Registration Dossier

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD guideline 471 for testing chemicals (July 21, 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
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 resultsopen allclose all
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
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
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
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
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.