Sage, Salvia lavandulifolia, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

GLP study conducted according to OECD test guideline No. 471 without deviations

Adequacy of study:

key study

Study period:

4 june to 28 september 2018

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sage, Salvia lavandulifolia, ext.

EC Number:

290-272-9

EC Name:

Sage, Salvia lavandulifolia, ext.

Cas Number:

90106-49-3

Molecular formula:

no relevant UVCB

IUPAC Name:

Sage, Salvia lavandulifolia, ext.

Test material form:

liquid

Details on test material:

UVCB

Specific details on test material used for the study:

Name : SPANISH SAGE OIL (Salvia lavandulifolia) Batch n° LS104585
Container : glass flask
Quantity : 19.78696 g (content + container)
Category : Chemical substance
Date of reception* : 24.05.2018
LEMI Code : LM-18/0169
Purity : 100%
Batch production date* : 08.2017
Expiry date* : 08.2021

Method

Species / strain

Species / strain / cell type:

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

Test concentrations with justification for top dose:

The test item is tested at these doses (5 000, 1 500, 500, 150 and 50 µg/plate).

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride

Details on test system and experimental conditions:

sterility tests : Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45°C, and poured after homogenization on the bottom agar (20 ml) onto a Petri plate (90 mm in diameter) (n = 3). Plates are incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.
WITHOUT METABOLIC ACTIVATION :
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x10 9 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).
Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 10 9 bacteria/mL and 0.1 mL (in aqueous or oily vehicle) / 50 µL (in non-aqueous or non-oily vehicule as DMSO ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL. Plates are incubated at 37°C over a 48-72 hour period. The number of revertant colonies per plate is counted.
WITH METABOLIC ACTIVATION :
Two protocols can be used:
 either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ;
 or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate.
This method is known to increase the detection sensitivity of a number of promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471).
For the first assay direct incorporation method is used.

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:

no 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:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Applicant's summary and conclusion

Conclusions:

Doses (5 000, 1 500, 500, 150 and 50 µg/plate) performed from solutions of the test item SPANISH SAGE OIL (Salvia lavandulifolia) Batch n° LS104585 (LEMI code : LM-18/0169), provided by Esencias Martínez Lozano, S.A. 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.