Registration Dossier

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:
2018-02-15 till 2018-02-22
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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; 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: in agar plate incorporation

DURATION:
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 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:
positive
Remarks:
strain TA 98 with and without S9 mix
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
strain WP2 uvrA with and without S9 mix
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: Thick suspension

Other confounding effects: Due to the intense color of the test item the colonies were partly counted manually.

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with and without S9 mix at 5000 µg/plate. In the remaining strains no toxic effects were observed.

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1888600

Study Code: Envigo 1888600

Experiment: 1888600 VV Plate

Date Plated: 15.02.2018

Assay Conditions:

Date Counted: 22.02.2018

Metabolic

Activation

Test

Group

Dose Level

(per plate)

TA 1535

Revertant Colony Counts (Mean ±SD)

TA 1537

Revertant Colony Counts (Mean ±SD)

TA 98

Revertant Colony Counts (Mean ±SD)

TA 100

Revertant Colony Counts (Mean ±SD)

WP2 uvrA

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

Without

DMSO

 

10 ± 4

9 ± 3

26 ± 6

118 ± 6

36 ± 9

Activation

Untreated

 

9 ± 3

13 ± 4

25 ± 3

169 ± 21

33 ± 2

 

Aluminium

3 µg

9 ± 3

9 ± 2

26 ± 6

119 ± 21

30 ± 4

 

Black CRO

10 µg

12 ± 4

10 ± 4

22 ± 8

133 ± 5

32 ± 10

 

 

33 µg

12 ± 5

8 ± 2

24 ± 6

131 ± 13

33 ± 9

 

 

100 µg

10 ± 5

11 ± 1

23 ± 5

101 ± 4

32 ± 3

 

 

333 µg

7 ± 3D

11 ± 1D

61 ± 6D M

123 ± 5D M

33 ± 11D

 

 

1000 µg

6 ± 1M D

11 ± 5M D

87 ± 5M D

129 ± 12M D

20 ± 8M D

 

 

2500 µg

6 ± 1M D

13 ± 2M D

125 ± 7M D

126 ± 15M D

20 ± 4M D

 

 

5000 µg

6 ± 3M D

10 ± 1M D

152 ± 14M D

75 ± 15M D

11 ± 4M D

 

NaN3

10 µg

1070 ± 73

 

 

1740 ± 120

 

 

4-NOPD

10 µg

 

 

340 ± 9

 

 

 

4-NOPD

50 µg

 

61 ± 7

 

 

 

 

MMS

2.0 µL

 

 

 

 

742 ± 59

 

 

 

 

 

 

 

 

With

DMSO

 

12 ± 3

13 ± 5

37 ± 1

109 ± 2

42 ± 6

Activation

Untreated

 

16 ± 3

13 ± 2

48 ± 9

146 ± 13

44 ± 1

 

Aluminium

3 µg

14 ± 0

11 ± 1

46 ± 3

112 ± 4

35 ± 8

 

Black CRO

10 µg

9 ± 2

13 ± 3

64 ± 3

119 ± 8

42 ± 1

 

 

33 µg

8 ± 3

18 ± 2

94 ± 2

135 ± 12

36 ± 6

 

 

100 µg

11 ± 4

19 ± 3

151 ± 30

133 ± 11

36 ± 7

 

 

333 µg

7 ± 2D

14 ± 2D

202 ± 9D M

114 ± 12D M

31 ± 5D

 

 

1000 µg

8 ± 2M D

15 ± 2M D

86 ± 15M D

125 ± 16M D

27 ± 3M D

 

 

2500 µg

8 ± 3M D

13 ± 2M D

119 ± 8M D

120 ± 18M D

26 ± 2M D

 

 

5000 µg

6 ± 1M D

9 ± 2M D

131 ± 13M D

125 ± 9M D

12 ± 4M D

 

2-AA

2.5 µg

355 ± 9

220 ± 14

3739 ± 231

2586 ± 60

 

 

2-AA

10.0 µg

 

 

 

 

467 ± 36

 

 

 

 

 

 

 

 


Key to Plate Postfix Codes:              

M: Manuel Count

D:  Densely Colored Plate

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:
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 metabolic activation (S9 mix).
Executive summary:

This study was performed to investigate the potential of Aluminium Black CRO to induce gene mutations according to the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

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

No precipitation of the test item occurred up to the highest investigated dose.

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.

Toxiceffects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2uvrAwith and without S9 mix at 5000µg/plate. In the remaining strains no toxic effects were observed.

A substantial increase in revertant colony numbers was observed following treatment with Aluminium Black CRO in strain TA 98 in the presence and absence of metabolic activation (S9 mix).The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 333 µg/plate and above without S9 mix and at concentrations of 33 µg/plate and above with S9 mix. In strain TA 98 with S9 mix the revertant colony count decreases at concentrations of 1000 µg/plate and above, but the values did not fall below the threshold of twice the number of the corresponding solvent control.

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