Eucalyptus radiata australiana, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 March 2022 to 07 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and under compliance with GLP (GLP deviation: the homogeneity test on the test item was not supplied by the Sponsor. Without impact on the conclusion of the study).

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 2022-05-18

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

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 (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, loss of cell wall LPS (rfa mutation), ampicillin resistance, UVB and UVA sensitivity, spontaneous revertant rate and sensitivity 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) (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).
- 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:

- Assay 1 (+/- S9): 5, 15, 50, 150, 500 and 1000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2 (uvr A-)(pKM101) with S9 and without S9 under the direct plate incorporation method.
- Assay 2 (+/- S9): 5, 15, 50, 150, 500 and 1000 µg/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(uvr A-)(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: The test item is soluble in ethanol. A stock solution for each assay was prepared extemporaneously at 20 mg/mL in ethanol. The aspect of solution for assay n°1 and n°2 was a limpid colorless solution. Due to the toxicity, the highest dose tested was 1000 µg/plate.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

In the presence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (for 2-Nitrofluorene and 9-Aminoacridine) and NaCl 0.15 M (for Sodium Azide and Methyl methanesulfonate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

In the absence of S9-mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: 3 plates/dose for all groups

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 TA 100)
In a test tube, 0.1 mL of the bacterial suspension (1-9 x 103 bacteria/mL) and 50 µL 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:
->Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1 9 x109 bacteria/mL and 50 µL 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 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-Tryptophan 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 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 assay 2, as the first assay is negative in presence of test item, 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.

This method is known to increase the detection sensitivity of a number of promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471).

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 : DMSO, NaCl 0.15 M
- Vehicle used to solubilize test item: Ethanol
- Positive control

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)

Rationale for test conditions:

Tested up to cytoxicity concentration (1000 µg/plate).

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).

Statistics:

Not reported

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate with and without S9 in first assay // At 1000 µg/plate without S9 and from 500 to 1000 µg/plate with S9 in second assay

Vehicle controls validity:

valid

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 500 to 1000 µg/plate with and without S9 in first assay // From 500 to 1000 µg/plate without S9 and from 150 to 1000 µg/plate with S9 in second assay

Vehicle controls validity:

valid

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate without S9 and from 500 to 1000 µg/plate with S9 in first assay // At 1000 µg/plate without S9 and from 150 to 1000 µg/plate with S9 in second assay

Vehicle controls validity:

valid

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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate without S9 and from 500 to 1000 µg/plate with S9 in first assay // At 1000 µg/plate without S9 and from 150 to 1000 µg/plate with S9 in second assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate with and without S9

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 toxicity in presence of the test item for the three highest doses in the assay 1 (1500, 2500 and 5000 µg/plate), and toxicity for the highest dose in the assays 2 and 3 (1000 µg/plate). Therefore, the test item was tested at the following doses: 1 000, 500, 150, 50, 15 and 5 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.
- 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 strains and Escherichia coli WP2(uvrA¯) (pKM 101) strain without or with metabolic activation in the presence of the dose range from 5 to 1000 µg/plate.
- According to the toxicity measured in the bacteriostatic assay we can observe a light to high 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 assay.

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

Conclusions:

In the assay conditions, in presence of doses from 5 to 1000 µg/plate prepared from the test item EUCALYPTUS RADIATA OIL BATCH 406007, there is no evidence of any increase in the number of revertant colonies. Therefore, the test item does 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 with or without metabolic activation, according to the OECD Guideline n° 471.

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 EUCALYPTUS RADIATA OIL BATCH 406007 (Identification code: PH-22/0140) 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 and showed toxicity in presence of the test item for the three highest doses in the assay 1 (1500, 2500 and 5000 µg/plate), and toxicity for the highest dose in the assays 2 and 3 (1000 µg/plate). Therefore, two independent assays were carried out at the following doses: 1 000, 500, 150, 50, 15 and 5 µg/plate.

 

For assay n° 1, dose range from 5 to 1000 µg/plate was 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, similar dose range was 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. These results validate the two assays.

 

Evidence of toxicity, demonstrated by a light to high 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 strains and Escherichia coli WP2(uvrA¯)(pKM 101) strain without or with metabolic activation in the presence of the dose range from 5 to 1000 µg/plate.

 

In the assay conditions, in presence of dose range from 5 to 1000 µg/plate prepared from the test item EUCALYPTUS RADIATA OIL BATCH 406007 (Identification code: PH-22/0140), there is no evidence of any increase in the number of revertant colonies. Therefore, the test item does 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 with or without 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1/ ICARE, 2022	Ames Test (OECD 471) K, rel. 1	Gene mutation	S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)(pKM101)	-S9+S9	5, 15, 50, 150, 500 and 1000 µg/plate Up to cytotoxic  concentration	-S9 : non mutagenic +S9 : non mutagenic

 

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, solutions obtained from EUCALYPTUS RADIATA OIL BATCH 406007 (Identification code: PH-22/0140) 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 and showed toxicity in presence of the test item for the three highest doses in the assay 1 (1500, 2500 and 5000 µg/plate), and toxicity for the highest dose in the assays 2 and 3 (1000 µg/plate). Therefore, two independent assays were carried out at the following doses: 1 000, 500, 150, 50, 15 and 5 µg/plate.

 

For assay n° 1, dose range from 5 to 1000 µg/plate was 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, similar dose range was 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. These results validate the two assays.

 

Evidence of toxicity, demonstrated by a light to high 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 strains and Escherichia coli WP2(uvrA¯)(pKM 101) strain without or with metabolic activation in the presence of the dose range from 5 to 1000 µg/plate.

 

In the assay conditions, in presence of dose range from 5 to 1000 µg/plate prepared from the test item EUCALYPTUS RADIATA OIL BATCH 406007 (Identification code: PH-22/0140), there is no evidence of any increase in the number of revertant colonies. Therefore, the test item does 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 with or without 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.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No.1272/2008.

 

Self-classification:

Based on the available information, no additional classification is proposed.