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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)
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:
2018
Report date:
2018

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:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
EC Number:
278-207-2
EC Name:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
Cas Number:
75431-69-5
Molecular formula:
C20H12N2O5S.1/3Al
IUPAC Name:
aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)
Constituent 2
Chemical structure
Reference substance name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
EC Number:
213-879-2
EC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Cas Number:
1047-16-1
Molecular formula:
C20H12N2O2
IUPAC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Specific details on test material used for the study:
The dose selection was adjusted to purity

Method

Species / strain
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3.3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Vehicle / solvent:
Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
exposure duration: 72 hours
Replication: 3 plates for each concentration including the controls
DETERMINATION OF CYTOTOXICITY: Evident as 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
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA 98 and TA 1537 at 5000 µg/plate
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: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in the absence and presence of S9 mix in experiment I, from 100 to 5000 µg/plate in the absence of Sp mix in experiment II, and from 333 to 5000 µg/plate in the presence of S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 µg/plate onward under all experimental conditions. The undissolved particles had no influence on the data recording.


Other confounding effects:none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation, except with strain TA 98 in the absence of S9 mix at 5000 µg/plate in experiment I and with strain 1537 in the presence of S9 mix at 5000 µg/plate in experiment I.
Remarks on result:
other:
Remarks:
the test item did not induce gene mutations

Any other information on results incl. tables

Summary of Experiment I

Date plated: 14.11.2017

Date counted: 20.11.2017

 

 

 

Revertant Colony Counts (Mean ± SD)

 

Test Group

Dose Level

(per plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

9 ± 2

8 ± 3

22 ± 4

145 ± 6

29 ± 8

 

Untreated

 

7 ± 1

7 ± 3

26 ± 4

189 ± 5

34 ± 3

 

HPP-Additive Q10

3 μg

11 ± 3

7 ± 2

24 ± 3

145 ± 29

38 ± 5

 

 

10 μg

9 ± 1

10 ± 5

21 ± 3

151 ± 20

35 ± 3

 

 

33 μg

9 ± 2

9 ± 3

24 ± 7

154 ± 10

31 ± 7

 

 

100 μg

13 ± 2

7 ± 3

22 ± 4

146 ± 9

31 ± 8

 

 

333 μg

10 ± 4

7 ± 2

16 ± 3

147 ± 4

32 ± 5

 

 

1000 μg

11 ± 4P

9 ± 2P

22 ± 3P

145 ± 7P

28 ± 1P

 

 

2500 μg

10 ± 3P

7 ± 3P

21 ± 5P

155 ± 14P

34 ± 8P

 

 

5000 μg

13 ± 2P

5 ± 2P M

9 ± 1P M

123 ± 14P M

24 ± 6P

 

NaN3

10 μg

1375 ± 53

 

 

2415 ± 133

 

 

4-NOPD

10 μg

 

 

344 ± 8

 

 

 

4-NOPD

50 μg

 

155 ± 20

 

 

 

 

MMS

2.0 μL

 

 

 

 

1003 ± 12

 

 

 

 

 

 

 

 

With Activation

DMSO

 

12 ± 3

12 ± 3

28 ± 7

143 ± 4

42 ± 5

 

Untreated

 

13 ± 4

9 ± 3

35 ± 5

175 ± 12

50 ± 10

 

HPP-Additive Q10

3 μg

11 ± 5

13 ± 2

33 ± 4

140 ± 5

42 ± 9

 

 

10 μg

12 ± 2

14 ± 4

27 ± 5

138 ± 14

38 ± 6

 

 

33 μg

12 ± 5

11 ± 3

28 ± 2

141 ± 22

36 ± 14

 

 

100 μg

9 ± 3

12 ± 4

32 ± 5

146 ± 16

46 ± 3

 

 

333 μg

11 ± 1

13 ± 2

47 ± 5

134 ± 3

42 ± 6

 

 

1000 μg

10 ± 2P

12 ± 3P

38 ± 10P

149 ± 6P

35 ± 9P

 

 

2500 μg

9 ± 3P

6 ± 1P M

31 ± 6P

114 ± 9P M

36 ± 5P

 

 

5000 μg

10 ± 3P M

2 ± 1P M

18 ± 6P M

89 ± 4P M

43 ± 13P

 

2-AA

2.5 μg

547 ± 56

150 ± 6

3998 ± 289

4679 ± 113

 

 

2-AA

10.0 μg

 

 

 

 

416 ± 33

 

           

Summary of Experiment II

Date plated: 23.11.2017

Date counted: 30.11.2017

 

 

 

Revertant Colony Counts (Mean ± SD)

 

Test Group

Dose Level

(per plate

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

Without Activation

DMSO

 

8 ± 2

8 ± 1

21 ± 2

109 ± 2

30 ± 8

 

Untreated

 

9 ± 3

8 ± 2

21 ± 4

199 ± 13

42 ± 10

 

HPP-Additive Q10

10 μg

11 ± 1

9 ± 2

16 ± 3

88 ± 10

29 ± 8

 

 

33 μg

11 ± 5

8 ± 3

20 ± 6

101 ± 18

30 ± 7

 

 

100 μg

9 ± 5

8 ± 3

17 ± 4

99 ± 7

39 ± 3

 

 

333 μg

11 ± 3

7 ± 3

24 ± 2

96 ± 12

32 ± 9

 

 

1000 μg

9 ± 4P

9 ± 2P

24 ± 6P

101 ± 12P

30 ± 7P

 

 

2500 μg

12 ± 2P

9 ± 3P

18 ± 6P

78 ± 8P

24 ± 7P

 

 

5000 μg

10 ± 2P

8 ± 2P

15 ± 3P

77 ± 20P

24 ± 6P

 

NaN3

10 μg

1170 ± 3

 

 

1474 ± 77

 

 

4-NOPD

10 μg

 

 

328 ± 8

 

 

 

4-NOPD

50 μg

 

188 ± 13

 

 

 

 

MMS

2.0 μL

 

 

 

 

890 ± 13

 

 

 

 

 

 

 

 

With Activation

DMSO

 

12 ± 6

14 ± 2

33 ± 3

95 ± 22

34 ± 6

 

Untreated

 

13 ± 2

18 ± 7

39 ± 4

201 ± 22

49 ± 7

 

HPP-Additive Q10

10 μg

12 ± 3

13 ± 1

30 ± 3

77 ± 11

45 ± 8

 

 

33 μg

13 ± 6

13 ± 5

30 ± 6

76 ± 5

48 ± 6

 

 

100 μg

11 ± 3

11 ± 1

27 ± 4

77 ± 21

38 ± 2

 

 

333 μg

13 ± 2

15 ± 1

34 ± 10

78 ± 19

49 ± 10

 

 

1000 μg

10 ± 5P

12 ± 3P

32 ± 5P

62 ± 6P

45 ± 3P

 

 

2500 μg

11 ± 4P

12 ± 6P

30 ± 3P

77 ± 11P

34 ± 3P

 

 

5000 μg

8 ± 3P

10 ± 2P

23 ± 4P

65 ± 11P

27 ± 4P M

 

2-AA

2.5 μg

413 ± 36

125 ± 5

2919 ± 81

2099 ± 108

 

 

2-AA

10.0 μg

 

 

 

 

389 ± 27

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

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.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using 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:

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

Experiment II:                             10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in the absence and presence of S9 mix in experiment I, from 100 to 5000 µg/plate in the absence of Sp mix in experiment II, and from 333 to 5000 µg/plate in the presence of S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 µg/plate onward under all experimental conditions. The undissolved particles had no influence on the data recording.

 

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.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation, except with strain TA 98 in the absence of S9 mix at 5000 μg/plate in experiment I and with strain TA 1537 in the presence of S9 mix at 5000 μg/plate in experiment I

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.