Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
1-(methylamino)anthraquinone
EC Number:
201-417-2
EC Name:
1-(methylamino)anthraquinone
Cas Number:
82-38-2
Molecular formula:
C15H11NO2
IUPAC Name:
1-(methylamino)anthraquinone
Test material form:
solid

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
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: best suitable solvent, because of its solubility proper¬ties 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:
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)


- 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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH 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 all strains with and without metabol¬ic activation.
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: 1778902

Study Code: Envigo 1778902

Experiment: 1778902 VV Plate

Date Plated: 04.07.2016

Assay Conditions:

Date Counted: 07.07.2016

 

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

DMSO

 

 

17 ± 1

13 ± 4

19 ± 3

110 ± 10

45 ± 7

Untreated

 

 

15 ± 1

9 ± 2

27 ± 1

133 ± 9

44 ± 8

Test item

3 µg

 

12 ± 3

12 ± 3

19 ± 4

112 ± 15

46 ± 5

10 µg

 

13 ± 4

15 ± 1

23 ± 4

110 ± 13

45 ± 5

 

33 µg

 

16 ± 5

14 ± 2

31 ± 6

122 ± 5

39 ± 3

 

100 µg

 

16 ± 3

16 ± 4

30 ± 4

115 ± 8

39 ± 4

 

333 µg

 

12 ± 1

11 ± 1

23 ± 4

113 ± 8

40 ± 4

 

1000 µg

 

17 ± 3P

16 ± 6P

38 ± 4P

124 ± 14P

37 ± 6P

 

2500 µg

 

14 ± 3P

17 ± 2P

31 ± 9P

124 ± 7P

38 ± 4P

 

5000 µg

 

18 ± 3P

18 ± 1P M

36 ± 2P M

125 ± 9P

33 ± 7P

NaN3

10 µg

 

1150 ± 20

 

 

1985 ± 123

 

4-NOPD

10 µg

 

 

 

392 ± 33

 

 

4-NOPD

50 µg

 

 

68 ± 10

 

 

 

MMS

2.0 µL

 

 

 

 

 

1053 ± 40

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

13 ± 7

12 ± 3

24 ± 8

107 ± 12

60 ± 3

Untreated

 

 

13 ± 3

16 ± 6

31 ± 7

118 ± 19

52 ± 3

Tets item

3 µg

 

13 ± 3

16 ± 3

39 ± 9

104 ± 3

57 ± 5

10 µg

 

14 ± 3

23 ± 4

29 ± 7

117 ± 11

59 ± 8

 

33 µg

 

10 ± 4

41 ± 4

34 ± 12

123 ± 21

55 ± 4

 

100 µg

 

12 ± 2

50 ± 3

35 ± 5

113 ± 16

57 ± 7

 

333 µg

 

12 ± 2

59 ± 10

43 ± 2

116 ± 13

45 ± 4

 

1000 µg

 

14 ± 5P

49 ± 9P

58 ± 3P

120 ± 13P

53 ± 7P

 

2500 µg

 

10 ± 3P

42 ± 8P

51 ± 9P

96 ± 12P

46 ± 5P

 

5000 µg

 

12 ± 5P

49 ± 3P

57 ± 4P M

108 ± 14P

40 ± 15P

2-AA

2.5 µg

 

382 ± 12

207 ± 13

3229 ± 284

4629 ± 124

 

2-AA

10.0 µg

 

 

 

 

 

318 ± 18

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

M

Precipitate

Manual count

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strains TA 1537 and TA 98 with metabolic activation.
Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

Discussion von Bericht

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and theEscheri­chia colistrain WP2 uvrA.

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

3; 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. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 µg/plate. 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 all strains with and without metabol­ic activation.

A substantial increase in revertant colony numbers was observed following treatment with the test item in strains TA 1537 and TA 98 with metabolic activation.The number of colonies exceeded the threshold of twice (strain TA 98) the number of the corresponding solvent control at concentrations from 1000 to 5000 µg/plate and thrice (strain TA 1537) the number of the corresponding solvent control at concentrations from 33to 5000 µg/plate.

A minor increase in revertant colony numbers was observed in strain TA 98 without metabolic activation, reaching, but not exceeding the threshold of twice the number of the corresponding solvent control at 1000µg/plate. At the next higher concentrations the revertant colony numbers did not reach or exceed the threshold of twicethe number of the corresponding solvent control. Therefore this effect is judged as a borderline effect based on biological fluctuation, but a mutagenic potential can be stated.

 

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

 

1      Conclusion

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 strains TA 1537 and TA 98 with metabolic activation.