Sage, Salvia officinalis, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one

EC Number:

208-912-2

EC Name:

1-isopropyl-4-methylbicyclo[3.1.0]hexan-3-one

Cas Number:

546-80-5

Molecular formula:

C10H16O

IUPAC Name:

(1S,4R,5R)-4-methyl-1-propan-2-ylbicyclo[3.1.0]hexan-3-one

Constituent 2

Reference substance name:

DL-bornan-2-one

EC Number:

244-350-4

EC Name:

DL-bornan-2-one

Cas Number:

21368-68-3

Molecular formula:

C10H16O

IUPAC Name:

1,7,7-trimethylbicyclo[2.2.1]heptan-2-one

Constituent 3

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 4

Reference substance name:

(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one

Cas Number:

471-15-8

Molecular formula:

C10H16O

IUPAC Name:

(1S, 4S, 5R)-4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one

Constituent 5

Reference substance name:

Camphene

EC Number:

201-234-8

EC Name:

Camphene

Cas Number:

79-92-5

Molecular formula:

C10H16

IUPAC Name:

2,2-diméthyl-3-méthylène-bicyclo[2,2,1]heptane

Constituent 6

Reference substance name:

Humulene

EC Number:

229-816-7

EC Name:

Humulene

Cas Number:

6753-98-6

Molecular formula:

C15H24

IUPAC Name:

(1E,4E,8E)-2,6,6,9-tetramethylcycloundeca-1,4,8-triene

Constituent 7

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 8

Reference substance name:

Caryophyllene

EC Number:

201-746-1

EC Name:

Caryophyllene

Cas Number:

87-44-5

Molecular formula:

C15H24

IUPAC Name:

(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene

Constituent 9

Reference substance name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

EC Number:

207-353-1

EC Name:

(1S-endo)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Cas Number:

464-45-9

Molecular formula:

C10H18O

IUPAC Name:

endo-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol

Constituent 10

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Test material form:

liquid

Details on test material:

-Name: SAGE DALMATIAN OIL
-IUPAC Name: Essential oil of Salvia officinalis (Lamiaceae) obtained from leaves, flowers and stalks by steam distillation.
-CAS: 84082-79-1
-EINECS: 282-025-9
-Batch no.: BU201707
-Purity: 100 % wt – UVCB substance
-Appearance: pale to slightly yellow
-Expiry date: 03/07/2019
-Storage: Sample should be stored tightly closed, below ambient temperature, preferably under inert gas atmosphere in dark, lightproofed bottles.

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 50, 150, 500, 1500 and 5000 μg/plate

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with S9 mix): 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

paraffin oil.
The item is soluble. An extemporaneous stock solution for each assay was prepared at 50 mg/mL in paraffin oil (not soluble in water, NaCl 0.15M).

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

sodium azide

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cis-Platinum (II) Diammine Dichloride, 2-Nitrofluorene and 2-Anthramine

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
Strains of Salmonella typhimurium and Escherichia coli are purchased from MOLTOX

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 37 °C for 30 minutes (with shaking)
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48-72 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity)

Rationale for test conditions:

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely
 There should be a mimimum of four non-toxic test item dose levels
 the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %.
 The spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
 the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
 the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
 Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Results and discussion

Test results

Key result

Species / strain:

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

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

Positive controls validity:

valid

Additional information on results:

STERILITY CONTROLS:
absence of any bacterial growth in the presence of the various concentrations of the test item.
absence of any bacterial growth in the presence of "S9-mix".

BACTERIOSTATIC ACTIVITY CONTROL:
neither original solution nor dilutions on TA100 have bacteriostatic effect. The test item is tested at these doses (5 000, 1 500, 500, 150 and 50 μg/plate).

MUTATION ASSAY
All controls (positive and negative with and without metabolic activation) showed the expected response when compared with the corresponding historical values obtained in the laboratory .
In the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 and 50 μg/plate) without and with metabolic activation (direct and indirect incorporation methods) we measure no increase in the number of revertant colonies at least two-fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three-fold for TA 1535 and TA 1537 in the presence of the test item stock solution and dilutions (5 000, 1 500, 500, 150 and 50 μg/plate).
The test item (code as) GQP021117 not show any excessive toxic effect and precipitate formation in any strain at all doses studied.
Results are confirmed in an independent experiment.

Applicant's summary and conclusion

Conclusions:

Under the test condition, test substance is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

Executive summary:

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays (plate incorporation and pre-incubation methods) were carried out

For assay n° 1, plate incorporation method, various concentrations of the test item were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2,pre-incubation method, various concentrations of the test item were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For both assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions in negative and vehicle controls, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory. The vehicle control plates gave counts of revertant colonies within the normal and historical range.

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (5 000, 1 500, 500, 150 and 50 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101).The test item (code as) GQP021117 not show any excessive toxic effect and precipitate formation in any strain at all doses studied.

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) performed from solutions of the test item , do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.