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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November - 31 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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

Constituent 1
Reference substance name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
EC Number:
945-898-3
Cas Number:
97675-63-3
Molecular formula:
not relevant for a UVCB substance
IUPAC Name:
Essential oil of Canarium commune (Burseraceae) obtained from gum by steam distillation
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Elemi oil
- Batch no.: 15/01509
- Analytical purity: 100% wt UVCB substance
- CAS No.: 8023-89-0
- EINECS-No.: 232-557-2
- Expiry date: Nov. 2016
- Date of Receipt: 16. Oct. 2015
- Appearance: courless to pale yellow liquid
- Homogeneity: Homogeneous
- Storage condition of test material: Room temperature 20 ± 5 °C, keep away from light/ humidity, heep under inert gas. The test item was stored in a closed aluminium vessel at 15.2-21 °C away from light and under inert gas.

Method

Target gene:
His+ for S. typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
10% (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST:
- Test concentrations: 0 (vehicle: DMSO), 10, 100, 500, 1000, 2500 and 5000 μg/plate, with and without S9 mix in TA 98, TA 100 and TA 102 strains (direct plate incorporation method)
- Justification for top dose: Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the top dose selected for the preliminary test was 5000 μg/plate.

MUTAGENICITY TESTS:
- Test concentrations without S9 mix
Experiment 1: 0, 0.8, 2.5, 7.4, 22.2, 66.7 and 200 μg/plate in all 5 strains (direct plate incorporation method)
Experiment 2: 0, 2.5, 7.4, 22.2, 66.7, 200 and 600 μg/plate in all 5 strains (direct plate incorporation method)
- Test concentrations with S9 mix:
Experiment 1: 0, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains; 0, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 100 strain (direct plate incorporation method)
Experiment 2: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 102 strain; 0, 19.53, 39.06, 78.13, 156.3, 312.5 and 625 μg/plate for the TA 1535, TA 1537 and TA 98 strains; 0, 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 μg/plate for the TA 100 strain (pre-incubation method)
- Justification for top dose: Since the test item was found freely soluble but toxic in the preliminary test, mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Vehicle was selected based on the available solubility data.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Anthramine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All five strains of Salmonella typhimurium were supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) or Culture Collections (Public Health England, Porton Down, Salisbury, SP4 0JG, UK).

METHOD OF APPLICATION: In agar (direct plate incorporation and pre-incubation method)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Incubation period: 48-72 h at 37 °C for both direct plate incorporation and pre-incubation methods

NUMBER OF REPLICATIONS:
- Preliminary experiment: 1 plate/dose
- Main experiments: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- After 48 to 72 hours of incubation at 37°C, the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated.
Rationale for test conditions:
Rationale for test concentrations:
- Preliminary toxicity test: Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the top dose selected for the preliminary test was 5000 μg/plate.
- Main tests: Since the test item was found freely soluble but toxic in the preliminary test, mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item was considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls was observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship was evidenced.

The test item was considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, was observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship was noted.
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
refer 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable.
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels ≥ 100 μg/plate towards the three strains used in the absence of S9 mix.
- In the presence of S9 mix, a strong toxicity was noted at 5000 μg/plate in the TA 98 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 102 strain.

MUTAGENICITY TESTS:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- Without S9 mix: In the first experiment, only a slight toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 200 μg/plate in all the tested strains. In the second experiment, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 μg/plate in the TA 1535, TA 1537 and TA 100 strains, and ≥ 200 μg/plate in the TA 98 and TA 102 strains.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 98 and TA 102 strains. In the second experiment, using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537 and TA 98 strains, ≥ 555.6 μg/plate in the TA 100 strain and ≥ 1250 μg/plate in the TA 102 strain.
- The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment, with or without S9 mix. These results with and without S9 mix met the criteria of a negative response.
- The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

OTHERS:
- Sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Any other information on results incl. tables

See attached document

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test substance is not considered as mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

An in vitro gene mutation study in bacteria was performed according to the OECD Guideline 471 (bacteria reverse gene mutation assay) and in compliance with GLP.

A preliminary toxicity test was performed to define the dose-levels of the test substance dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.  Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).  Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48-72 hours of incubation at 37°C, the revertant colonies were scored.  The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. Since the test item was found freely soluble but toxic in the preliminary test mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.

Main experiments without S9 mix

Selected dose-levels ranged from 0.8 to 600 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, only a slight toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 200 μg/plate in all the tested strains. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains in this first experiment.

In the second experiment, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 μg/plate in the TA 1535, TA 1537 and TA 100 strains, and ≥ 200 μg/plate in the TA 98 and TA 102 strains. These observations were consistent with the toxicity observed in the preliminary toxicity test. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in this second experiment. These data were consistent with results of the first experiment performed under the same experimental conditions despite the use of a higher range of dose-levels.

The overall results without S9 mix met the criteria of a negative response.

Main experiments with S9 mix

Selected dose-levels ranged from 6.9 to 5000 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 98 and TA 102 strains.

In the second experiment, using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537 and TA 98 strains, ≥ 555.6 μg/plate in the TA 100 strain and ≥ 1250 μg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results with S9 mix met the criteria of a negative response.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Under the test conditions, the test substance is not considered as mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.