Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

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:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 Jul 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

909-092-5

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Test material form:

solid: particulate/powder

Details on test material:

Batch 22426
100% UVCB
storage at room temperature
storage stability until 10 Dec 2025

Specific details on test material used for the study:

purity: 100% UVCB
physical state: solid, violet to brown

Method

Target gene:

his+ / trp+

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 specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA

Evaluation criteria:

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

In the preincubation assay

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 100 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+
or trp+ revertants) was observed in the standard plate test up to the highest concentration.
In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ and trp+
revertants) was occasionally observed depending on the strain and test conditions from about
1000 μg/plate onward.

Any other information on results incl. tables

Standard plate test, without metabolic activation
strain		Test item	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535		Water	-	12.0	4.0	-	12, 16, 8
		Test item	33	9.3	3.5	0.8	9, 6, 13
			100	12.7	1.2	1.1	14 P, 12 P, 12 P
			333	14.0	4.6	1.2	15 P, 18 P, 9 P
			1000	10.3	0.6	0.9	10 P, 10 P, 11 P
			2500	12.0	3.0	1.0	12 P, 9 P, 15 P
			5000	14.0	2.6	1.2	17 P, 13 P, 12 P
		MNNG	5.0	5209.3	223.5	434.1	5207, 4987, 5434
TA 100		Water	-	91.7	7.6	-	90, 85, 100
		Test item	33	103.7	14.5	1.1	87, 113, 111
			100	109.7	15.0	1.2	101 P, 127 P, 101 P
			333	96.7	7.6	1.1	100 P, 88 P, 102 P
			1000	108.7	6.7	1.2	107 P, 103 P, 116 P
			2500	104.7	6.7	1.1	112 P, 99 P, 103 P
			5000	98.3	13.0	1.1	111 P, 85 P, 99 P
		MNNG	5.0	3985.0	185.9	43.5	3927, 4193, 3835
TA 1537		Water	-	10.0	2.6	-	11, 12, 7
		Test item	33	8.3	1.2	0.8	7, 9, 9
			100	10.0	3.6	1.0	14 P, 9 P, 7 P
			333	9.0	3.0	0.9	12 P, 6 P, 9 P
			1000	8.3	3.5	0.8	5 P, 8 P, 12 P
			2500	7.7	2.5	0.8	8 P, 10 P, 5 P
			5000	10.0	1.7	1.0	8 P, 11 P, 11 P
		AAC	100	520.0	288.3	52.0	534, 225, 801
TA 98		Water	-	21.3	3.2	-	25, 20, 19
		Test item	33	24.3	7.6	1.1	26, 31, 16
			100	24.0	7.5	1.1	17 P, 32 P, 23 P
			333	21.0	6.0	1.0	21 P, 27 P, 15 P
			1000	25.0	0.0	1.2	25 P, 25 P, 25 P
			2500	20.7	6.5	1.0	27 P, 14 P, 21 P
			5000	20.0	4.4	0.9	15 P, 22 P, 23 P
		NOPD	10	657.3	39.7	30.8	653, 699, 620
E. coli		Water	-	26.7	4.2	-	22, 28, 30
		Test item	33	29.0	10.1	1.1	38, 31, 18
			100	22.3	3.2	0.8	20 P, 26 P, 21 P
			333	32.7	5.1	1.2	37 P, 34 P, 27 P
			1000	26.3	3.8	1.0	29 P, 22 P, 28 P
			2500	22.3	4.9	0.8	28 P, 20 P, 19 P
			5000	29.7	7.6	1.1	23 P, 28 P, 38 P
		4-NQO	5	1273.3	4.2	47.8	1270, 1278, 1272

P = precipitation

Standard plate test, with metabolic activation
strain		Test item	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535		Water	-	15.7	2.3	-	17, 13, 17
		Test item	33	12.0	3.6	0.8	15, 8, 13
			100	10.3	3.5	0.7	10 P, 7 P, 14 P
			333	13.7	2.1	0.9	16 P, 13 P, 12 P
			1000	11.0	6.2	0.7	18 P, 9 P, 6 P
			2500	12.0	4.6	0.8	17 P, 8 P, 11 P
			5000	16.3	2.1	1.0	17 P, 14 P, 18 P
		2-AA	2.5	277.7	43.0	17.7	324, 270, 239
TA 100		Water	-	105.7	11.0	-	117, 95, 105
		Test item	33	101.0	7.8	1.0	96, 110, 97
			100	119.7	10.7	1.1	129 P, 108 P, 122 P
			333	113.7	13.3	1.1	105 P, 129 P, 107 P
			1000	120.7	3.2	1.1	123 P, 117 P, 122 P
			2500	128.0	16.8	1.2	109 P, 141 P, 134 P
			5000	130.7	2.5	1.2	133 P, 131 P, 128 P
		2-AA	2.5	2360.0	121.6	22.3	2220, 2439, 2421
TA 1537		Water	-	10.7	2.9	-	9, 14, 9
		Test item	33	12.7	4.0	1.2	15, 8, 15
			100	16.0	5.6	1.5	10 P, 17 P, 21 P
			333	13.3	1.2	1.3	14 P, 12 P, 14 P
			1000	12.0	3.6	1.1	15 P, 8 P, 13 P
			2500	11.0	1.0	1.0	10 P, 11 P, 12 P
			5000	12.0	4.4	1.1	15 P, 14 P, 7 P
		2-AA	2.5	169.0	2.6	15.8	171, 166, 170
TA 98		Water	-	29.7	9.0	-	39, 29, 21
		Test item	33	25.3	2.5	0.9	23, 25, 28
			100	32.3	5.7	1.1	34 P, 37 P, 26 P
			333	34.0	7.5	1.1	26 P, 35 P, 41 P
			1000	34.7	4.0	1.2	39 P, 34 P, 31 P
			2500	30.7	2.9	1.0	34 P, 29 P, 29 P
			5000	32.0	1.0	1.1	31 P, 32 P, 33 P
		2-AA	2.5	2063.7	63.7	69.6	2090, 1991, 2110
E. coli		Water	-	28.3	10.1	-	23, 22, 40
		Test item	33	36.3	1.5	1.3	36, 35, 38
			100	23.7	1.2	0.8	25 P, 23 P, 23 P
			333	26.3	5.8	0.9	23 P, 23 P, 33 P
			1000	25.0	8.7	0.9	35 P, 19 P, 21 P
			2500	27.3	2.5	1.0	30 P, 25 P, 27 P
			5000	22.7	7.4	0.8	20 P, 17 P, 31 P
		2-AA	60	126.0	19.5	4.4	148, 119, 111

P = Precipitation

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in bacteria.