 22

164 ± 25

55 ± 3

 

33 µg

 

22 ± 5

29 ± 6

170 ± 22

163 ± 15

69 ± 9

 

100 µg

 

19 ± 8

24 ± 4

160 ± 26

165 ± 4

47 ± 6

 

333 µg

 

22 ± 2P

22 ± 5P

161 ± 10P

166 ± 15P

48 ± 2P

 

1000 µg

 

22 ± 4P

19 ± 2P

132 ± 10P

164 ± 8P

58 ± 5P

 

2500 µg

 

13 ± 2P

21 ± 1P

155 ± 9P

170 ± 11P

65 ± 2P

 

5000 µg

 

17 ± 4P

25 ± 7P

156 ± 11P

180 ± 16P

65 ± 6P

2-AA

2.5 µg

 

235 ± 15

226 ± 12

1558 ± 66

1628 ± 515

 

2-AA

10.0 µg

 

 

 

 

 

379 ± 133

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

Precipitate

- Experiment I a

Metabolic

Activation

Test

Group

Dose Level

(µg/plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

TA 98

 

 

 

 

 

Without Activation

DMSO

 

 

27 ± 7

Untreated

 

 

40 ± 4

FAT 40842/ATE

3 µg

 

35 ± 2

 

10 µg

 

36 ± 5

 

33 µg

 

39 ± 2

 

100 µg

 

46 ± 6

 

333 µg

 

59 ± 7P

 

1000 µg

 

61 ± 6P

 

2500 µg

 

63 ± 2P

 

5000 µg

 

66 ± 5P M

4-NOPD

10 µg

 

482 ± 38

 

 

 

 

 

With Activation

DMSO

 

 

38 ± 3

Untreated

 

 

38 ± 4

FAT 40842/ATE

3 µg

 

131 ± 10

 

10 µg

 

133 ± 11

 

33 µg

 

143 ± 7

 

100 µg

 

157 ± 19

 

333 µg

 

165 ± 14P

 

1000 µg

 

172 ± 23P

 

2500 µg

 

161 ± 5P

 

5000 µg

 

176 ± 7P M

2-AA

2.5 µg

 

1683 ± 52

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

4-NOPD

2-AA

4-nitro-o-phenylene-diamine

2-aminoanthracene

P

M

Precipitate

Manual count

Applicant's summary and conclusion

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with and without of metabolic activation.
Therefore, FAT 40842/A TE is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The present study (Sokolowski 2009) was performed to investigate the potential of FAT 40842/A TE to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. A confirmatory experiment was performed with and without rat S9 mix with strain TA 98 (reported as experiment Ia). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate

The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation.

A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 40842/A TE in strain TA 98 with and without rat S9 mix.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with and without of metabolic activation.

Therefore, FAT 40842/A TE is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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