Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

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

Study period:

2016-11-08 till 2016-11-29

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:

2017

Report date:

2017

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:

Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

EC Number:

220-678-3

EC Name:

Disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2,6-disulphonate

Cas Number:

2861-02-1

Molecular formula:

C14H10N2O10S2.2Na

IUPAC Name:

disodium 4,8-diamino-1,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2,6-disulfonate

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; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 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

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:

no cytotoxicity

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 both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 1000 to 5000 µg/plate and in experiment II from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.
In experiment I the incubated agar plates showed a dense color at the highest investigated concentration. This had no impact on evaluation of the plates.

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.

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: 08.11.2016

Date counted: 11.11.2016

	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		11 ± 1	8 ± 3	21 ± 1	139 ± 12	40 ± 3
Activation	Untreated		14 ± 4	8 ± 3	29 ± 8	141 ± 14	39 ± 3
	test item	3 µg	11 ± 4	5 ± 2	22 ± 1	135 ± 22	40 ± 5
		10 µg	11 ± 6	6 ± 1	25 ± 8	124 ± 3	41 ± 6
		33 µg	9 ± 4	9 ± 3	29 ± 4	135 ± 27	42 ± 3
		100 µg	12 ± 2	7 ± 3	20 ± 2	126 ± 9	42 ± 11
		333 µg	12 ± 4	5 ± 1	20 ± 5	137 ± 10	35 ± 2
		1000 µg	6 ± 1^P	7 ± 3^P	28 ± 4^P	110 ± 13^P	35 ± 11^P
		2500 µg	9 ± 3^P	7 ± 2^P	27 ± 9^P	110 ± 11^P	32 ± 8^P
		5000 µg	8 ± 2^D M P	6 ± 2^P D M	22 ± 4^P D M	113 ± 15^P D M	32 ± 5^P D M
	NaN3	10 µg	1360 ± 15			2457 ± 59
	4-NOPD	10 µg			510 ± 34
	4-NOPD	50 µg		68 ± 13
	MMS	2.0 µL					1003 ± 21
With	DMSO		9 ± 2	9 ± 2	31 ± 4	122 ± 3	40 ± 3
Activation	Untreated		12 ± 6	11 ± 5	40 ± 5	152 ± 5	52 ± 5
	test item	3 µg	9 ± 3	11 ± 3	35 ± 7	105 ± 7	47 ± 11
		10 µg	9 ± 3	7 ± 3	33 ± 3	118 ± 9	45 ± 7
		33 µg	10 ± 4	9 ± 3	30 ± 2	101 ± 8	48 ± 1
		100 µg	10 ± 3	8 ± 5	31 ± 7	116 ± 9	49 ± 12
		333 µg	12 ± 3	11 ± 3	39 ± 9	105 ± 21	45 ± 11
		1000 µg	8 ± 2^P	7 ± 2^P	28 ± 7^P	83 ± 8^P	44 ± 1^P
		2500 µg	13 ± 3^P	9 ± 2^P	23 ± 8^P	103 ± 2^P D M	48 ± 9^P
		5000 µg	9 ± 2^P D M	7 ± 1^P D M	19 ± 5^P D M	106 ± 9^P D M	43 ± 10^P D M
	2-AA	2.5 µg	429 ± 15	123 ± 9	4108 ± 693	4654 ± 167
	2-AA	10.0 µg					383 ± 24

 

               

Summary of Experiment II

Date plated: 25.11.2016

Date counted: 29.11.2016

	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 ± 0	8 ± 2	22 ± 4	147 ± 23	36 ± 1
Activation	Untreated		14 ± 3	7 ± 2	24 ± 5	190 ± 5	32 ± 1
	test item	33 µg	12 ± 3	9 ± 2	31 ± 4	135 ± 7	37 ± 1
		100 µg	9 ± 2	9 ± 3	26 ± 7	147 ± 5	30 ± 9
		333 µg	12 ± 2	11 ± 3	26 ± 5	161 ± 7	41 ± 9
		1000 µg	11 ± 1	8 ± 2	26 ± 6	137 ± 11	27 ± 3
		2500 µg	11 ± 1^P	7 ± 2^P	21 ± 3^P	133 ± 18^P	34 ± 5^P
		5000 µg	13 ± 2^P	5 ± 2^P	15 ± 2^P	120 ± 6^P	34 ± 3^P
	NaN3	10 µg	1180 ± 200			2373 ± 31
	4-NOPD	10 µg			478 ± 22
	4-NOPD	50 µg		84 ± 9
	MMS	2.0 µL					920 ± 14
With	DMSO		9 ± 1	8 ± 2	34 ± 9	145 ± 8	45 ± 5
Activation	Untreated		10 ± 0	7 ± 1	37 ± 9	194 ± 12	48 ± 9
	test item	33 µg	13 ± 6	9 ± 2	39 ± 11	134 ± 29	38 ± 10
		100 µg	10 ± 6	10 ± 3	35 ± 9	130 ± 20	48 ± 7
		333 µg	6 ± 2	8 ± 2	34 ± 2	129 ± 16	46 ± 5
		1000 µg	12 ± 4	14 ± 3	40 ± 5	154 ± 27	41 ± 8
		2500 µg	8 ± 2^P R	9 ± 0^P R	33 ± 4^P R	138 ± 41^P R	40 ± 5^P
		5000 µg	11 ± 4^P M R	9 ± 2^P R	20 ± 2^P M R	111 ± 5^P M R	33 ± 4^P M
	2-AA	2.5 µg	344 ± 74	104 ± 14	4410 ± 38	3983 ± 152
	2-AA	10.0 µg					407 ± 27

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

R:  Reduced Background growth

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:

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:                            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 both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 1000 to 5000 µg/plate and in experiment II from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

In experiment I the incubated agar plates showed a dense color at the highest investigated concentration. This had no impact on evaluation of the plates.

 

The plates incubated with the test item showed reduced background growth in experiment II in strains TA 1535, TA 1537, TA 98, and TA 100 with metabolic activationfrom 2500 to 5000 µg/plate.

 

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.

 

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.