Elemol-rich fraction of essential oil of Canarium commune (Burseraceae) obtained from exudate by fractional distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic based on classified constituents

Ames test positive in TA98 with metabolic activation according to OECD Guideline 471

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Key result

Species / strain:

other: not relevant

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: not relevant

Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the mutagenic potential of the registered substance.

The registered substance has not been tested itself in appropriate in vitro or in vivo tests but one of its constituents is classified as mutagenic Cat. 2 (methyl eugenol) and potentially present above the CLP generic concentration limit of 1% that triggers classification of the mixture. Therefore, the registered substance is classified for mutagenicity Category 2 according to the Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May- August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method:
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 10, 30, 100, 300, 500 and 1000 μg/plate

Test for Mutagenicity (Experiment 2) :
Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA (with and without S9 mix): 10, 30, 100, 300, 500 and 1000 μg/plate except for TA98 with metabolica activation : 30, 100, 300, 500, 750 and 1000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cis-Platinum (II) Diammine Dichloride, 2-Nitrofluorene and 2-Anthramine

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM:
Strains of Salmonella typhimurium and Escherichia coli are purchased from MOLTOX

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period:
37 °C for 30 minutes (with shaking)
- Exposure duration:
Plates were incubated at 37 °C ± 3 °C for approximately 48-72 hours


NUMBER OF REPLICATIONS:
Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity)

Rationale for test conditions:

The highest dose tested was 1 000 μg / plate.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely
 There should be a mimimum of four non-toxic test item dose levels
 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,
 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).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

BACTERIOSTATIC ACTIVITY CONTROL:
Results show a high toxicity from 1500 to 5000 μg/plate. Due to the toxicity of the test item a supplementary dose was studied. The test item is tested at these doses (1 000, 500, 300, 100, 30 and 10 μg/plate).

MUTATION ASSAY
- 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 an increase in the number of revertant colonies in the presence of the test item stock solution and dilutions (750 and 500 μg/plate) with metabolic activation in Salmonella typhimurium, TA 98.
- One can observe a thinning of the bacterial lawn in the presence of the highest doses correlated with the toxicity measured.
- Results are confirmed in an independent experiment.

Conclusions:

Doses (750 and 500 μg/plate) prepared from solutions of the test item, induce a significant increase in the number of revertants in Salmonella typhimurium, TA 98, with metabolic activation, according to the OECD Guideline n° 471.

Executive summary:

The test item was tested for it 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. Two independent assays were carried out.

For assay n° 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 n° 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.

There is an increase in the number of revertant colonies in the presence of the various concentrations of the test item (750 and 500 μg/plate), with metabolic activation in Salmonella typhimurium, TA 98.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item without metabolic activation in Salmonella typhimurium TA 98 as well as without and with metabolic activation in Salmonella typhimurium TA1535, TA1537, TA100 and in Escherichia coli WP2(uvrA-)(pKM101)

Doses (750 and 500 μg/plate) prepared from solutions of the test item, induce a significant increase in the number of revertants in Salmonella typhimurium, TA 98, with metabolic activation, according to the OECD Guideline n° 471.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the mutagenic potential of the registered substance.

The registered substance has not been tested itself in appropriate in vitro or in vivo tests but one of its constituents is classified as mutagenic Cat. 2 (methyl eugenol) and potentially present above the CLP generic concentration limit of 1% that triggers classification of the mixture. Therefore, the registered substance is classified for mutagenicity Category 2 according to the Regulation (EC) No 1272/2008.

In addition in an Ames test, performed according to OECD 471 Guideline, in GLP condition the test item induce a significant increase in the number of revertants in Salmonella typhimurium, TA 98, with metabolic activation.

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 boundary composition provided by the Lead Registrant, and the result of the OECD 471 test, the registered substance is classified for mutagenicity Category 2 (H341) according to the Regulation (EC) No. 1272/2008 (CLP).