Silybum marianum, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

The reported data of this mutagenicity assay show (see Appendix 2 to 6) that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Extract of Chardon marie without support (Batch Number: VDO06205A) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

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:

21 August 2018 - 01 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The storage conditions of test item were changed than indicated in the Study Plan by the Sponsor request. This fact had no impact on the study on the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

The storage conditions of test item were changed than indicated in the Study Plan by the Sponsor request. This fact had no impact on the study on the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The storage conditions of test item were changed than indicated in the Study Plan by the Sponsor request. This fact had no impact on the study on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine and tryptophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Salmonella typhimurium TA98 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA100 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1535 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1537 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

Additional strain / cell type characteristics:

other: see "Any other information on materials and methods incl. tables" for details.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli WP2 uvrA 07 December 2017, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA

Additional strain / cell type characteristics:

other: see "Any other information on materials and methods incl. tables" for details.

Metabolic activation:

with and without

Metabolic activation system:

Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.

Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 28 June 2018 (Citoxlab code: E12924, Expiry date: 28 June 2020).
The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 28.1 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

The S9 Mix (containing 10 % (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix (see above) 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Contraction Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Confirmatory Mutation Test different concentrations were used.

Vehicle / solvent:

The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. Test item was insoluble (oil drops) at 100 mg/mL concentration using Distilled water. Partial dissolution was observed using DMSO and DMF at this concentration but the test item was soluble using Acetone. Therefore, Acetone was selected as vehicle (solvent) for the study.

In the study three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The following chemicals were used for vehicle (solvent) control groups:
Acetone:
Supplier: VWR
Batch No.: 17J024003
Expiry date: 02 October 2022
Dimethyl sulfoxide (DMSO):
Supplier: VWR
Batch No.: 17F294008* / 18C294015
Expiry date: 13 June 2022 / 20 March 2023
Purity: 100%
Distilled water:
Manufacturer: Hungaro-Gal Kft. / Magilab Kft. / Magilab Kft.
Batch No.: 8090418* / 180615** / 180708
Expiry date: 23 October 2018 / 21 December 2018 / 24 January 2019
*Note: These lots were used in the Preliminary Range Finding Test.
** Note: This lot was used in the Initial Mutation Test and Confirmatory Mutation Test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD), Sodium azide (SAZ),9-aminoacridine (9AA), Methyl-methanesulfonate (MMS), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, a 100 mg/mL stock solution was prepared in Acetone. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Tests)
Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in Acetone, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg/plate.

Procedure for Exposure in the Initial Mutation Test
The Initial Mutation Test followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for approximately 48 hours.

Procedure for Exposure in the Confirmatory Mutation Tests
The Confirmatory Mutation Test and the Complementary Confirmatory Mutation Test followed the standard pre-incubation procedure [1][2][5][6][7][8] since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2. ) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.

Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 5.3.6.) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Rationale for test conditions:

In accordance with test guidelines

Evaluation criteria:

The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines [5][6][7][8], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

No statistical procedure was applied.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).

Slight precipitate was detected on the plates in the in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000 and 2500 μg/plate concentration.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.

Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Tests, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Tests were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Tests were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg test item/plate.

In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation at several concentrations. In these cases, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains without metabolic activation was considered invalid and was repeated in an additional experimental period (Experimental Period III) using the pre-incubation method and a modified concentration range (in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation at 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg test item/plate). Results of the invalid experiment are not reported; however, all data are kept and archived in the raw data binder.

Slight precipitate was detected on the plates in the in the Initial Mutation Test in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
Reduced background lawn* was observed in the Initial Mutation Test in Salmonella typhimurium TA100 bacterial strain on the plates at 5000 μg/plate concentration without metabolic activation, in the Confirmatory Mutation Test in Salmonella typhimurium TA100 bacterial strain on the plates at 5000 and 1581 μg/plate concentration with metabolic activation and in the Complementary Confirmatory Mutation Test, reduced/slightly reduced background lawn was observed in all examined bacterial strains on the plates at 50 and 15.81 μg/plate concentrations without metabolic activation.
Note: The lower numbers of revertant colonies (MF<0.50) are considered to indicate an inhibitory effect.

VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.12). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In the Confirmatory Mutation Tests (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.50). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the Confirmatory Mutation Tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Summary Table of the Range Finding Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	16.3	21.3	91.7	102.0
	MF	0.92	0.91	1.02	0.98
DMSO control	Mean	16.3	22.0	--	100.3
	MF	0.92	0.94	--	0.96
Distilled water control	Mean	--	--	93.0	--
	MF	--	--	1.03	--
Acetone control	Mean	17.7	23.3	90.0	104.0
	MF	1.00	1.00	1.00	1.00
5000	Mean	19.0	25.7	65.0	76.3
	MF	1.08	1.10	0.72	0.73
2500	Mean	26.0	26.3	67.0	72.7
	MF	1.47	1.13	0.74	0.70
1000	Mean	23.7	24.3	82.3	75.7
	MF	1.34	1.04	0.91	0.73
316	Mean	21.7	29.0	95.3	113.0
	MF	1.23	1.24	1.06	1.09
100	Mean	23.7	27.7	99.3	107.3
	MF	1.34	1.19	1.10	1.03
31.6	Mean	24.0	33.7	99.7	110.0
	MF	1.36	1.44	1.11	1.06
10	Mean	24.3	26.7	96.3	110.3
	MF	1.38	1.14	1.07	1.06
NPD (4μg)	Mean	400.0	--	--	--
	MF	24.49	--	--	--
2AA (2μg)	Mean	--	2365.3	--	2402.7
	MF	--	107.52	--	23.95
SAZ	Mean	--	--	1053.3	--
	MF	--	--	11.33	--

 

 

Summary Table of the Initial Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.3	26.0	88.3	99.7	11.3	13.3	7.0	9.0	42.3	41.3
	MF	0.97	0.95	1.00	0.99	0.85	1.05	0.95	1.17	1.09	0.96
DMSO control	Mean	21.7	27.7	86.0	95.3	12.7	11.3	6.7	7.0	42.0	40.3
	MF	0.94	1.01	0.97	0.95	0.95	0.89	0.91	0.91	1.08	0.94
Distilled water control	Mean	--	--	92.7	95.7	12.7	12.7	--	--	39.7	43.7
	MF	--	--	1.05	0.95	0.95	1.00	--	--	1.02	1.02
Acetone control	Mean	23.0	27.3	88.3	100.3	13.3	12.7	7.3	7.7	39.0	43.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	18.3	22.3	38.0	63.7	11.3	10.0	7.0	7.3	39.3	38.0
	MF	0.80	0.82	0.43	0.63	0.85	0.79	0.95	0.96	1.01	0.88
1581	Mean	19.0	28.0	87.7	83.3	11.3	13.0	7.0	7.0	40.7	43.3
	MF	0.83	1.02	0.99	0.83	0.85	1.03	0.95	0.91	1.04	1.01
500	Mean	20.0	26.7	82.0	94.3	11.0	13.0	7.3	7.7	39.3	41.0
	MF	0.87	0.98	0.93	0.94	0.83	1.03	1.00	1.00	1.01	0.95
158.1	Mean	22.7	29.0	97.7	98.7	13.3	13.3	6.7	8.0	41.7	44.0
	MF	0.99	1.06	1.11	0.98	1.00	1.05	0.91	1.04	1.07	1.02
50	Mean	21.7	30.0	93.7	110.0	10.7	11.3	7.3	7.0	42.3	41.7
	MF	0.94	1.10	1.06	1.10	0.80	0.89	1.00	0.91	1.09	0.97
15.81	Mean	25.0	28.0	99.0	108.3	12.3	13.7	7.7	6.7	42.3	40.3
	MF	1.09	1.02	1.12	1.08	0.93	1.08	1.05	0.87	1.09	0.94
NPD (4μg)	Mean	410.7	--	--	--	--	--	--	--	--	--
	MF	18.95	--	--	--	--	--	--	--	--	--
2AA (2μg)	Mean	--	2477.3	--	2464.0	--	209.0	--	218.0	--	258.3
	MF	--	89.54	--	25.85	--	18.44	--	31.14 42.3	--	6.40
2AA (50μg)	Mean	--	--	--	--	--	--	--	--	--
	MF	--	--	--	--	--	--	--	--	--
SAZ (2μg)	Mean	--	--	1137.3	--	1186.7	--	--	--	--	--
	MF	--	--	12.27	--	93.68	--	--	--	--	--
9AA (50μg)	Mean	--	--	--	--	--	--	430.7	--	--	--
	MF	--	--	--	--	--	--	64.60	--	--	--
MMS (2μL)	Mean	--	--	--	--	--	--	--	--	1048.0	--
	MF	--	--	--	--	--	--	--	--	26.42	--

Summary Table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	18.0	19.0	88.7	95.3	14.3	12.7	8.7	6.7	46.0	48.7
	MF	1.00	0.92	1.06	1.00	1.10	1.12	1.18	1.18	1.00	1.01
DMSO control	Mean	19.0	20.0	--	92.3	--	12.7	7.3	6.7	45.0	48.0
	MF	1.06	0.97	--	0.97	--	1.12	1.00	1.18	0.98	0.99
Distilled water control	Mean	--	--	94.3	--	13.0	--	--	--	48.3	48.3
	MF	--	--	1.13	--	1.00	--	--	--	1.05	1.00
Acetone control	Mean	18.0	20.7	83.7	95.0	13.0	11.3	7.3	5.7	46.0	48.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	--	12.3	--	37.3	--	11.0	--	5.3	35.7	47.0
	MF	--	0.60	--	0.39	--	0.97	--	0.94	0.78	0.97
1581	Mean	--	15.0	--	40.7	--	7.0	--	3.7	34.0	44.3
	MF	--	0.73	--	0.43	--	0.62	--	0.65	0.74	0.92
500	Mean	--	24.7	--	73.3	--	12.7	--	6.7	32.7	48.7
	MF	--	1.19	--	0.77	--	1.12	--	1.18	0.71	1.01
158.1	Mean	--	23.3	--	103.3	--	11.3	--	6.7	36.3	47.0
	MF	--	1.13	--	1.09	--	1.00	--	1.18	0.79	0.97
50	Mean	9.0	24.0	53.7	108.3	9.7	14.3	3.0	4.3	36.7	46.7
	MF	0.50	1.16	0.64	1.14	0.74	1.26	0.41	0.76	0.80	0.97
15.81	Mean	12.7	23.7	57.7	111.0	11.3	17.0	5.3	5.7	43.0	45.3
	MF	0.70	1.15	0.69	1.17	0.87	1.50	0.73	1.00	0.93	0.94
5	Mean	17.3	22.0	78.0	106.0	12.0	8.7	7.0	6.0	44.3	49.0
	MF	0.96	1.06	0.93	1.12	0.92	0.76	0.95	1.06	0.96	1.01
1.581	Mean	17.3	--	87.7	--	12.3	--	9.0	--	--	--
	MF	0.96	--	1.05	--	0.95	--	1.23	--	--	--
0.5	Mean	19.0	--	89.7	--	12.0	--	7.7	--	--	--
	MF	1.06	--	1.07	--	0.92	--	1.05	--	--	--
0.1581	Mean	17.3	--	85.7	--	11.7	--	8.7	--	--	--
	MF	0.96	--	1.02	--	0.90	--	1.18	--	--	--
0.05	Mean	17.7	--	89.3	--	13.3	--	7.7	--	--	--
	MF	0.98	--	1.07	--	1.03	--	1.05	--	--	--
0.01581	Mean	17.7	--	92.0	--	13.3	--	10.0	--	--	--
	MF	0.98	--	1.10	--	1.03	--	1.36	--	--	--
NPD (4μg)	Mean	413.3	--	--	--	--	--	--	--	--	--
	MF	21.75	--	--	--	--	--	--	--	--	--
2AA (2μg)	Mean	--	2361.3	--	2477.3	--	226.7	--	208.3	--	--
	MF	--	118.07	--	26.83	--	17.89	--	31.25	--	--
2AA (50μg)	Mean	--	--	--	--	--	--	--	--	--	258.0
	MF	--	--	--	--	--	--	--	--	--	5.38
SAZ (2μg)	Mean	--	--	1136.0	--	1228.0	--	--	--	--	--
	MF	--	--	12.04	--	94.46	--	--	--	--	--
9AA (50μg)	Mean	--	--	--	--	--	--	409.3	--	--	--
	MF	--	--	--	--	--	--	55.82	--	--	--
MMS (2μL)	Mean	--	--	--	--	--	--	--	--	1128.0	--
	MF	--	--	--	--	--	--	--	--	23.34	--

 

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

The reported data of this mutagenicity assay show (see Appendix 2 to 6) that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Extract of Chardon marie without support (Batch Number: VDO06205A) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli(Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

 

Based on the results of the Compatibility Test, the test item was dissolved in Acetone at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg test item/plate.

Slight precipitate was detected on the plates in Preliminary Range Finding Test and in the Initial Mutation Test in all examined bacterial strains with and without metabolic activation at some concentrations.

 

Reduced background lawn* was observed in the Initial Mutation Test in Salmonella typhimurium TA100 bacterial strain on the plates at 5000 μg/plate concentration without metabolic activation, in the Confirmatory Mutation Test in Salmonella typhimurium TA100 bacterial strain on the plates at 5000 and 1581 μg/plate concentration with metabolic activation and in the Complementary Confirmatory Mutation Test, reduced/slightly reduced background lawn was observed in all examined bacterial strains on the plates at 50 and 15.81 μg/plate concentrations without metabolic activation.

 

Note: The lower numbers of revertant colonies (MF<0.50) are considered to indicate an inhibitory effect.

In the Initial Mutation Test and Confirmatory Mutation Tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Extract of Chardon marie without support (Batch Number: VDO06205A) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium(Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

 

Based on the results of the Compatibility Test, the test item was dissolved in Acetone at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strainsSalmonella typhimuriumTA98 and TA100 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate and in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5, 0.1581, 0.05 and 0.01581 μg test item/plate.

Slight precipitate was detected on the plates in Preliminary Range Finding Test and in the Initial Mutation Test in all examined bacterial strains with and without metabolic activation at some concentrations.

 

Reduced background lawn* was observed in the Initial Mutation Test inSalmonella typhimuriumTA100 bacterial strain on the plates at 5000 μg/plate concentration without metabolic activation, in the Confirmatory Mutation Test inSalmonella typhimuriumTA100 bacterial strain on the plates at 5000 and 1581 μg/plate concentration with metabolic activation and in the Complementary Confirmatory Mutation Test, reduced/slightly reduced background lawn was observed in all examined bacterial strains on the plates at 50 and 15.81 μg/plate concentrations without metabolic activation.

 

Note: The lower numbers of revertant colonies (MF<0.50) are considered to indicate an inhibitory effect.

In the Initial Mutation Test and Confirmatory Mutation Tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Extract of Chardon marie without support (Batch Number: VDO06205A) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Justification for classification or non-classification

Based on the available information for the substance, the substance does not meet the criteria for classifiying as mutagenic under Regulation (EC) 1272/2008.