1,4-bis(butylamino)anthraquinone

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:

2014-08-26 till 2014-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

1,4-bis(butylamino)anthraquinone

EC Number:

241-379-4

EC Name:

1,4-bis(butylamino)anthraquinone

Cas Number:

17354-14-2

Molecular formula:

C22H26N2O2

IUPAC Name:

1,4-bis(butylamino)anthraquinone

Details on test material:

Purity: 99.2 % (w/w)
Expiry Date: 25 June 2024

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: Better than others

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
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, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in strain TA 98 with metabolic activation

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: unsoluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed
The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground 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 metabol¬ic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Study Name: 1652800	Study Code: Harlan CCR 1652800
Experiment: 1652800 VV Plate	Date Plated: 26/08/2014
Assay Conditions:	Date Counted: 29/08/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			26 ± 4^B M	7 ± 2	21 ± 7	174 ± 10	35 ± 13
	Untreated			28 ± 3^B M	10 ± 3	22 ± 6	162 ± 30	48 ± 5
	Fat Blue B 01	3 µg		27 ± 4^B M	10 ± 4	28 ± 1	177 ± 2	42 ± 0
		10 µg		26 ± 2^B M	8 ± 2	19 ± 4	166 ± 9	39 ± 5
		33 µg		30 ± 2^B M	8 ± 1	22 ± 3	191 ± 2	44 ± 6
		100 µg		26 ± 1^B M	7 ± 1	17 ± 2	216 ± 16	41 ± 4
		333 µg		28 ± 2^B M	9 ± 1	19 ± 9	185 ± 16	54 ± 9
		1000 µg		28 ± 1^B D M	7 ± 1^D	21 ± 1^D	173 ± 10^D	44 ± 9^D
		2500 µg		26 ± 5^B D M	6 ± 2^D M	21 ± 4^D M	174 ± 11^D M	32 ± 4^D M
		5000 µg		28 ± 2^B D M	6 ± 1^D M	17 ± 2^D M	176 ± 13^D M	27 ± 4^D M
	NaN3	10 µg		2459 ± 235			1835 ± 57
	4-NOPD	10 µg				395 ± 13
	4-NOPD	50 µg			80 ± 8
	MMS	2.0 µL						1107 ± 49
With Activation	DMSO			13 ± 6	15 ± 2	30 ± 10	123 ± 23	55 ± 4
	Untreated			14 ± 7	12 ± 5	38 ± 6	175 ± 4	57 ± 14
	Fat Blue B 01	3 µg		19 ± 5	20 ± 1	41 ± 1	155 ± 7	41 ± 4
		10 µg		18 ± 2	18 ± 5	32 ± 3	146 ± 5	54 ± 6
		33 µg		17 ± 0	19 ± 4	37 ± 13	155 ± 4	53 ± 2
		100 µg		16 ± 4	16 ± 1	42 ± 2	163 ± 16	51 ± 5
		333 µg		13 ± 5	22 ± 4	73 ± 3	148 ± 16	50 ± 2
		1000 µg		14 ± 3^D	20 ± 5^D	117 ± 13^D	151 ± 18^D	46 ± 5^D
		2500 µg		13 ± 2^D M	21 ± 2^D M	172 ± 33^D M	168 ± 6^D M	49 ± 10^D M
		5000 µg		13 ± 3^D M	18 ± 2^D M	192 ± 7^D M	179 ± 5^D M	34 ± 3^D M
	2-AA	2.5 µg		454 ± 13	310 ± 18	2167 ± 35	4272 ± 129
	2-AA	10.0 µg						212 ± 10

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	D M B	Densely coloured plate Manual count Extensive bacterial growth

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive in strain TA 98 with metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix.

Executive summary:

The test item Fat Blue B 01 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and theEscheri­chia colistrain WP2 uvrA.

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

3; 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. No precipitation of the test item was observed on the incubated agar plates. 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.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Fat Blue B 01 at any dose level in the absence of metabolic activation (S9 mix).

In the presence of metabolic activation a substantial and dose dependent increase was observed in strain TA 98.The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations from 333 µg/plate to 5000 µg/plate

 

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

The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.