Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 2015 to 09 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed accoring to OECD Guideline 471 and the GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

EC Number:

946-253-9

Molecular formula:

Not applicable

IUPAC Name:

Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Spearmint Essential Oil, ex Mentha spicata
- Physical state: Clear colourless to pale yellow liquid
- Analytical purity: confidental
- Impurities (identity and concentrations): confidental
- Purity test date: confidental
- Lot/batch No.: confidental
- Expiration date of the lot/batch: confidental
- Storage condition of test material: at room temperature

Method

Species / strain

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 specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital – 5,6-Benzoflavone induced S9 from Sprague Dawley rat liver

Test concentrations with justification for top dose:

Preliminary toxicity test: 50, 158, 500, 1580, 5000 µg/plate

Exp 1 (plate incorporation)
TA1535 - S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 +S9 3200, 1600, 800, 400, 200 and 100 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 ±S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1537 ±S9 200, 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate
WP2 uvrA –S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
WP2 uvrA +S9 5000, 2500, 1250, 625 and 313 µg/plate

Exp. 2 (pre incubation)
WP2 uvrA –S9 5000, 2500, 1250, 625, 313, 156 and 78.1 µg/plate
WP2 uvrA + S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
TA1535 + S9 3200, 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 − S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA100 + S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 − S9 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA1537 ± S9 400, 200, 100, 50.0, 25.0 and 12.5 µg/plate

Exp. 3 (pre incubation)
TA1535 −S9 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 µg/plate
TA1535 +S9 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity

Controls

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
Experiment I: Plate incorporation, Experiment II and III: preincubation
DURATION
- Preincubation period: 30 min
- Exposure duration: 72 h at 37 C

NUMBER OF REPLICATIONS: Three replicates

Solubility was determined in a preliminary test.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies
with increasing dose levels.

Statistics:

regression analysis, t test

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium 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

Test results 5

Key result

Species / strain:

E. coli WP2

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

Additional information on results:

- Precipitation: not detected

RANGE-FINDING/SCREENING STUDIES: the results of the preliminary toxicity test revealed cytotoxicity in some concentration levels, mentioned as thinning of the background lawn. Details can be found below, in section 'Any other information on results including tables'.

COMPARISON WITH HISTORICAL CONTROL DATA: some exceptions, but were considered acceptable
Sterility: confirmed based on absent clonies on additional agar plates.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: PRELIMINARY TOXICITY TEST WITHOUT METABOLIC ACTIVATION

Dose level (µg/plate)	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	WP2uvrA Rev/pl.
untreated	21	20	37	169	26
0.00	24	23	31	118	24
50.0	39	16*	35	121	24
158	25*	13*	36	108	28
500	59*	12*	26*	104	24
1580	65*	12*	40*	76*	18*
5000	50*	M	34*	M	13*

*: thinning of the background lawn
M: microcolony formation

Table 2: PRELIMINARY TOXICITY TEST WITH METABOLIC ACTIVATION

Dose level (µg/plate)	TA-1535 Rev/pl.	TA-1537 Rev/pl.	TA-98 Rev/pl.	TA-100 Rev/pl.	WP2uvrA Rev/pl.
untreated	19	21	35	169	34
0.00	22	27	32	132	31
50.0	23	15*	28	119	20
158	22	23*	46	133	27
500	28	16*	31*	123	30
1580	31*	12*	23*	75*	22
5000	13*	M*	18*	13*	17*

*: thinning of the background lawn
M: microcolony formation

**Tables of the main mutagenicity experiments 1, 2 and 3 can be found in the attached document (Tables_Results_Spearmint Spicata) below, section 'Attached background material'.

Applicant's summary and conclusion

Conclusions:

negative with and without S9. Spearmint Spicata did not induce reverse mutations in S. typhimurium or E.coli, in the presence and absence of metabolic activation, under the conditions of this test.

Executive summary:

In a bacterial reverse mutation assay, Spearmint Spicata was tested in order to examine its potential to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli. The following strains were used: TA1535, TA1537, TA98, TA100 and WP2uvrA. Experiments were conducted both in the absence and presence of metabolic activation (liver S9 fraction, induced with phenobarbitone and betanaphthoflavone). A preliminary test was performed in order to determine cytotoxicity and define appropriate concentration levels for the main experiments. Both the plate incorporation and pre incubation methods were used. The study was performed according to the OECD Guideline 471.

No precipitation was seen at all concentrations tested, while some toxicity was detected. Concentrations levels were adapted based on these reults. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The positive controls induced marked increases in the No of revertant colonies, which show the validity of this result.

It was concluded that Spearmint Spicata does not show any mutagenic activity under the conditions of this test. Therefore, the test substance does not need to be classified under the conditions of this test according to the criteria outlined in Annex I of 1272/2008/EC (CLP).