Savory, Satureja hortensis, 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:

23.04.2019 – 23.07.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD guideline 471 for testing chemicals (July 21, 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Savory, Satureja hortensis, ext.

EC Number:

283-922-8

EC Name:

Savory, Satureja hortensis, ext.

Cas Number:

84775-98-4

IUPAC Name:

Essential oil of Savory, Satureja hortensis, obtained from whole, dried herb by steam distillation

Test material form:

liquid

Specific details on test material used for the study:

ssentÍal oil of Summer Savory/batch No. SSVI.ES.sample
Date of manufacture: 08.01.2019
Expration date of the batch: 08.01.2021
UVCB substance;
Purity test date: 18.01.2019

Method

Target gene:

Salmonella typhimurium TA 98 – his D
Salmonella typhimurium TA 1537 – his C
Salmonella typhimurium TA 100 – his G
Salmonella typhimurium TA 1535 – his G
Escherichia coli WP (uvr A_(pKM101) - tryp E

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 applicable

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

S9-mix
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOXTM

Test concentrations with justification for top dose:

Results show a very high toxicity in presence of the doses from 1 500 to 5 000 µg/plate.
Therefore, the test item was tested at the following doses: 1 500, 450, 150, 45 and 15 µg/plate.

Vehicle / solvent:

Negative control Solvent:
Absolute Ethanol;
Dimethyl sulfoxide (DMSO);
Acetone;
NaCl 0.15 M

Positive control Solvent or Vehicle:
DMSO for 2-Nirofluorene, 9-Aminoacredine, 2 Anthramine;
NaCL0.15:M for Sodium Azide and for cis-Platinum (II) Diammine Dichloride
Acetone for 7,12-dimethylbenzanthracene

Moreover the following controls were carried out:
Negative controls:

- absolute negative control containing no test item corresponding to the spontaneous reversion rate,
- solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M.
Vehicle used to solubilize test item: Absolute Ethanol
Positive control (see table 2 of the study report)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine; cis-Platiniun (II) Diammine Dichloride

Details on test system and experimental conditions:

Bacterial strains
Strains of Salmonella typhimurium and Escherichia coli were purchased from MOLTOXTM. They are maintained in PHYCHER Bio développement.

Control of strains
The genotype of bacterial strains was checked:
Histidine and tryptophane requirements with cultures in presence and in absence of L-histidine and L-triptophane for Salmonella typhimurium and Escherichia coli strains respectively.
Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains.
Ampicillin resistance for the strains which have the pKM 101 plasmide.
uvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and uvr A mutation i.e. U.V.A sensitivity for Escherichia coli.
Spontaneous revertant rate.
Sensitivity to reference mutagens (see Table Reference mutagens (Positive controls))

Solutions preparation
The test item was found soluble in Absolute Ethanol at this concentration. A stock solution for each assay was prepared at 30 mg/mL in Ethanol. The highest dose tested was 1 500 µg / plate.


Assay No 1 Assay No 2
LEMI code : 19/0064-100519-S1 19/0064-240519-S1
Aspect-Color : Colorless clear solution Colorless clear solution
Solubility : completely soluble completely soluble
Stability : prepared extemporaneously prepared extemporaneously

Preparation of the metabolic activation system
Obtention of S9 fraction
S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) ((S9 Moltox-11101-5-39 validated on 06.2018 – expiry date: 07.02.2020).

Preparation of S9-mix 10 % (v/v)

Sterility tests
Test item and the corresponding dilutions were 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 were incubated for 48 - 72 hours at 37°C and then examined. There should be no bacterial growth on any plate. S9-mix sterility was checked using the same protocol.

Preliminary cytotoxicity testing (strain TA100)
In a test tube, 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of the stock solution and dilutions, were successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone was run in parallel.

In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.

Mutagenic assay
Assay without metabolic activation
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x109 bacteria/mL ) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer were 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 109 bacteria/mL and ) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer were successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth No 2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.

Plates were incubated at 37°C over a 48-72-hour period. The number of revertant colonies per plate was counted.
Moreover the following controls were carried out:

Negative controls : absolute negative control containing no test item corresponding to the spontaneous reversion rate, solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M
Vehicle used to solubilize test item : Absolute Ethanol Positive control.
Assay with metabolic activation
Two methodologies 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 471).
For the first assay direct incorporation method was used.
Assay repetition
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of test item, is negative, the pre-incubation test is performed for the second assay.

Presentation of data
After a 48-72-hour incubation period at 37° C, revertant colonies were manually counted in each plate.
Data are presented as the number of revertant colonies (mean standard deviation) per plate.

The following ratio is calculated:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Stable under the storage conditions, room temperature (15C0-25C0)

NUMBER OF REPLICATIONS: Triplicate.

Rationale for test conditions:

According to the OECD guideline 471 for testing chemicals Bacterial Reverse Mutation Test (July 21, 1997)

Evaluation criteria:

The following validity criteria were checked to validate each experiment:
Tthe 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).

Statistics:

According to the guideline 471, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Sterility controls
Test items

Results show the absence of any bacterial growth in the presence of the various concentrations of the test item.
"S9-mix"
Results show the absence of any bacterial growth in the presence of "S9-mix".

Bacteriostatic activity control

Results presented show a very high toxicity in presence of the doses from 1 500 to 5 000 µg/plate. Therefore, the test item was tested at the following doses: 1 500,450, 150, 45 and 15 µg/plate.

Mutation assay interpretation
There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.

There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (1 500, 450, 150, 45 and 15 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).We can observe a thinning of the bacterial lawn for the highest dose and a decrease in the number of revertants colonies correlated with the toxicity measured.

Results are confirmed in an independent experiment.

Any other information on results incl. tables

Results of sterility control of test item show the absence of any bacterial growth in the presence of the various concentrations of the test item and in the presence of "S9-mix".

Results of Bacteriostatic activity control show a very high toxicity in presence of the doses from 1 500 to 5 000 µg/plate. Therefore, the test item was tested at the following doses: 1 500, 450, 150, 45 and 15 µg/plate.

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.

 

There is no evidence of any increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (1 500, 450, 150, 45 and 15 µg/plate) without and with metabolic activation in (Salmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 and inEscherichia coli WP2(uvrA^-) (pKM 101).We can observe a thinning of the bacterial lawn for the highest dose and a decrease in the number of revertants colonies correlated with the toxicity measured.

 

Results are confirmed in an independent experiment.

Two independent assays were carried out.

For assay no 1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay no 2, various concentrations 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 the two 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, 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.

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 concentrations of the test item (1 500, 450, 150, 45 and 15 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).

Table: Sterility control

Serie	Doses	Colony number/plate	Colony number/plate	Colony number/plate
ControlNo1		1	2	3
Solution of Summer Savory oil BATCH: SSV1.ES.sample (LEMI code :19/0064-100519-S1)	1500 μg /plate   450 μg /plate   150 μg /plate   45 μg /plate   15 μg /plate	0   0   0   0   0	0   0   0   0   0	0   0   0   0   0
S9-mix	500 μL/plate	0	0	0
Control No 2		1	2	3
Solution of Summer Savory oil BATCH: SSV1.ES.sample (LEMI code :19/0064-240519-S1)	1500 μg /plate   450 μg /plate   150 μg /plate   45 μg /plate   15 μg /plate	0   0   0   0   0	0   0   0   0   0	0   0   0   0   0
S9-mix	500 μL/plate	0	0	0

Table : Bacteriostatic activity controls no 1

		Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)
		0 (negative control)	Absolute Ethanol	50 µg	150 µg	500 µg	1 500 µg	2 500 µg	5 000 µg
Solution of Summer Savory oil BATCH: SSV1.ES.sample	N1   N2   N3   N	627   572   543   581 ± 43	594   603   529   575± 40	526   590   536   551 ±34	579   580   586   582 ± 4	623   630   535   596 ± 53	130   135   98   121 ± 20	0   0   0   0 ± 0	0   0   0   0 ± 0
LEMI code: 19/0064-230419-S1	%	-	99%	95%	100%	103%	21%	0%	0%

Table: Bacteriostatic activity controls no 2

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

Doses (/plate)

0 (negative control)

Absolute Ethanol

1.5 μg

5 μg

15 μg

50 µg

150 µg

500 µg

1 500 µg

2 500 µg

5 000 µg

Solution of Summer Savory oil BATCH: SSV1.ES.sample

N1   N2   N3   N

526   571   543   547 ± 23

521   526   532   526 ± 6

570   519   521   537 ±29

560   526   537   541 ± 17

513   555   548   539 ±23

560   548   547   552±7

569   559   511   569±10

517   539   511   522 ±15

160   150   139   150±11

0   0   0   0±0

0   0   0   0±0

LEMI code: 19/0064-260419-S1

%

-

96%

98%

99%

99%

101%

104%

96%

27%

0%

0%

Table: Bacteriostatic activity controls no 3

		Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)
		0 (negative control)	Absolute Ethanol	500µg	800µg	1200µg	1 500 µg	2 500 µg	5 000 µg
Solution of Summer Savory oil BATCH: SSV1.ES.sample	N1   N2   N3   N	560   550   543   551±9	580   554   548   561±17	525   552   541   539 ±14	431   432   415   426±10	201   304   312   272±62	126   131   152   136 ±14	0   0   0   0±0	0   0   0   0±0
LEMI code: 19/0064-060519-S1	%	-	102%	98%	77%	49%	25%	0%	0%

Table: Bacteriostatic activity controls no 4

		Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)	Doses (/plate)
		0 (negative control)	Absolute Ethanol	15µg	45µg	150µg	450µg	1 500 µg
Solution of Summer Savory oil BATCH: SSV1.ES.sample	N1   N2   N3   N	528   499   521   516±15	520   515   489   508±17	557   524   523   535±19	535   546   511   531±18	574   535   563   557±20	550   618   512   560±54	130   119   142   130±12
LEMI code: 19/0064-100519-S1	%	-	98%	104%	103%	108%	109%	25%

N1    Number of colonies in plate 1

N2    Number of colonies in plate 2

N3    Number of colonies in plate 3

N     Mean per plate

%       Percent of survival compared to negative control

TA 1535 – Assay no1 of mutagenic activity

Table: Assay no1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	8	13	11	10.67	2.52	-
Positive control solvent	5 µL	8	12	13	11.00	2.65	-
Positive control : Sodium azide	5 µg in 5 µL	802	818	793	804.33	12.66	73.12
Vehicle	50µL	10	14	9	11.00	2.65	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	2   15   12   10	3   11   9   13	1   8   8   10	2.00   11.33   9.67   11.00	1.00   3.51   2.08   1.73	0.18   1.03   0.88   1.00
LEMI code: 19/0064-100519-S1	15µg	11	10	15	12.00	2.65	1.09

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	18	17	16	17.00	1.00	-
Positive control solvent	20µL	18	12	15	15.00	3.00	-
Positive control : 2-Anthramine	2µg in20µL	119	121	103	114.33	9.87	7.62
Vehicle	50µL	8	12	10	10.00	2.00	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	4   7   12   20	3   7   11   12	2   11   13   13	3.00   8.33   12.00   15.00	1.00   2.31   1.00   4.36	0.30   0.83   1.20   1.50
LEMI code: 19/0064-100519-S1	15µg	13	16	12	13.67	2.08	1.37

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 1535 – Assay no2 of mutagenic activity

Table : Assay no 2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	14	11	17	14.00	3.00	-
Positive control solvent	5 µL	14	20	10	14.67	5.03	-
Positive control : Sodium azide	5 µg in 5 µL	810	1048	856	904.67	126.24	61.68
Vehicle	50µL	12	9	10	10.33	1.53	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	3   7   12   9	7   9   11   14	3   11   13   11	4.33   9.00   12.00   11.33	2.31   2.00   1.00   2.52	0.42   0.87   1.16   1.10
LEMI code: 19/0064-240519-S1	15µg	11	9	13	11.00	2.00	1.06

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	11	16	15	14.00	2.65	-
Positive control solvent	10µL	13	15	15	14.33	1.15	-
Positive control : Sodium azide	1µg in10µL	84	135	91	103.33	27.65	7.21
Vehicle	50µL	10	17	19	15.33	4.73	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	1   4   11   11	0   6   12   15	2   5   19   18	1.00   5.00   14.00   14.67	1.00   1.00   4.36   3.51	0.07   0.33   0.91   0.96
LEMI code: 19/0064-240519-S1	15µg	18	15	14	15.67	2.08	1.02

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 1537 – Assay no1 of mutagenic activity

Table: Assay no 1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	5	4	5	4.67	0.58	-
Positive control solvent	20µL	6	5	4	5.00	1.00	-
Positive control : 9-Aminoacridine	50µg in 20 µL	1673	1675	1479	1609.00	112.59	321.80
Vehicle	50µL	6	4	5	5.00	1.00	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	1   4   4   5	1   3   5   7	2   5   4   8	1.33   4.00   4.33   6.67	0.58   1.00   0.58   1.53	0.27   0.80   0.87   1.33
LEMI code: 19/0064-100519-S1	15µg	5	6	4	5.00	1.00	1.00

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Assay n°1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	7	6	5	6.00	1.00	-
Positive control solvent	20µL	5	6	4	5.00	1.00	-
Positive control : 2-Anthramine	2 µg in 20 µL	32	46	58	45.33	13.01	9.07
Vehicle	50µL	8	6	6	6.67	1.15	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	4   7   10   10	3   5   8   8	1   5   5   11	2.67   5.67   7.67   9.67	1.53   1.15   2.52   1.53	0.40   0.85   1.15   1.45
LEMI code: 19/0064-100519-S1	15µg	6	8	6	6.67	1.15	1.00

* Light thinning of the bacterial lawn

 ** Thinning of the bacterial lawn

TA 1537 – Assay no2 of mutagenic activity

Table: Assay no 2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	5	10	3	6.00	3.61	-
Positive control solvent	20µL	9	11	5	8.33	3.06	-
Positive control : 9-Aminoacridine	50µg in 20 µL	1392	1213	1079	1228.00	157.04	147.36
Vehicle	50µL	4	4	6	4.67	1.15	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	1   4   11   6	2   1   7   3	1   6   10   8	1.33   3.67   9.33   5.67	0.58   2.52   2.08   2.52	0.29   0.79   2.00   1.21
LEMI code: 19/0064-240519-S1	15µg	14	4	6	8.00	5.29	1.71

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no 2 – with metabolic activation (10% S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	12	11	7	10.00	2.65	-
Positive control solvent	10µL	6	12	9	9.00	3.00	-
Positive control : 2-Anthramine	1 µg in 10 µL	37	61	57	51.67	12.86	5.74
Vehicle	50µL	10	6	5	7.00	2.65	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	2   5   8   8	2   1   10   14	1   4   4   9	1.67   3.33   7.33   10.33	0.58   2.08   3.06   3.21	0.24   0.48   1.05   1.48
LEMI code: 19/0064-240519-S1	15µg	9	13	10	10.67	2.08	1.52

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 98 – Assay no1 of mutagenic activity

Table: Assay no1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	21	19	20	20.00	1.00	-
Positive control solvent	20µL	25	21	23	23.00	2.00	-
Positive control : 2-Nitrofluorene	2 µg in 20 µL	197	208	235	213.33	19.55	9.28
Vehicle	50µL	20	18	21	19.67	1.53	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	0   16   13   19	9   11   15   14	1   15   13   18	3.33   14.00   13.67   17.00	4.93   2.65   1.15   2.65	0.17   0.71   0.69   0.86
LEMI code: 19/0064-100519-S1	15µg	20	18	15	17.67	2.52	0.90

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	21	27	22	23.33	3.21	-
Positive control solvent	20µL	25	23	26	24.67	1.53	-
Positive control : 2-Anthramine	2 µg in 20 µL	662	523	511	565.33	83.93	22.92
Vehicle	50µL	24	21	23	22.67	1.53	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	13   22   27   25	15   23   29   20	8   25   23   21	12.00   23.33   26.33   22.00	3.61   1.53   3.06   2.65	0.53   1.03   1.16   0.97
LEMI code: 19/0064-100519-S1	15µg	19	25	26	23.33	3.79	1.03

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 98 – Assay no2 of mutagenic activity

Table : Assay no2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	17	10	22	16.33	6.03	-
Positive control solvent	20µL	17	19	18	18.00	1.00	-
Positive control : 2-Nitrofluorene	2 µg in 20 µL	316	195	294	268.33	64.45	14.91
Vehicle	50µL	13	12	18	14.33	3.21	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	1   18   21   15	2   15   23   21	4   14   20   25	2.33   15.67   21.33   20.33	1.53   2.08   1.53   5.03	0.16   1.09   1.49   1.42
LEMI code: 19/0064-240519-S1	15µg	18	19	26	21.00	4.36	1.47

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	27	25	26	26.00	1.00	-
Positive control solvent	10µL	28	25	23	25.33	2.52	-
Positive control : 2-Anthramine	1 µg in 10 µL	461	618	419	499.33	104.89	19.71
Vehicle	50µL	24	24	21	23.00	1.73	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	2   19   32   28	5   16   39   34	7   13   28   22	4.67   16.00   33.00   28.00	2.52   3.00   5.57   6.00	0.20   0.70   1.43   1.22
LEMI code: 19/0064-240519-S1	15µg	35	34	31	33.33	2.08	1.45

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 100 – Assay no1 of mutagenic activity

Table: Assay no1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	68	63	67	66.00	2.65	-
Positive control solvent	20µL	71	68	68	69.00	1.73	-
Positive control : Sodium azide	20µg in 20 µL	1653	1562	1492	1569.00	80.73	22.74
Vehicle	50µL	76	83	86	81.67	5.13	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	15   50   77   80	21   44   71   77	18   52   65   68	18.00   48.67   71.00   75.00	3.00   4.16   6.00   6.24	0.22   0.60   0.87   0.92
LEMI code: 19/0064-100519-S1	15µg	74	83	71	76.00	6.24	0.93

 Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no1 – with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	87	81	85	84.33	3.06	-
Positive control solvent	20µL	91	85	80	85.33	5.51	-
Positive control : 2-Anthramine	2 µg in 20 µL	784	767	803	784.67	18.01	9.20
Vehicle	50µL	98	89	93	93.33	4.51	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	32   66   98   80	29   71   85   81	45   74   91   83	35.33   70.33   91.33   81.33	8.50   4.04   6.51   1.53	0.38   0.75   0.98   0.87
LEMI code: 19/0064-100519-S1	15µg	83	90	79	84.00	5.57	0.90

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

TA 100 – Assay no2 of mutagenic activity

Table: Assay no2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	73	69	74	72.00	2.65	-
Positive control solvent	20µL	64	79	73	72.00	7.55	-
Positive control : Sodium azide	20µg in 20 µL	1583	1459	1570	1537.33	68.15	21.35
Vehicle	50µL	104	76	71	83.67	17.79	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	10   59   78   77	9   70   65   78	7   63   70   79	8.67   64.00   71.00   78.00	1.53   5.57   6.56   1.00	0.10   0.76   0.85   0.93
LEMI code: 19/0064-240519-S1	15µg	67	76	69	70.67	4.73	0.84

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay no2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	96	88	104	96.00	8.00	-
Positive control solvent	10µL	95	105	97	99.00	5.29	-
Positive control : 2-Anthramine	1 µg in 10 µL	381	303	348	344.00	39.15	3.47
Vehicle	50µL	103	104	91	99.33	7.23	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	5   82   63   85	10   23   72   81	3   39   82   77	6.00   48.00   72.33   81.00	3.61   30.51   9.50   4.00	0.06   0.48   0.73   0.82
LEMI code: 19/0064-240519-S1	15µg	72	74	68	71.33	3.06	0.72

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

E. COLI – Assay no1 of mutagenic activity

Table : Assay no1 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	149	158	139	148.67	9.50	-
Positive control solvent	10µL	163	152	149	154.67	7.37	-
Positive control : cis-Platinum (II)	1 µg in 10 µL	603	595	563	587.00	21.17	3.80
Vehicle	50µL	139	142	153	144.67	7.37	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	42   88   130   129	53   78   122   144	39   77   111   143	44.67   81.00   121.00   138.67	7.37   6.08   9.54   8.39	0.31   0.56   0.84   0.96
LEMI code: 19/0064-100519-S1	15µg	161	154	139	151.33	11.24	1.05

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Table: Assay n°1– with metabolic activation (10 % S9-mix) – without pre-incubation

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	203	173	182	186.00	15.39	-
Positive control solvent	5 µL	199	211	189	199.67	11.02	-
Positive control : Dimethylbenzanthracene	5 µg in 5 µL	720	830	634	728.00	98.24	3.65
Vehicle	50µL	205	216	181	200.67	17.90	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	61   147   189   178	42   152   219   203	51   139   201   191	51.33   146.00   203.00   190.67	9.50   6.56   15.10   12.50	0.26   0.73   1.01   0.95
LEMI code: 19/0064-100519-S1	15µg	195	208	191	198.00	8.89	0.99

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

E. COLI – Assay no2 of mutagenic activity

Table : Assay no2 – without metabolic activation (-S9-mix)

Serie	Dose/Plate	Plate no1	Plate no2	Plate no3	Mean	Standard deviation	R
Negative control	100 µL	130	152	119	133.67	16.80	-
Positive control solvent	10µL	130	147	148	141.67	10.12	-
Positive control : cis-Platinum (II)	1 µg in 10 µL	510	412	423	448.33	53.69	3.16
Vehicle	50µL	141	111	144	132.00	18.25	-
Solution of S ummer S avory oil BATCH: SSV1.ES.sample	1500 µg**   450µg*   150 µg   45µg	47   96   130   148	58   72   128   129	51   63   120   130	52.00   77.00   126.00   135.67	5.57   17.06   5.29   10.69	0.39   0.58   0.95   1.03
LEMI code : 19/0064-240519-S1	15µg	140	134	136	136.67	3.06	1.04

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Assay no2 – with metabolic activation (10 % S9-mix) – with pre-incubation

Serie	Dose/Plate	Plate no1	Plateno2	Plateno3	Mean	Standard deviation	R
Negative control	100 µL	225	202	188	205.00	18.68	-
Positive control solvent	5 µL	218	183	179	193.33	21.46	-
Positive control : Dimethylbenzanthracene	2.5µg in 5 µL	612	704	656	657.33	46.01	3.40
Vehicle	50µL	181	177	190	182.67	6.66	-
Solution of Summer Savory oil BATCH: SSV1.ES.sample	1500 µg**   1500 µg*   150 µg   45µg	19   118   154   190	15   150   141   149	10   123   148   150	14.67   130.33   147.67   163.00	4.51   17.21   6.51   23.39	0.08   0.71   0.81   0.89
LEMI code: 19/0064-240519-S1	15µg	210	202	189	200.33	10.60	1.10

* Light thinning of the bacterial lawn

** Thinning of the bacterial lawn

Historical control values (2009 - 2018)

2009 to 2018

Without metabolic activation

With metabolic activation (without pre-incubation)

With metabolic activation (with pre-incubation)

n

Mean

min/max

n

Mean

min/max

n

Mean

min/max

Salmonella typhimurium TA 1535

Spontaneous reversion

921

11.1±3.7

4.0/23.0

Spontaneous reversion

492

12.3±4.1

3.0/23.0

Spontaneous reversion

486

13.0±4.3

5.0/25.0

Salmonella typhimurium TA 1535

Sodium Azide

921

749.3±220.1

190.0/1487.0

2-Anthramine

492

111.6±57.0

26.0/269.0

2-Anthramine

486

76.6±38.1

25.0/217.0

Salmonella typhimurium TA 1537

Spontaneous reversion

921

6.0±2.6

1.0/20.0

Spontaneous reversion

492

8.0±3.6

1.0/24.0

Spontaneous reversion

486

8.1±3.4

1.0/21.0

Salmonella typhimurium TA 1537

9-Aminoacridine

921

898.9±460.1

224.0/1967.0

2-Anthramine

492

54.6±24.3

24.0/170.0

2-Anthramine

486

46.8±23.7

21.0/182.0

Salmonella typhimurium TA 98

Spontaneous reversion

921

15.9±3.9

6.0/29.0

Spontaneous reversion

492

23.2±5.0

11.0/38.0

Spontaneous reversion

486

23.1±5.5

10.0/36.0

Salmonella typhimurium TA 98

2-Nitrofluorene

921

492.4±224.5

187.0/1667.0

2-Anthramine

492

579.2±223.2

220.0/1499.0

2-Anthramine

486

458.7±193.6

174.0/1368.0

Salmonella typhimurium TA 100

Spontaneous reversion

921

59.2±11.3

40.0/112.0

Spontaneous reversion

489

91.7±17.8

55.0/152.0

Spontaneous reversion

486

91.0±19.3

51.0/147.0

Salmonella typhimurium TA 100

Sodium Azide

921

1019.7±331.9

381.0/1690.0

2-Anthramine

489

839.2±362.6

361.0/2163.0

2-Anthramine

489

634.1±275.9

309.0/1687.0

Salmonella typhimurium TA 102

Spontaneous reversion

339

167.9±39.1

109.0/285.0

Spontaneous reversion

174

290.0±42.8

196.0/384.0

Spontaneous reversion

168

284.5±44.3

195.0/402.0

Salmonella typhimurium TA 102

Mitomycin C

339

722.7±177.5

447.0/1632.0

Benzo[a]pyrene

174

1054.5±284.9

490.0/2510.0

Benzo[a]pyrene

168

1000.4±264.1

501.0/2450.0

Escherichia coli WP2 (pKM 101) (uvr A-)

Spontaneous reversion

753

85.0±35.3

41.0/188.0

Spontaneous reversion

402

154.6±36.5

64.0/263.0

Spontaneous reversion

402

158.2±37.4

69.0/250.0

Escherichia coli WP2 (pKM 101) (uvr A-)

cis-Platinium (II) Diamine Dichloride

723

477.9±170.8

248.0/1089.0

Dimethyl Benzanthracene

372

675.2±247.6

365.0/1680.0

Dimethyl Benzanthracene

372

661.4±228.8

281.0/1680.0

Escherichia coli WP2 (pKM 101)

Spontaneous reversion

0

-±-

-/-

Spontaneous reversion

0

-±-

-/-

Spontaneous reversion

0

-±-

-/-

Escherichia coli WP2 (pKM 101)

Mitomycin C

30

101.5±36.1

56.0/185.0

Benzo[a]pyrene

30

154.8±55.8

85.0/262.0

Benzo[a]pyrene

30

166.8±43.6

96.0/240.0

Applicant's summary and conclusion

Conclusions:

Doses ( 1 500, 450, 150, 45 and 15 µg/plate) prepared from solutions of the test item Summer Savory oil BATCH: SSV1.ES.sample (LEMI code : LM-19/0064), 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 Guideline n°471.
There is no mutagenic effect in the conditions of the conducted experiment.

Executive summary:

In a reverse gene mutation assay in bacteria (16_399-007M), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to solutions of the test item Summer Savory oil (BATCH: SSV1.ES.sample.). Solutions obtained from LM-19/0064 have been tested for their capacity to induce reverse mutation in four Salmonella typhimuriumstrains and one Escherichia coli WP2(uvr A^¯)(pKM101)strain. This study was performed in the absence and presence of metabolic activation.The study was performed according to the method of Guideline OECD 471(acterial Reverse Mutation Assay) in compliance with Good Laboratory Practice.

Two independent assays were carried out

For assay no1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay no 2, various concentrations 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 the two 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, 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.

These results validate the two tests.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (1 500, 450, 150, 45 and 15 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) in the conditions of the conducted experiment.

There is no mutagenic effect in the conditions of the conducted experiment.

Therefore, solution of Summer Savory oil is not considered as mutagenic according to CLP regulation (EC) N° 1272/2008 under the conditions of the conducted experiment.