1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

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:

8 February 2021 to 4 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

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:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

EC Number:

241-443-1

EC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)anthraquinone

Cas Number:

17418-59-6

Molecular formula:

C22H17NO5

IUPAC Name:

1-amino-4-hydroxy-2-(2-phenoxyethoxy)-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Details on test material:

Physical state: Red powder
Storage condition: at room temperature

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Type and composition of metabolic activation system:
- source of S9: ORIENTAL YEAST, CO., LTD.
- Species and strain: Sprague-Dawley rat
- Sex: male
- Age: 7 weeks
- Weight: 216.8±8.4 g
- Inducing substance: Phenobarbital (PB) and 5,6-benzoflavone (BF)
- Administration route: i.p. injection
- Dose: PB = 30 + 60 + 60 + 60 mg/kg bw (4 days); BF = 80 mg/kg bw (on 3rd day)
- Storage: Below -80 °C
- Composition of S9 mix (per 1 mL): Water (0.9 mL), S9 (0.1 mL), MgCl2 (8.0 µmol), KCl (33.0 µmol), glucose-6-phosphate (5.0 µmol), NADPH (4.0 µmol), NADH (4.0 µmol), Na-phosphate buffer (pH 7.4) (100.0 µmol)

Test concentrations with justification for top dose:

- Preliminary test: 1.2, 4.9, 20, 78, 313, 1 250 and 5 000 µg/plate

In the preliminary test, the growth inhibition by the test material was observed at 78 µg/plate and more in all strains without metabolic activation, and at 313 µg/plate and more in S. typhimurium TA strains with metabolic activation, and at 1250 µg/plate and more in E. Coli WP2 uvrA with metabolic activation. And the precipitate of the test material on the plates was observed at 313 µg/plate both with and without metabolic activation.
Therefore, as the highest dose level of the test material in the main tests, the 78 µg/plate dose was selected for all strains without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurium strains with metabolic activation and the 1250 µg/plate dose was selected for E. Coli WP2 uvrA with metabolic activation, and these highest doses were diluted 5 times (using a common ratio of 2) to provide a total of 6 dose levels.

- Main test (first and second):
2.4, 4.9, 10, 20, 39 and 78 µg/plate (-S9 mix)
10, 20, 39, 78, 156, 313, 625 and 1250 µg/plate (+S9 mix)

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test material was best suspended in DMSO, therefore DMSO was used as solvent for preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl and 2-Aminoanthracene

Details on test system and experimental conditions:

PREPARATION OF THE TEST SOLUTION
Test solution of the maximum concentration was prepared, which was fully stirred with mixer and suspended with ultrasonication (2 mins) after the test material was weighed and the solvent was added. The lower doses were prepared by diluting stepwise from the test solution of the maximum concentration. Preparation of the test solution was carried out just prior to use under lamps with ultraviolet absorbent filter. The test solution was prepared without correcting the purity of the test material.

METHOD OF APPLICATION: pre-incubation

DURATION
- Pre-culture period procedure: A bacterial suspension of each strain (20 µL of S. typhimurium TA strains, 5 µL of E. coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until starting incubation, and then incubated while shaking (100 rpm) in a water bath at 37 °C for 8 hours. After incubation, the optical density was measured and the number of viable cell was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until starting the test. The number of bacteria in the culture was estimated from the optical density at the end of the pre-culture.

- Pre-incubation method test procedure: For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37 °C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. And 0.1 mL of the positive control solution was carried out equally.
One plate was used for each dose level in the preliminary test and three plates were used for each dose level in the two main tests which were performed at the same doses.
For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2.0 mL of top agar were then added to the tube, and the contents of each tube were poured over the surface of the plate.
These operations were conducted under lamps with ultraviolet absorbent filter. As top agar, the 0.5 mM biotin-0.5 mM L-histidine solution and the 0.5 mM L-tryptophan solution were added to the soft agar solution (0.6 % Agar and 0.5 % NaCl) by volume of 1/10, for the S. typhimurium TA strains and the E. coli strain, respectively.
All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope. The presence or absence of precipitate of the test material on the plates was visually observed and recorded.

COUNTING PROCEDURE
- The number of revertant colonies was counted visually due to the colour of the test material on the plates. But the revertant colonies of positive controls were counted with a colony counter.

Evaluation criteria:

In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test material was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition at and above 78 µg/plate in -S9mix and 1250 µg/plate in +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition at and above 78 µg/plate in -S9mix and 313 µg/plate in +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

cytotoxicity

Remarks:

Growth inhibition at and above 78 µg/plate in -S9mix and 313 µg/plate in +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition at and above 78 µg/plate in -S9mix and 313 µg/plate in +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition at and above 78 µg/plate in -S9mix and 313 µg/plate in +S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3 S.D.) in historical data, indicating that this study was performed correctly.
From these results, mutagenicity of the test material was judged negative.
Precipitate of the test material on the plates was observed at 313 µg/plate and more without metabolic activation and at 156 µg/plate and more with metabolic activation.
In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Remarks on result:

other: Not mutagenic

Any other information on results incl. tables

Summary of Main Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-S9 mix	DMSO	98	14	23	20	8
	2.4	99	12	20	18	9
	4.9	115	14	23	20	10
	10	106	15	24	17	8
	20	104	17	20	20	9
	39	105*	12*	21*	17*	7*
	78	122*	12*	26*	18*	7*
+S9 mix	DMSO	130	11	25	26	17
	10	116	9	NT	26	13
	20	112	9	NT	29	14
	39	107	9	23	28	19
	78	107	12	27	27	17
	156	111	10	27	26	17
	313	94*	5*	28	28*	17*
	625	NT	NT	24	NT	NT
	1250	NT	NT	25*	NT	NT
Positive Controls
-	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Mean no. colonies/plate	533	420	135	512	1292
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Mean no. colonies/plate	778	247	310	187	69

2AA = 2-aminoanthracene

B[a]P = benzo(a)pyrene

NaN3 = sodium azide

AF-2 = 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide

ICR-191 = 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

* = Growth inhibition of the tested bacterium by the test material was observed.

NT = not tested

 

Summary of Main Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-S9 mix	DMSO	121	13	23	19	8
	2.4	129	15	20	20	7
	4.9	117	12	24	19	6
	10	120	11	23	17	6
	20	112	15	26	18	9
	39	115*	13*	24*	20*	9*
	78	114*	12*	22*	19*	5*
+S9 mix	DMSO	131	12	29	28	13
	10	119	11	NT	24	13
	20	115	9	NT	27	18
	39	128	10	24	25	16
	78	127	10	27	29	16
	156	136	9	29	30	13
	313	125*	10*	24	28	12
	625	NT	NT	22	NT	NT
	1250	NT	NT	24*	NT	NT
Positive Controls
-	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Mean no. colonies/plate	535	385	153	518	1365
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Mean no. colonies/plate	751	242	321	184	59

2AA = 2-aminoanthracene

B[a]P = benzo(a)pyrene

NaN3 = sodium azide

AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

ICR-191 = 2-methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

* = Growth inhibition of the tested bacterium by the test material was observed.

NT = not tested

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 471 and under GLP conditions.

The mutagenicity potential of the test material was assessed with Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA in the bacterial reverse mutation assay.

Bacteria were exposed to the test material using a pre-incubation method both in the presence and absence of metabolic activation in the form of S9 mix. All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies were counted. Positive and solvent controls were also included in the study.

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in historical data, indicating that this study was performed correctly. In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Under the conditions of this study, the test material was not mutagenic in the bacterial reverse mutation assay.