1,3,4,6,7,9,9b-heptaazaphenalene-2,5,8-triamine

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	576^c	17^c	22.75^c	874^c	18^c	9.54^c	170^c	25^c	13.10^c	182^c	19^c	17.58^c	565^d	22^d	4.08^d
^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/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
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	277^c	11^c	10.81^c	578^d	12^d	6.54^d	169^d	12^d	12.68^d	178^e	13^e	18.38^e	576^f	22^f	4.03^f
^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.