Essential oil of Matricaria recutita (Asteraceae) obtained from flower tops by steam distillation of European origin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

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:

09 to 27 Februray 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 471 without any deviation

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

dated 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ENV/EPOC(97)4 OECD Guideline for the Testing of Chemicals, Proposal for Replacement of Guidelines 471 and 472 Bacterial Reverse Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 2000/32, L1362000, Annex 4D, dated May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 September 2015

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Storage Conditions:Room temperature

Target gene:

he histidine dependent strains are derived from Salmonella typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain Escherichia coli WP2 and its derivatives carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

According to the standard limit concentrations recommended in the regulatory guidelines 5000 µg/plate was applied as the highest concentration in the pre-experiment.
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strains TA 100: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in DMSO (purity > 99 %). The solvent has been chosen according to its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

METHOD OF APPLICATION:
EXPERIMENT: Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
Experiment II (Pre-Incubation)
In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

CONTROLS:
- Vehicle/solvent control: DMSO
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation

NUMBER OF REPLICATIONS: Triplicate

OTHER: PRE-EXPERIMENT: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed in experiment I, a minimum of six concentrations was tested in experiment II. Based on the observed toxic effects 1000 µg/plate were chosen as maximum concentration for strain TA 100. For the remaining strains the maximum concentration was 5000 µg/plate. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
Strain TA 100: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate
The remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
•regular background growth in the negative and solvent control;
•the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above 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;
•a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5;
•if precipitation occurs a minimum of four concentrations showing no precipitation should be analysed.

Statistics:

None

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:

valid

Untreated 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:

valid

Untreated 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:

cytotoxicity

Remarks:

5000 µg/plate in Exp 1 without S9, 5000 µg/plate in Exp 1 with S9, 2500 to 5000 µg/plate in Exp 2 without S9, 2500 to 5000 µg/plate in Exp 2 with S9,

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

333 to 5000 µg/plate in Exp 1 without S9, 1000 to 5000 µg/plate in Exp 1 with S9, 100 to 1000 µg/plate in Exp 2 without S9, 333 to 1000 µg/plate in Exp 2 with S9,

Vehicle controls validity:

valid

Untreated 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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. Based on a lower top dose of 1000 µg/plate no precipitation of the test substance was observed on the incubated agar plates in strain TA 100 in experiment II. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix ith S9 mix
TA 1535 / / / /
TA 1537 1000 – 5000 1000 – 5000 / /
TA 98 1000 – 5000 1000 – 5000 1000 – 5000 1000 – 5000
TA 100 333 – 5000 1 000 – 5000 3 33 – 1000 333 – 1000
WP2 uvrA / / / /

MUTAGENICITY
- The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Blue chamomilla - European origin 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 in-crease in induced revertant colonies.
In experiment II the number of colonies did not quite reach the lower limit of our historical control data in the negative control strain Wp2 uvrA without S9 mix. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study

- Refer Tables 7.6.1/1 and 7.6.1/2 for more details and attached background for individual results.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Positive historical control data: Refer Table 7.6.1/3
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/3

Table 7.6.1 /1 Summary of Experiment I

Study Name: 1875200	Study Code: Envigo 1875200
Experiment: 1875200 VV Plate	Date Plated: 09.02.2018
Assay Conditions:	Date Counted: 13.02.2018

 

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		12 ± 3	12 ± 2	27 ± 8	173 ± 20	28 ± 2
	Untreated		7 ± 3	12 ± 3	30 ± 8	179 ± 10	37 ± 11
	Blue	3 µg	11 ± 3	13 ± 6	24 ± 3	183 ± 6	28 ± 3
	chamomilla-	10 µg	12 ± 2	9 ± 3	23 ± 4	180 ± 21	29 ± 9
	European	33 µg	9 ± 3	9 ± 1	23 ± 4	161 ± 9	30 ± 11
	origin	100 µg	9 ± 1	10 ± 2	30 ± 6	144 ± 1	27 ± 6
		333 µg	12 ± 5	14 ± 5	24 ± 3	47 ± 1^R	32 ± 8
		1000 µg	12 ± 2	14 ± 4^R	24 ± 2^R	17 ± 5^M R	32 ± 3
		2500 µg	12 ± 4^P M	14 ± 2^P R	15 ± 3^P R	3 ± 1^P M R	26 ± 8^P
		5000 µg	8 ± 3^P M	9 ± 1^P M R	7 ± 1^P M R	1 ± 1^P M R	30 ± 8^P M
	NaN3	10 µg	868 ± 74			2222 ± 194
	4-NOPD	10 µg			384 ± 11
	4-NOPD	50 µg		59 ± 2
	MMS	2.0 µL					808 ± 4
With Activation	DMSO		11 ± 2	12 ± 1	42 ± 8	185 ± 9	37 ± 10
	Untreated		12 ± 2	12 ± 4	49 ± 6	189 ± 36	44 ± 10
	Blue	3 µg	12 ± 3	16 ± 1	44 ± 6	149 ± 3	36 ± 4
	chamomilla-	10 µg	14 ± 3	12 ± 4	36 ± 5	146 ± 11	28 ± 8
	European	33 µg	14 ± 3	13 ± 3	42 ± 9	165 ± 12	35 ± 11
	origin	100 µg	14 ± 3	17 ± 3	37 ± 2	132 ± 16	44 ± 10
		333 µg	15 ± 5	13 ± 3	30 ± 3	87 ± 6	37 ± 4
		1000 µg	12 ± 3	16 ± 1^R	25 ± 8^R	25 ± 6^R	30 ± 9
		2500 µg	11 ± 4^P M	11 ± 3^P M R	30 ± 9^P R	6 ± 2^P M R	29 ± 10^P M
		5000 µg	6 ± 1^P M	12 ± 3^P M R	18 ± 3^P M R	2 ± 2^P M R	30 ± 8^P M
	2-AA	2.5 µg	506 ± 8	143 ± 18	3923 ± 200	5457 ± 247
	2-AA	10.0 µg					475 ± 38

Table 7.6.1/2 Summary of Experiment II

Study Name: 1875200	Study Code: Envigo 1875200
Experiment: 1875200 HV2 Pre	Date Plated: 20.02.2018
Assay Conditions:	Date Counted: 27.02.2018

 

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		9 ± 3	8 ± 2	26 ± 10	136 ± 17	29 ± 2
	Untreated		10 ± 3	8 ± 3	29 ± 7	196 ± 40	28 ± 4
	Blue	0.3 µg				139 ± 8
	chamomilla-	1 µg				125 ± 15
	European	3 µg				122 ± 7
	origin	10 µg				109 ± 6
		33 µg	9 ± 4	7 ± 2	27 ± 8	84 ± 18	23 ± 0
		100 µg	8 ± 4	10 ± 2	17 ± 4	32 ± 3	26 ± 4
		333 µg	8 ± 3	8 ± 1	15 ± 5	29 ± 2^R	27 ± 6
		1000 µg	9 ± 3	10 ± 1	15 ± 2^R	16 ± 2^M R	26 ± 8
		2500 µg	7 ± 2^P	7 ± 3^P	8 ± 0^P M R		28 ± 2^P
		5000 µg	9 ± 1^P M	5 ± 1^P M	2 ± 1^P M R		26 ± 7^P M
	NaN3	10 µg	1145 ± 70			2142 ± 148
	4-NOPD	10 µg			370 ± 30
	4-NOPD	50 µg		52 ± 15
	MMS	2.0 µL					680 ± 56
With Activation	DMSO		13 ± 4	12 ± 2	35 ± 9	107 ± 8	39 ± 12
	Untreated		13 ± 4	13 ± 2	41 ± 4	177 ± 7	32 ± 1
	Blue	0.3 µg				108 ± 3
	chamomilla-	1 µg				110 ± 14
	European	3 µg				124 ± 13
	origin	10 µg				124 ± 17
		33 µg	13 ± 6	13 ± 4	34 ± 7	113 ± 6	43 ± 10
		100 µg	13 ± 1	9 ± 3	31 ± 6	118 ± 6	41 ± 8
		333 µg	12 ± 4	12 ± 1	24 ± 1	39 ± 5^R	37 ± 11
		1000 µg	12 ± 2	13 ± 3	26 ± 9^R	23 ± 12^R	32 ± 10
		2500 µg	12 ± 4^P	12 ± 4^P M	15 ± 4^P M R		25 ± 8^P M
		5000 µg	8 ± 2^P	14 ± 3^P M	3 ± 2^P M R		27 ± 5^P M
	2-AA	2.5 µg	365 ± 7	197 ± 14	4121 ± 575	4029 ± 522
	2-AA	10.0 µg					245 ± 33

 

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	R M P	Reduced background growth Manual count Precipitate

Table 7.6.1/3/ Historical data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
	Solvent control	12	2.5	6	25	12	2.5	7	26
TA 1535	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
	Solvent control	10	2.2	6	19	13	3.5	7	30
TA1537	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
	Solvent control	25	4.4	13	43	34	6.2	15	58
TA 98	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
	Solvent control	156	26.0	78	209	148	32.3	73	208
TA 100	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
	Solvent control	41	5.6	27	63	50	6.8	28	72
WP2uvrA	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Mean = mean value of revertants/plate
SD = standard deviation

Min = minimal value/Max = maximal value

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.

Executive summary:

This study was performed to investigate the potential of Blue chamomilla - European origin to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was 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:

Strains TA 100:                              0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate

The remaining strains:                   33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in experiment I and from 333 µg/plate up to the highest investigated dose in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in both experiments. Based on a lower top dose of 1000 µg/plate no precipitation of the test substance was observed on the incubated agar plates in strain TA 100 in experiment II. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background in strains TA 1537, TA 98, and TA 100.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 98 and TA 100 with and without S9 mix in both experiments .

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Blue chamomilla - European origin at any dose 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 and showed a distinct increase of induced revertant colonies.

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, Blue chamomilla - European origin is considered to be non-mutagenic in thisSalmonella typhimuriumandEscherichia colireverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Schultz, 2018	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA	-S9 +S9	Up to cytotoxic or highest recommended concentration	-S9 : non mutagenic +S9 : non mutagenic

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no classification is proposed.