Coriander, 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:

From 12 JULY 2021 to 29 JULY 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21st, 1997 and corrected June 26th, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2021-10-27

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Coriander, ext.

EC Number:

283-880-0

EC Name:

Coriander, ext.

Cas Number:

84775-50-8

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Essential oil of Coriandrum sativum L. (Apiaceae) obtained from seeds of coriander by steam distillation

Test material form:

liquid

Details on test material:

- Name of test substance: CORIANDRE GRAINES ESS C43451
- Batch No.: 5030061915
- Storage: room temperature, keep away from light
- Expiration date of the lot/batch: 15 september 2022

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

Details on mammalian cell type (if applicable):

Strains of Salmonella typhimurium and Escherichia coli were purchased from MOLTOX (TM) and maintained in the laboratory. The genotype of bacterial strains was checked for histidine and tryptophane requirements, loss of cell wall LPS (rfa mutation), ampicillin resistance, UVB and UVA sensitivity, spontaneous revertant rate and sensitivuty to reference mutagens.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOX (TM).
- method of preparation of S9 mix:
The final concentration of co-factors and salts was as follows:
S9 fraction 10%
MgCl2-6H2O 8 mM
KCl 33 mM
Glucose-6-phosphate Na2 5 mM
NADP Na2 4 mM
Phosphate buffer (pH 7.4) 0.1 M

Test concentrations with justification for top dose:

- Experiment 1 (+/- S9): 25, 75, 250, 750, 1500 and 2500 µg/plate (up to cytotoxic concentration) for plate incorporation method.
- Experiment 2 (+/- S9): 25, 75, 250, 750, 1500 and 2500 µg/plate (up to cytotoxic concentration) for pre-incubation method

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Absolute ethanol
- Justification for choice of solvent/vehicle: The test item is soluble in Absolute ethanol. A stock solution for each assay was prepared extemporaneously at 100 mg/mL in Absolute ethanol. The aspect of solution for assay n°1 and n°2 was a colorless solution. The highest dose tested was 2 500 µg / plate because a high toxicity was showed from 2500 to 5000 µg/plate.

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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

In the presence of S9-mix

Positive control substance:

other: 2-Anthramine 2 µg/plate for TA98, TA100, TA1535 and TA 1537 without pre-incubation 1 µg/plate for TA98, TA100, TA1535 and TA 1537 with pre-incubation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO or NaCLO 15 M

True negative controls:

no

Positive controls:

yes

Remarks:

In the absence of S9-mix

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Sodium azide 20 µg/plate for TA 100 and 5 µg/plate for TA 1535

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

PRELIMINARY CYTOTOXICITY TESTING
In a test tube, 0.1 mL of the bacterial suspension (1-9 x 10^3 bacteria/mL) and 50 µL (as absolute 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 24-72 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.

METHOD OF TREATMENT/ EXPOSURE:
- Assay without S9:
For each Salmonella Typhimurium strains, 0.1 mL of the bacterial suspension containing 1-9 x10^9 bacteria/mL and 50 µL (as aboslute 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).
For Escherichia coli strain, in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 10^9 bacteria/mL and 50 µL 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 n° 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 .

- Assay with S9:
Two methodologies were used:
- In assay n°1, 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;
- As the first assay, in presence of test item negative, the pre-incubation assay is performed for the second assay. 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.

Moreover the following controls were carried out:
- Negative controls :
o absolute negative control containing no test item corresponding to the spontaneous reversion rate,
o solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M
- Vehicle used to solubilize test item: Absolute ethanol
- Positive control

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not reported
- Exposure duration/duration of treatment: 48-72 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Tested up to cytoxicity concentration (2500 µg/plate).

Evaluation criteria:

EVALUATION CRITERIA
The following validity criteria were checked to validate each experiment:

- 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,
- In presence of the solvent the spontaneous reversion rate shall comply with the historical values of the absolute negative control of the laboratory.
- 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).


CRITERIA CONCLUSION
The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains without and with metabolic activation.

The result of the test is considered positive if a dose-reponse relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is 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.

All results must be confirmed in an independent experiment.

Statistics:

Not reported

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 2500 µg/plate

Vehicle controls validity:

valid

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 2500 µg/plate

Vehicle controls validity:

valid

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

up to 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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

BACTERIOSTATIC ACTIVITY CONTROL
Results show a high toxicity equal to 100 % in presence of the highest dose 5 000 µg/plate. In presence of 2 500 µg/plate one can observe a toxicity compatible with the maximum tolerated 75%. Therefore, the test item was tested at the following doses: 2 500, 1 500, 750, 250, 75 and 25 µg/plate.

MUTATION ASSAY INTERPRETATION
- There is no 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 without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2(uvr A¯)(pKM101) strain.
- Evidence of toxicity, demonstrated by a thinning of the bacterial lawn of non-revertant in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains was shown. For Escherichia coli WP2(uvr A¯)(pKM101) strain, a decrease of the number of spontaneous reversions in presence of the highest doses is observed.

Results are confirmed in an independent experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: cf. attached document
- Negative (solvent/vehicle): cf. attached document

Any other information on results incl. tables

Tables of results were attached in the section "Attached background material".

Applicant's summary and conclusion

Conclusions:

In the assay conditions, in presence of doses (range from 25 to 2500 µg/plate) prepared from the test item CORIANDRE GRAINES ESS C43451 BATCH: 5030061915 provided by Linalool Consortium, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response for Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2 (uvrA-)(pKM101) strain without, or with metabolic activation, according to the OECD Guideline n°471.

Executive summary:

In a reverse gene mutation assay in bacteria, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to cytotoxic concentration, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

To determine the dose-range concentrations, a preliminary cytotoxicity testing was performed. A high toxicity from 2500 to 5000 μg/plate was showed, therefore, two independent mutagenic assays were carried out at the following doses: 2 500, 1 500, 750, 250, 75 and 25 µg/plate.

For assay n° 1, dose range 25 to 2500 µg/plate 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, the same dose range 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 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.

 

Evidence of toxicity, demonstrated by a thinning of the bacterial lawn of non-revertant in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains was shown. For Escherichia coli WP2(uvr A-)(pKM101) strain, a decrease of the number of spontaneous reversions in presence of the highest doses is observed.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (2 500, 1 500, 750, 250, 75 and 25 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 this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the test item was considered to be non-mutagenic.

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