Matricaria recutita, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to the maximum limit in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

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:

27 September to 23 October, 2017

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

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:

Version / remarks:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Chemikaliengesetz (Chemicals Act) of the Federal Republic of Germany (inspec ted on July 13-16, 2015 / Signed on September 14, 2015)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: All formulations were prepared freshly before treatment and used within two hours of preparation.
- Solubility and stability of the test substance in the solvent/vehicle: Miscible in alcohol and other oils. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively.

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

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male Wistar rats treated with Phenobarbital/ß-naphthoflavone (80 mg/kg bw/day by oral route).

Test concentrations with justification for top dose:

Experiment I (plate incorporation test):
In the pre-experiment, eight concentrations of the test item between 3 and 5000 μg/plate were tested as followed:
- All strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
The experimental conditions in this pre-experiment were the same as described for the experiment I.
Since the required number of non-toxic concentrations was not obtained in strain TA 100 without S9 mix, this part of experiment I had to be repeated (reported as experiment Ia).
Experiment Ia (plate incorporation test):
The repeated experiment was performed as a plate incorporation assay with the following concentrations: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate.
Experiment II (pre-incubation test):
Since toxic effects were observed in experiment I a minimum of seven concentrations was tested in experiment II.
- Strains TA 1537 and TA 100: 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
- The remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
In experiment II the expected cytotoxicity was not detected in strain TA 1537 with and without S9 mix: Therefore this part had to be repeated as a pre-incubation assay (reported as experiment IIa)
Experiment IIa (pre-incubation test):
Strain TA 1537: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used for test item preparation: Ethanol (purity 99.99%).
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Preparation of test formulation: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

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).
The 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.

METHOD OF APPLICATION:
Experiment I and Ia: in agar (plate incorporation)
Experiment II and IIa: pre-incubation

DURATION
- Preincubation period: at 37°C for 60 minutes
- Exposure duration: plates were incubated upside down for at least 48 hours at 37°C

SELECTION AGENT (mutation assays): Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHERS:
- The colonies were counted using a validated computer system, which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test item the colonies were partly counted manually.

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.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
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.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, it is acceptable to conclude an equivocal response.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 1537 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: Not reported
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 μg/plate up to the highest investigated concentration. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 μg/plate up to the highest investigated concentration. The undissolved particles had no influence on the data recording.
- Other confounding effects: Not reported

HISTORICAL CONTROL DATA: The colony counts remain within the historical range of negative and solvent controls.
The laboratory´s historical control data from November 2014 until November 2016 represent approx. 600 experiments (WP2 uvrA the historical data are based on approx. 350 experiments).
- Positive historical control data as Mean value of revertants/plate (SD):
TA 1535: 1130 (143.1) -S9-mix ; 388 (58.2) +S9-mix
TA 1537: 82 (12.7) -S9-mix ; 191 (60.8) +S9-mix
TA 98: 378 (73.7) -S9-mix ; 3949 (771.8) +S9-mix
TA 100: 1966 (293.2) -S9-mix ; 3798 (830.4) +S9-mix
WP2 uvrA: 798 (362.7) -S9-mix ; 378 (112.6) +S9-mix
- Negative (solvent) historical control data as Mean value of revertants/plate (SD):
TA 1535: 12 (2.5) -S9-mix ; 12 (2.5) +S9-mix
TA 1537: 10 (2.2) -S9-mix ; 13 (3.5) +S9-mix
TA 98: 25 (4.4) -S9-mix ; 34 (6.2) +S9-mix
TA 100: 156 (26.0) -S9-mix ; 148 (32.3) +S9-mix
WP2 uvrA: 41 (5.6) -S9-mix ; 50 (6.8) +S9-mix
- Negative (untreated) historical control data as Mean value of revertants/plate (SD):
TA 1535: 12 (3.1) -S9-mix ; 12 (2.9) +S9-mix
TA 1537: 11 (2.7) -S9-mix ; 14 (4.0) +S9-mix
TA 98: 27 (4.9) -S9-mix ; 37 (6.5) +S9-mix
TA 100: 176 (23.6) -S9-mix ; 172 (25.4) +S9-mix
WP2 uvrA: 42 (5.8) -S9-mix ; 52 (6.8) +S9-mix

ADDITIONAL INFORMATION ON CYTOTOXICITY
See 'Any other information on results incl. tables'

MUTAGENICITY RESULTS
See 'Any other information on results incl. tables'

Table 7.6.1/2: Reduced normal background observed at the following concentrations (μg/plate):

Strain	Experiment I		Experiment Ia	Experiment II
	without S9 mix	with S9 mix	without S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	n.p.	/	/
TA 1537	1000 – 5000	1000 – 5000	n.p.	/	/
TA 98	/	/	n.p.	2500 – 5000	/
TA 100	1000 – 5000	1000 – 5000	333 – 2500	/	/
WP2 uvrA	/	/	n.p.	/	/

/ = normal background growth

In the repeated experiment IIa normal background growth was observed with and without S9 mix.

Table 7.6.1/3: Reduction in the number of revertants (below the induction factor of 0.5) observed at the following concentrations (μg/plate):

Strain	Experiment I		Experiment Ia
	without S9 mix	with S9 mix	without S9 mix
TA 1535	5000	/	n.p.
TA 1537	2500 – 5000	2500 – 5000	n.p.
TA 98	2500 – 5000	2500 – 5000	n.p.
TA 100	100 – 5000	333 – 5000	333 – 2500
WP2 uvrA	/	2500 – 5000	n.p.

Strain	Experiment II		Experiment IIa
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	n.p.	n.p.
TA 1537	/	/	2500 – 5000	2500 – 5000
TA 98	2500 – 5000	2500 – 5000	n.p.	n.p.
TA 100	100 – 2500	333 – 2500	n.p.	n.p.
WP2 uvrA	/	/	n.p.	n.p.

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

n.p. = not performed

Conclusions:

Under the test condition, test item is not mutagenic with and without metabolic activation in S.typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the range-finding test (plate incorporation, Experiment I) was predetermined as 3 to 5000 μg/plate.

The assay was performed in four independent experiments. Experiments I, II and IIa were performed with and without liver microsomal activation, experiment Ia was performed without liver microsomal activation. 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 Ia: Strain TA 100 without S9 mix: 0.3; 1; 3; 10; 33; 100; 333; 1000; and 2500μg/plate

Experiment II:

- Strains TA 1537 and TA 100 with and without S9 mix: 1; 3; 10; 33; 100; 333; 1000; and 2500μg/plate

- The remaining strains with and without S9 mix: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment IIa: Strain TA 1537 with and without S9 mix: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 μg/plate up to the highest investigated concentration. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 μg/plate up to the highest investigated concentration. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth 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), were observed in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item 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.

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation assay:

A bacterial reverse gene mutation assay (Ames test) was performed according to the OECD test guideline No. 471 and in compliance with GLP (Envigo, 2018). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

The test item precipitated in the overlay agar in the test tubes from 1000 μg/plate up to the highest investigated concentration. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 μg/plate up to the highest investigated concentration. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed reduced background growth 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), were observed in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item 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.

The substance is therefore considered as non-mutagenic according to the Ames Test.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.