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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
EC Number:
216-122-4
EC Name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
Cas Number:
1502-47-2
Molecular formula:
C6H6N10
IUPAC Name:
1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine
Test material form:
solid
Details on test material:
Test Item Name: Exolit 775
Sample Number: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
CAS No.: 1502-47-2

Chemical name (IUPAC): 1,3,4,6,7,9,9b-Heptaazaphenalene-2,5,8-triamine/Melem
CAS No.: 1502-47-2
Sample No.: Ba-Ha-19-025-1; Deflam 20 20151106 JOB D02441
Manufactured Date: 2019-09-06
Expiry Date: 2034-09-06
Purity as per Certificate of Analysis: 95.75%
Physical Appearance: Solid White
Storage Conditions: Cool and Dry (+2 to +8 °C)

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli
Rationale for test conditions:
The results of the study from both the initial and confirmatory mutation assay showed that, the test item did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.
Evaluation criteria:
Tester strain integrity
All Salmonella typhimurium tester strains and the Escherichia coli tester strain used in this assay must
demonstrate their growth potential in the presence of histidine and tryptophan, respectively.
All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and ultraviolet light to
demonstrate the presence of rfa mutation and uvrB mutation, respectively.
Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of
uvrA mutation.
Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) mu
st exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.
• The spontaneous reversion rates in the vehicle control must be in the range of in-house historical
data.
• There must be at least three non-toxic dose levels.
• The top dose selected should demonstrate toxicity. In case of non-toxic test items, the top dose
tested should be 5000 μg/plate.
• All tester strain culture titers must be in the range of 1-2 x 109 cells/mL to ensure that appropriate
numbers of bacteria are used for plating.
• The positive control substances should produce a more than 3-fold increase in mutant colony
frequencies when compared to the respective vehicle control plates

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

TABLE 1.   Summary Results of Initial Bacterial Reverse Mutation Assay in the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

DMSO

25

3

NA

92

14

NA

13

1

NA

10

1

NA

138

9

NA

 

50

24

3

0.96

84

6

0.92

13

1

0.97

10

1

0.97

139

7

1.01

 

158

24

2

0.95

88

13

0.96

13

1

0.97

9

1

0.87

135

9

0.98

 

500

25

4

1.00

86

6

0.93

12

1

0.95

9

2

0.90

139

9

1.01

 

1581

24

2

0.95

79

9

0.87

13

1

1.00

10

2

0.94

132

8

0.95

 

5000

22

1

0.88

73

2

0.80

13

1

0.97

10

1

0.97

131

4

0.95

 

Positive control

576c

17c

22.75c

874c

18c

9.54c

170c

25c

13.10c

182c

19c

17.58c

565d

22d

4.08d

aValues are means of three replicatescalculated from individual values of Appendix 3and are rounded off to the nearest whole number           

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.  

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)

dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate)                 SD: Standard deviation  NA: Not applicable     

TABLE 2.       Summary Results of Initial Bacterial Reverse Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

DMSO

26

2

NA

88

6

NA

13

2

NA

10

1

NA

143

7

NA

 

50

25

2

0.96

82

13

0.93

13

1

0.98

10

1

1.03

133

9

0.93

 

158

23

2

0.91

85

6

0.96

12

1

0.93

10

1

1.00

133

8

0.93

 

500

23

1

0.90

80

9

0.91

12

1

0.93

10

2

1.00

138

5

0.97

 

1581

23

2

0.90

84

8

0.95

13

1

0.95

10

1

1.03

136

6

0.95

 

5000

23

1

0.88

76

7

0.86

12

1

0.93

9

1

0.90

131

8

0.92

 

Positive control

277c

11c

10.81c

578d

12d

6.54d

169d

12d

12.68d

178e

13e

18.38e

576f

22f

4.03f

aValues are means of three replicatescalculated from individual values of Appendix 4and are rounded off to the nearest whole number           

bRatio of treated/Vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table.

cTA98: 2-Nitrofluorene (2 µg/plate),                                                                         

dTA100, TA1535: Sodium azide (1 µg/plate)                                 eTA1537: 9-Aminoacridine (50 µg/plate)                                    

fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)          SD: Standard deviation    NA: Not applicable                

Applicant's summary and conclusion

Conclusions:
The registration substance was investigated for its mutagenicity according to the OECD Guideline 471 (Bacterial reverse mutation assay). No mutagenicity was found.
Executive summary:

The registration substance was investigated for its mutagenicity according to the OECD Guideline 471(Bacterial reverse mutation assay). Tester strains S. typhimurium TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA (pKM 101) were treated with the registration substance up to the concentration of 5000 µg/plate without and with metabolic activation (S9 mix). No increase of mutation rate was found. The registration substance is not mutagenic in the bacterial reverse mutation assay.

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