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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:
2016 -04-19 till 2016-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)
EC Number:
276-538-7
EC Name:
Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)
Cas Number:
72252-58-5
Molecular formula:
C23H8Cr2N6O16S3.4Na
IUPAC Name:
tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9 in experiment I and non-induced hamster liver S9 in experiment II
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I and experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [deionised water
- Justification for choice of solvent/vehicle: best suitable solvent
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 30 min
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
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:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in strain TA 1535, TA 1537 and TA 98
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: yes
- Precipitation:No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 / / / 5000
TA 1537 / 5000 5000 /
TA 98 / 5000 / /
TA 100 / / / /
WP2 uvrA / / / /

Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1763502

Study Code: Envigo 1763502

Experiment: 1763502 VV Plate

Date Plated: 19.04.2016

Assay Conditions:

Date Counted: 26.04.2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

Without Activation

Deion. water

 

11 ± 4

9 ± 2

29 ± 4

186 ± 16

54 ± 6

Untreated

 

12 ± 3

9 ± 4

32 ± 6

157 ± 14

52 ± 11

Sanodal-

3 µg

9 ± 1

10 ± 3

34 ± 4

172 ± 4

49 ± 11

Schwarz 2LW

10 µg

15 ± 5

7 ± 2

26 ± 5

172 ± 17

51 ± 7

 

33 µg

11 ± 1

9 ± 1

27 ± 6

157 ± 6

41 ± 4

 

100 µg

13 ± 6

11 ± 5

28 ± 6

166 ± 28

61 ± 6

 

333 µg

12 ± 3

6 ± 1

29 ± 3

168 ± 12

48 ± 5

 

1000 µg

10 ± 2D M

8 ± 4D M

26 ± 7D M

149 ± 4D M

43 ± 6D M

 

2500 µg

10 ± 3D M

8 ± 3D M

19 ± 5D M

146 ± 7D M

36 ± 4D M

 

5000 µg

8 ± 2D M

7 ± 1D M

15 ± 1D M

135 ± 5D M

36 ± 3D M

NaN3

10 µg

1382 ± 52

 

 

2415 ± 69

 

4-NOPD

10 µg

 

 

403 ± 23

 

 

4-NOPD

50 µg

 

75 ± 7

 

 

 

MMS

2.0 µL

 

 

 

 

1075 ± 78

 

 

 

 

 

 

 

 

With Activation

Deion. water

 

10 ± 2

9 ± 3

40 ± 6

170 ± 20

63 ± 5

Untreated

 

13 ± 4

10 ± 5

45 ± 7

165 ± 20

61 ± 14

Sanodal-

3 µg

10 ± 1

11 ± 4

44 ± 6

167 ± 8

62 ± 8

Schwarz 2LW

10 µg

12 ± 5

9 ± 3

48 ± 6

172 ± 18

53 ± 10

 

33 µg

8 ± 2

11 ± 2

37 ± 5

172 ± 16

52 ± 4

 

100 µg

10 ± 0

8 ± 3

40 ± 10

173 ± 21

69 ± 11

 

333 µg

9 ± 5

11 ± 4

34 ± 2

174 ± 25

54 ± 13

 

1000 µg

10 ± 1D M

8 ± 2D M

27 ± 4D M

135 ± 6D M

42 ± 1D M

 

2500 µg

7 ± 3D M

5 ± 2D M

23 ± 6D M

115 ± 8D M

40 ± 2D M

 

5000 µg

5 ± 2D M

3 ± 1D M

17 ± 2D M

106 ± 7D M

30 ± 4D M

 

2-AA

2.5 µg

415 ± 21

219 ± 43

6586 ± 361

4872 ± 201

 

 

2-AA

10.0 µg

 

 

 

 

414 ± 15

 

 

 

 

 

 

 

 

 

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

D

M

Densely coloured plate

Manual count

 


 

Table2            Summary of Experiment II

Study Name: 1763502

Study Code: Envigo 1763502

Experiment: 1763502 HV2 Pre

Date Plated: 12.05.2016

Assay Conditions:

Date Counted: 18.05.2016

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

Without Activation

Deion. water

 

10 ± 1

13 ± 3

29 ± 2

146 ± 22

51 ± 9

Untreated

 

12 ± 7

12 ± 2

32 ± 8

169 ± 11

52 ± 7

Sanodal-

3 µg

11 ± 1

11 ± 1

26 ± 9

161 ± 8

41 ± 1

Schwarz 2LW

10 µg

12 ± 6

10 ± 2

25 ± 6

167 ± 13

42 ± 2

 

33 µg

13 ± 3

12 ± 6

28 ± 2

148 ± 22

50 ± 5

 

100 µg

13 ± 3

11 ± 3

27 ± 12

154 ± 8

37 ± 3

 

333 µg

14 ± 4P

10 ± 1P

32 ± 9P

167 ± 7P

32 ± 8P

 

1000 µg

7 ± 3P M

8 ± 2P M

16 ± 2P M

159 ± 6P M

29 ± 4M P

 

2500 µg

7 ± 2P M

8 ± 2P M

14 ± 2P M

157 ± 14P M

28 ± 7M P

 

5000 µg

7 ± 2P D M

4 ± 1P D M

15 ± 3P D M

143 ± 6P D M

28 ± 9M P D

NaN3

10 µg

1140 ± 35

 

 

1849 ± 49

 

4-NOPD

10 µg

 

 

447 ± 25

 

 

4-NOPD

50 µg

 

72 ± 12

 

 

 

MMS

2 µL

 

 

 

 

879 ± 65

 

 

 

 

 

 

 

 

With Activation

Deion. water

 

18 ± 4

25 ± 3

50 ± 2

140 ± 32

46 ± 4

Untreated

 

18 ± 4

23 ± 9

57 ± 10

123 ± 19

50 ± 11

Sanodal-

3 µg

22 ± 0

29 ± 2

57 ± 3

147 ± 14

51 ± 3

Schwarz 2LW

10 µg

20 ± 4

30 ± 6

55 ± 13

150 ± 11

48 ± 3

 

33 µg

22 ± 6

31 ± 10

55 ± 6

177 ± 13

42 ± 4

 

100 µg

13 ± 1

30 ± 6

49 ± 8

167 ± 32

45 ± 11

 

333 µg

16 ± 5P

29 ± 4P

51 ± 4P

143 ± 7P

49 ± 4P

 

1000 µg

13 ± 3P M

30 ± 8P M

36 ± 3P M

139 ± 12P M

31 ± 5P M

 

 

2500 µg

11 ± 3P M

21 ± 3P M

29 ± 4P M

132 ± 11P M

33 ± 8P M

 

 

5000 µg

8 ± 2P D M

18 ± 3P D M

23 ± 3P D M

123 ± 2P D M

26 ± 9P D M

 

2-AA

2.5 µg

 

 

 

963 ± 99

 

 

2-AA

2.5 µg

329 ± 27

115 ± 14

 

 

 

 

2-AA

10 µg

 

 

 

 

1043 ± 60

 

Congo red

500 µg

 

 

328 ± 10

 

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

Congo red

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

Congo red

methyl methane sulfonate

P

M

D

Precipitate

Manual count

Densely coloured plate

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.

Furthermore in Experiment II precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

/

/

/

5000

TA 1537

/

5000

5000

/

TA 98

/

5000

/

/

TA 100

/

/

/

/

WP2 uvrA

/

/

/

/

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanodal-Schwarz 2LW at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

 

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

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