[No public or meaningful name is available]

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 23 November 2021 to 26 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 21 July 1997 and corrected 26 June 2020

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

litsea cubeba (fruit) extract – terpenes fraction

Molecular formula:

not applicable

IUPAC Name:

litsea cubeba (fruit) extract – terpenes fraction

Test material form:

liquid

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). They are maintained in the laboratory (lyophilized discs stored at 2-8°C from TRINOVA BIOCHEM) according to internal SOP.

The genotype of bacterial strains was checked for:
- 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 plasmid.
- U.V.B sensitivity for Salmonella typhimurium and U.V.A sensitivity for Escherichia coli.
- Spontaneous revertant rate.
- Sensitivity to reference mutagens.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOX(TM) (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11-101-5-4477 validated on 11.10.2021 – expiry date: 13.07.2023).

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:

First assay: 30, 90, 300, 900, 1500 and 3000 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Second assay: 30, 90, 300, 900, 1500 and 3000 μg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) without S9 and with S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A stock solution for each assay was prepared extemporaneously at 60 mg/mL in ethanol. The aspect of solution for assay n°1 and n°2 was an opalescent solution for stock solution and clear colorless for dilutions. Due to the toxicity, the highest dose tested was 3 000 µg/plate.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO for 9-aminoacridine and 2-nitrofluorene and NaCL0.15M for sodium azide and cis-Platinum (II) Diammine Dichloride

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO for 2-anthramine and acetone for Dimethyl-benzanthracene

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine: 2 and 1 µg/plate for TA98, TA100, TA1537 and TA1535 without and with preincubation respectively. Dimethyl-benzanthracene: 5 and 2.5 µg/plate for Escherichia coli WP2 (uvr A-) (pKM101) without and with preincubation respectively.

Remarks:

with S9 mix

Details on test system and experimental conditions:

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.

BACTERIOSTATIC ACTIVITY CONTROL (determination of cytotoxicity):
In a test tube, 0.1 mL of the bacterial suspension (1-9 x 10^3 bacteria/mL) and 50 µL (as ethanol is used as a solvent) 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.

MAIN TESTS:
- 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 ethanol is used as a solvent) 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 the 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 (as ethanol is used as a solvent) 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 and assessed for evidence of toxicity.

- with S9: In the 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; In the assay n°2, as the first assay, in presence of test item, is negative, the pre-incubation method 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.

NUMBER OF REPLICATIONS: 3 plates/dose for all groups

DATA PRESENTATION:
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)

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).

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 assay.

Statistics:

Data are presented as the number of revertant colonies (mean +/- standard deviation) per plate.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 300 to 3000 µg/plate without S9 and from 900 to 3000 µg/plate with S9 in first assay From 900 to 3000 µg/plate with and without S9 in second assay

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 90 to 3000 µg/plate without S9 and from 900 to 3000 µg/plate with S9 in both assay

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 300 to 3000 µg/plate without S9 and from 900 to 3000 µg/plate with S9 in both assay

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 900 to 3000 µg/plate with and without S9 in both assay

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 3000 µg/plate with and without S9 in first assay At 3000 µg/plate without S9 and from 900 to 3000 µg/plate with S9 in second assay

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

CYTOTOXICITY TEST
Results show a high toxicity in presence of the highest dose of the test item (5 000 µg/plate). In presence of 3 000 µg/plate the toxicity is high but compatible with the maximum acceptable higher of 25% of survival. Therefore, the test item was tested at the following doses: 3 000, 1 500, 900, 300, 90 and 30 µg/plate.

MUTAGENICITY TEST:
- 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.
- The numbers of revertant colonies in all cases did not exceed the criteria for a positive response and do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli WP2(uvr A¯)(pKM101) strain without, or with metabolic activation.
- According to the toxicity measured in the bacteriostatic assay we can observe a thinning of the bacterial lawn observed in presence of the highest doses tested in presence and in absence of metabolic activation.
- Results are confirmed in an independent experiment.


HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data of the test facility.

OTHERS:
- Sterility test showed absence of any bacterial growth in the presence of the various concentrations of the test item and in the presence of S9-mix.

Any other information on results incl. tables

See table of results attached below.

Applicant's summary and conclusion

Conclusions:

Under the test condition, doses (5, 15, 50, 150 and 500 µg/plate), prepared from the test item did not induce an increase in revertant colonies of all strains without or with metabolic activation, according to the OECD guideline No. 471.
Therefore, the test material is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, solutions obtained from Solution of LITSEA CUBEBA TERPENES L62840 BATCH:100190967 (Identification code : PH-21/01002) have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. To determine the dose-range concentrations, a preliminary cytotoxicity testing was performed. A high toxicity in presence of the highest dose of the test item (5 000 µg/plate) was showed, and in presence of 3 000 µg/plate the toxicity is high but compatible with the maximum acceptable higher of 25% of survival. Therefore, two independent assays were carried out at the following doses: 3 000, 1 500, 900, 300, 90 and 30 µg/plate.

 

For assay n° 1, dose range from 30 to 3000 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, dose range from 30 to 3000 µg/plate were put in contact with the strains in the absence of metabolic activation (30-minute at 37°C) 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. These results validate the two assays.

 

Evidence of toxicity, demonstrated by a thinning of the bacterial lawn of non-revertant bacteria was observed in presence of the highest doses tested in presence and in absence of metabolic activation, in both the plate incorporation and the pre-incubation method.

The numbers of revertant colonies in all cases did not exceed the criteria for a positive response and do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli  WP2(uvr A¯)(pKM101) strain without or with metabolic activation in the presence of the various concentrations of the test item (3 000, 1 500, 900, 300, 90 and 30 µg/plate).

 

In the assay conditions, in presence of doses (range from 30 to 3000 µg/plate) prepared from the test item, the numbers of revertant colonies in all cases did not exceed the criteria for a positive response and do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 strains and Escherichia coli  WP2(uvr A¯)(pKM101) strain without, or with metabolic activation, according to the OECD Guideline n° 471.

Therefore, the test material is not mutagenic in the presence and absence of metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains. This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.