2,3-dihydro-1,4-dihydroxyanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Dihydrochinizarin was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of rat liver S9 mix. The test item did induce frameshift mutations in TA 1537 in the presence of metabolic activation. Therefore under the conditions of the study, the test substance is considered to be mutagenic in vitro in the presence of metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-10 - 2016-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471:
Bacterial Reverse Mutation Test, adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008 B13/14, dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 95.6 %
Appearance: Solid, yellow to brown powder
Stability in solvent: Stable in DMSO over 4 and 24 hours
Expiry Date: 16 September 2017
Storage Conditions: At room temperature

Target gene:

Salmonella typhimurium (TA strains): histidine locus
Escherichia coli (WP2 uvrA strain): tryptophan locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I (plate incorporation test):
Experiment I 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment Ia (confirmatory test): 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Since a positive result was obtained in this experiment, a confirmatory experiment Ia was performed to verify the result.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (purity > 99 %)
- Justification for choice of solvent/vehicle: solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (without activation), 2-AA (with activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment Ia: plate incorporation confirmatory test;
DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours
NUMBER OF REPLICATIONS: In both experiments, each concentration, including the controls, was tested in triplicate.
DETERMINATION OF CYTOTOXICITY: reduction in the number of revertants (below the indication 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.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

induced frameshift mutations in TA 1537 in the presence of metabolic activation (S9 mix)

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Precipitation: test item precipitated in the overlay agar in the test tubes from 333 to 5000 μg/plate.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).

HISTORICAL CONTROL DATA:

Strain without S9 mix with S9 mix
Mean SD Min Max Mean SD Min Max
Solvent control 11 2.15 7 23 12 2.14 7 21
TA 1535 Untreated control 12 2.97 6 24 12 2.71 7 26
Positive control 1090 123.80 334 1372 392 62.85 176 549

Solvent control 10 1.83 6 18 13 3.27 7 27
TA1537 Untreated control 10 2.29 6 20 14 3.72 7 25
Positive control 83 12.28 55 131 175 44.44 82 327

Solvent control 24 3.75 16 36 33 5.55 18 51
TA 98 Untreated control 26 4.72 15 43 36 5.83 17 56
Positive control 344 51.13 211 599 3822 857.83 319 5048

Solvent control 155 24.19 84 194 145 31.81 81 204
TA 100 Untreated control 174 21.92 90 206 170 23.62 93 212
Positive control 1956 279.93 6 58 2528 3606 676.07 722 4940

Solvent control 41 5.72 27 63 51 6.91 37 72
WP2uvrA Untreated control 42 6.01 31 63 53 7.05 38 88
Positive control 732 161.66 322 1066 362 72.26 212 858

ADDITIONAL INFORMATION ON CYTOTOXICITY:
not mentioned

Summary of Experiment I Study Name: 1757301                                                  Study Code: Envigo 1757301 Experiment: 1757301 VV Plate                                        Date Plated: 10.06.2016 Assay Conditions:                                                       Date Counted: 13.06.2016 Metabolic      Test             Dose Level   Revertant Colony Counts (Mean ±SD) Activation     Group          (per plate)
			TA 1535	TA 1537	TA 98	TA 100	WP2
							uvrA
Without	DMSO		13 ± 1	11 ± 3	21 ± 2	192 ± 9	36 ± 4
Activation	Untreated		14 ± 2	10 ± 5	20 ± 5	190 ± 19	50 ± 8
	Dihydro-	3 µg	11 ± 6	9 ± 2	24 ± 2	170 ± 3	47 ± 8
	chinizarin	10 µg	10 ± 2	10 ± 2	24 ± 5	163 ± 9	41 ± 4
		33 µg	12 ± 2	12 ± 5	26 ± 1	170 ± 16	42 ± 11
		100 µg	13 ± 5	14 ± 2	37 ± 8	183 ± 17	35 ± 8
		333 µg	12 ± 4	17 ± 3	27 ± 5	165 ± 15	35 ± 9
		1000 µg	9 ± 4P	19 ± 3P M	23 ± 4P	176 ± 17^P	25 ± 6^P
		2500 µg	8 ± 3P	16 ± 2P M	16 ± 1P M	98 ± 18P M	28 ± 2^P
		5000 µg	10 ± 4P M	9 ± 3P M	12 ± 2P M	93 ± 24P M	22 ± 5P M
	NaN3	10 µg	1206 ± 58			1950 ± 76
	4-NOPD	10 µg			357 ± 59
	4-NOPD	50 µg		67 ± 3
	MMS	2.0 µL					1144 ± 34
With	DMSO		8 ± 3	9 ± 4	35 ± 4	169 ± 7	58 ± 1
Activation	Untreated		15 ± 6	13 ± 6	38 ± 8	200 ± 21	52 ± 3
	Dihydro-	3 µg	8 ± 1	14 ± 7	38 ± 6	185 ± 16	53 ± 5
	chinizarin	10 µg	9 ± 1	15 ± 5	41 ± 4	176 ± 17	48 ± 6
		33 µg	7 ± 4	24 ± 4	30 ± 4	165 ± 2	41 ± 7
		100 µg	9 ± 2	40 ± 3	40 ± 9	156 ± 16	47 ± 4
		333 µg	13 ± 2^P	42 ± 3^P	41 ± 7P	145 ± 17^P	53 ± 15^P
		1000 µg	8 ± 1P	35 ± 4P M	40 ± 2P	148 ± 9P	56 ± 11^P
		2500 µg	11 ± 3P M	31 ± 5P M	42 ± 3P	132 ± 8P	49 ± 4^P
		5000 µg	10 ± 2P M	23 ± 2P M	35 ± 5P M	125 ± 11P M	40 ± 6P M
	2-AA	2.5 µg	372 ± 32	251 ± 17	3902 ± 513	5302 ± 398
	2-AA	10.0 µg					439 ± 17

 

Key to Positive Controls                                              Key to Plate Postfix Codes

NaN3         sodium azide                                            P         Precipitate

2-AA          2-aminoanthracene                                    M        Manual count

4-NOPD     4-nitro-o-phenylene-diamine

MMS         methyl methane sulfonate

Summary of Experiment Ia

Study Name: 1757301                                                                            Study Code: Envigo 1757301

Experiment: 1757301 VVa Plate                                                              Date Plated: 16.06.2016

Assay Conditions:                                                                                 Date Counted: 20.06.2016

MetabolicActivation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)

 

TA 1537

With Activation

DMSO                        11 ± 3 Untreated                                  14 ± 3 Dihydrochinizarin 3 µg                  16 ± 8 10 µg                  19 ± 7 33 µg                  28 ± 12 100 µg                 48 ± 7 333 µg                 46 ± 5 1000 µg               54 ± 1^P 2500 µg               57 ± 7^P 5000 µg               45 ± 7P M 2-AA                2.5 µg               220 ± 30

 

Key to Positive Controls                                             Key to Plate Postfix Codes

2-AA 2-aminoanthracene                                             P         Precipitate

M        Manual count

Conclusions:

The test item Dihydrochinizarin was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of rat liver S9 mix. The test item did induce frameshift mutations in TA 1537 in the presence of metabolic activation. Under the conditions of the study, the test substance was thus considered mutagenic in vitro in the presence of metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli strain WP2 uvrA were exposed to Dihydrochinizarin in DMSO in concentrations of 0 (control), 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) in a plate incorporation test (experiment I) and in concentrations of 0 (control), 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat liver S9 mix) in confirmatory plate incoproration test (experiment Ia).

Each concentration and the controls were tested in triplicates.

Precipitation of the test item occurred in the overlay agar on the incubated agar plates from 1000 to 5000 µg/plate in the absence of S9 mix and from 333 to 5000 µg/plate in the presence of S9 mix in experiment I.

In experiment Ia precipitation of the test item on the incubated agar plates was observed from 1000 to 5000 μg/plate.

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.  

In Experiment I, a relevant increase in revertant colony numbers was observed following treatment with Dihydrochinizarin in strain TA 1537 in the presence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of thrice the number of the corresponding solvent control at concentrations ranging from 100 μg/ plate to 2500 μg/plate. This observed mutagenic effect was reproduced in Experiment Ia. After treatment with the test item at concentrations ranging from 100 to 5000 μg/plate, a relevant increase in revertant colony numbers exceeding the threshold of thrice was observed.

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

The test item did induce frameshift mutations in TA 1537 in the presence of metabolic activation (S9 mix)

Under the conditions of the study, the test substance is considered to be mutagenic in vitro.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Based on reliable, relevant and adequate data on the test substance is considered to be mutagenic in vitro. The test item did induce frameshift mutations in TA 1537 in the presence of metabolic activation. According to Regulation EC No. 1272/2008, no classification and labelling for mutagenicity is required since no in vivo study is available. Based on the positive in vitro data the endpoint genotoxicity is inconclusive.