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

Genetic toxicity: in vitro

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

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

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

Test material information

Constituent 1

Reference substance name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

EC Number:

278-207-2

EC Name:

Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)

Cas Number:

75431-69-5

Molecular formula:

C20H12N2O5S.1/3Al

IUPAC Name:

aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)

Constituent 2

Reference substance name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

213-879-2

EC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

1047-16-1

Molecular formula:

C20H12N2O2

IUPAC Name:

5,12-dihydroquino[2,3-b]acridine-7,14-dione

Specific details on test material used for the study:

The dose selection was adjusted to purity

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

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: 3.3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Experiment II: 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:
exposure duration: 72 hours
Replication: 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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 98 and TA 1537 at 5000 µg/plate

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 in the absence and presence of S9 mix in experiment I, from 100 to 5000 µg/plate in the absence of Sp mix in experiment II, and from 333 to 5000 µg/plate in the presence of S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 µg/plate onward under all experimental conditions. The undissolved particles had no influence on the data recording.


Other confounding effects:none

COMPARISON WIT 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 the test groups with and without metabolic activation, except with strain TA 98 in the absence of S9 mix at 5000 µg/plate in experiment I and with strain 1537 in the presence of S9 mix at 5000 µg/plate in experiment I.

Remarks on result:

other:

Remarks:

the test item did not induce gene mutations

Any other information on results incl. tables

Summary of Experiment I

Date plated: 14.11.2017

Date counted: 20.11.2017

			Revertant Colony Counts (Mean ± SD)
	Test Group	Dose Level (per plate	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		9 ± 2	8 ± 3	22 ± 4	145 ± 6	29 ± 8
	Untreated		7 ± 1	7 ± 3	26 ± 4	189 ± 5	34 ± 3
	HPP-Additive Q10	3 μg	11 ± 3	7 ± 2	24 ± 3	145 ± 29	38 ± 5
		10 μg	9 ± 1	10 ± 5	21 ± 3	151 ± 20	35 ± 3
		33 μg	9 ± 2	9 ± 3	24 ± 7	154 ± 10	31 ± 7
		100 μg	13 ± 2	7 ± 3	22 ± 4	146 ± 9	31 ± 8
		333 μg	10 ± 4	7 ± 2	16 ± 3	147 ± 4	32 ± 5
		1000 μg	11 ± 4^P	9 ± 2^P	22 ± 3^P	145 ± 7^P	28 ± 1^P
		2500 μg	10 ± 3^P	7 ± 3^P	21 ± 5^P	155 ± 14^P	34 ± 8^P
		5000 μg	13 ± 2^P	5 ± 2P M	9 ± 1P M	123 ± 14^P M	24 ± 6^P
	NaN3	10 μg	1375 ± 53			2415 ± 133
	4-NOPD	10 μg			344 ± 8
	4-NOPD	50 μg		155 ± 20
	MMS	2.0 μL					1003 ± 12
With Activation	DMSO		12 ± 3	12 ± 3	28 ± 7	143 ± 4	42 ± 5
	Untreated		13 ± 4	9 ± 3	35 ± 5	175 ± 12	50 ± 10
	HPP-Additive Q10	3 μg	11 ± 5	13 ± 2	33 ± 4	140 ± 5	42 ± 9
		10 μg	12 ± 2	14 ± 4	27 ± 5	138 ± 14	38 ± 6
		33 μg	12 ± 5	11 ± 3	28 ± 2	141 ± 22	36 ± 14
		100 μg	9 ± 3	12 ± 4	32 ± 5	146 ± 16	46 ± 3
		333 μg	11 ± 1	13 ± 2	47 ± 5	134 ± 3	42 ± 6
		1000 μg	10 ± 2^P	12 ± 3^P	38 ± 10^P	149 ± 6^P	35 ± 9^P
		2500 μg	9 ± 3^P	6 ± 1P M	31 ± 6^P	114 ± 9P M	36 ± 5^P
		5000 μg	10 ± 3P M	2 ± 1P M	18 ± 6P M	89 ± 4P M	43 ± 13^P
	2-AA	2.5 μg	547 ± 56	150 ± 6	3998 ± 289	4679 ± 113
	2-AA	10.0 μg					416 ± 33

 

           

Summary of Experiment II

Date plated: 23.11.2017

Date counted: 30.11.2017

			Revertant Colony Counts (Mean ± SD)
	Test Group	Dose Level (per plate	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		8 ± 2	8 ± 1	21 ± 2	109 ± 2	30 ± 8
	Untreated		9 ± 3	8 ± 2	21 ± 4	199 ± 13	42 ± 10
	HPP-Additive Q10	10 μg	11 ± 1	9 ± 2	16 ± 3	88 ± 10	29 ± 8
		33 μg	11 ± 5	8 ± 3	20 ± 6	101 ± 18	30 ± 7
		100 μg	9 ± 5	8 ± 3	17 ± 4	99 ± 7	39 ± 3
		333 μg	11 ± 3	7 ± 3	24 ± 2	96 ± 12	32 ± 9
		1000 μg	9 ± 4^P	9 ± 2^P	24 ± 6^P	101 ± 12^P	30 ± 7^P
		2500 μg	12 ± 2^P	9 ± 3^P	18 ± 6^P	78 ± 8^P	24 ± 7^P
		5000 μg	10 ± 2^P	8 ± 2^P	15 ± 3^P	77 ± 20^P	24 ± 6^P
	NaN3	10 μg	1170 ± 3			1474 ± 77
	4-NOPD	10 μg			328 ± 8
	4-NOPD	50 μg		188 ± 13
	MMS	2.0 μL					890 ± 13
With Activation	DMSO		12 ± 6	14 ± 2	33 ± 3	95 ± 22	34 ± 6
	Untreated		13 ± 2	18 ± 7	39 ± 4	201 ± 22	49 ± 7
	HPP-Additive Q10	10 μg	12 ± 3	13 ± 1	30 ± 3	77 ± 11	45 ± 8
		33 μg	13 ± 6	13 ± 5	30 ± 6	76 ± 5	48 ± 6
		100 μg	11 ± 3	11 ± 1	27 ± 4	77 ± 21	38 ± 2
		333 μg	13 ± 2	15 ± 1	34 ± 10	78 ± 19	49 ± 10
		1000 μg	10 ± 5^P	12 ± 3^P	32 ± 5^P	62 ± 6^P	45 ± 3^P
		2500 μg	11 ± 4^P	12 ± 6^P	30 ± 3^P	77 ± 11^P	34 ± 3^P
		5000 μg	8 ± 3^P	10 ± 2^P	23 ± 4^P	65 ± 11^P	27 ± 4P M
	2-AA	2.5 μg	413 ± 36	125 ± 5	2919 ± 81	2099 ± 108
	2-AA	10.0 μg					389 ± 27

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

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:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

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

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Experiment II:                             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 in the absence and presence of S9 mix in experiment I, from 100 to 5000 µg/plate in the absence of Sp mix in experiment II, and from 333 to 5000 µg/plate in the presence of S9 mix in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 µg/plate onward under all experimental conditions. 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 the test groups with and without metabolic activation, except with strain TA 98 in the absence of S9 mix at 5000 μg/plate in experiment I and with strain TA 1537 in the presence of S9 mix at 5000 μg/plate in experiment I

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

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