Essential oil of Boswellia carterii (Burseraceae) obtained from gum by distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Micronucleus test: on-going (OECD 487, GLP, K, Rel. 1)

HPRT test: on-going (OECD 476, GLP, K, Rel. 1)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April to 26 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

05 March 2015

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 TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% (v/v) S9 fraction; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix (plate incorporation method)
Mutagenicity experiments
Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)
Experiments with S9 mix:
312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),
62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),
31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle was ethanol.
- Dose formulation preparation: No correction factor was applied. The test item was diluted in the vehicle at a concentration of 200 mg/mL for the preliminary toxicity test and for both mutagenicity experiments. The stock solutions and their dilutions were prepared within 4 h of use, and then kept at room temperature and protected from light until use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Anthramine

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 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 (plate incorporation); preincubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS:
Preliminary toxicity test: one plate/dose-level
Mutagenicity experiments: three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed based on the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHERS:
- The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
- After 48 to 72 h 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:

Using a test item concentration of 200 mg/mL in the vehicle and a treatment volume of 25 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the dose-levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
Since the test item was found freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main experiments was 5000 μg/plate, according to the criteria specified in the international guidelines.

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 is 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 is observed, in any strain, at any dose-level, and/or a reproducible dose-response relationship is evidenced.

The test item is 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, is observed at any of the tested dose-levels, nor any evidence of a dose-response relationship is noted.

Statistics:

None

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

PRELIMINARY TOXICITY TEST
- No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any of the tested dose-levels, towards the three strains used, either with or without S9 mix.

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- A moderate toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used.
- 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 met the criteria of a negative response.
Experiments with S9 mix:
- 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, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains.
- In the second experiment using the pre-incubation method, a moderate to strong toxicity (thinning of the bacterial lawn) was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain, and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted 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 met the criteria of a negative response.

HISTORICAL CONTROL DATA
- Positive historical control data: 710 ± 133.0, 440 ± 401.6, 155 ± 31.9, 787 ± 108.6 and 1699 ± 291.3 for TA 1535 (NaN3), TA 1537 (9AA), TA 98 (2 NF), TA 100 (NaN3) and TA 102 (MMC) strains, respectively (without S9); 249 ± 63.8, 125 ± 32.5, 1594 ± 275.8, 912 ± 250.5 and 1791 ± 467.5 for TA 1535 (2 AM), TA 1537 (2 AM), TA 98 (2 AM), TA 100 (BaP) and TA 102 (2 AM) strains, respectively (with S9)
- Negative (solvent/vehicle) historical control data: 19 ± 4.3, 8 ± 1.9, 24 ± 4.2, 120 ± 15.1 and 340 ± 61.2 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (without S9); 18 ± 2.7, 12 ± 2.1, 36 ± 5.8, 136 ± 12.3 and 449 ± 89.8 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (with S9)
Note: sodium azide (NAN3); 9-Aminoacridine (9 AA); 2-Nitrofluorene (2 NF); Mitomycin C (MMC); 2-Anthramine (2 AM); Benzo(a)pyrene (BaP)

None

Conclusions:

Under the test conditions, test substance is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to test substance at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix (plate incorporation method)

Mutagenicity experiments

Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)

Experiments with S9 mix:

312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),

62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),

31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

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

Experiments without S9 mix:

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A moderate toxicity was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used. 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 met the criteria of a negative response.

Experiments with S9 mix:

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, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains. In the second experiment using the pre-incubation method, a moderate to strong toxicity was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted 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 met the criteria of a negative response.

 

Under the test conditions, test substance is not mutagenic in this bacterial system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to Section 13]

Reason / purpose for cross-reference:

read-across source

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

PRELIMINARY TOXICITY TEST
- No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any of the tested dose-levels, towards the three strains used, either with or without S9 mix.

MUTAGENICITY EXPERIMENTS
Experiments without S9 mix:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- A moderate toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used.
- 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 met the criteria of a negative response.
Experiments with S9 mix:
- 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, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains.
- In the second experiment using the pre-incubation method, a moderate to strong toxicity (thinning of the bacterial lawn) was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain, and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted 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 met the criteria of a negative response.

HISTORICAL CONTROL DATA
- Positive historical control data: 710 ± 133.0, 440 ± 401.6, 155 ± 31.9, 787 ± 108.6 and 1699 ± 291.3 for TA 1535 (NaN3), TA 1537 (9AA), TA 98 (2 NF), TA 100 (NaN3) and TA 102 (MMC) strains, respectively (without S9); 249 ± 63.8, 125 ± 32.5, 1594 ± 275.8, 912 ± 250.5 and 1791 ± 467.5 for TA 1535 (2 AM), TA 1537 (2 AM), TA 98 (2 AM), TA 100 (BaP) and TA 102 (2 AM) strains, respectively (with S9)
- Negative (solvent/vehicle) historical control data: 19 ± 4.3, 8 ± 1.9, 24 ± 4.2, 120 ± 15.1 and 340 ± 61.2 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (without S9); 18 ± 2.7, 12 ± 2.1, 36 ± 5.8, 136 ± 12.3 and 449 ± 89.8 for TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, respectively (with S9)
Note: sodium azide (NAN3); 9-Aminoacridine (9 AA); 2-Nitrofluorene (2 NF); Mitomycin C (MMC); 2-Anthramine (2 AM); Benzo(a)pyrene (BaP)

None

Conclusions:

Under the test conditions, test substance Pepper black oil is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains. Therefore the registered substance Olibanum oil is not mutagenic in these bacterial strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to test substance at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix (plate incorporation method)

Mutagenicity experiments

Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)

Experiments with S9 mix:

312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),

62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),

31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

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

Experiments without S9 mix:

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A moderate toxicity was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used. 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 met the criteria of a negative response.

Experiments with S9 mix:

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, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains. In the second experiment using the pre-incubation method, a moderate to strong toxicity was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted 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 met the criteria of a negative response.

 

Under the test conditions, test substance Pepper black oil is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains. Therefore the registered substance Olibanum oil is not mutagenic in these bacterial strains.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 476 Guideline without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

28 October 2016

Type of assay:

other: In vitro mammalian cell gene mutation test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Esseterre / FRC15.091117.H1
- Appearance: Pale yellow to yellow mobile liquid
- Expiration date of the lot/batch: 09 November 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8ºC), in the dark

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Cell cycle length, doubling time or proliferation index: The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Modal number of chromosomes: 22

MEDIA USED
- Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes; cells have a stable karyotype
- Periodically 'cleansed' against high spontaneous background: Yes; Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (2%) prepared from male rats, dosed with phenobarbital and β-Naphthaflavone

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: μg/mL, 4 h exposure without and with metabolic activation

Mutation tests:
4 h exposure with metabolic activation: μg/mL
4 h exposure with metabolic activation: μg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

CELL CULTURE
The stock of cells was stored in liquid nitrogen. For use, a sample of cells were removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.

METHOD OF APPLICATION: Eagles Minimal Essential (MEM) with 10% fetal bovine serum (FBS)
- Cell density at seeding: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 h being exposed to the test or control items.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
All cell cultures were incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG) at a final concentration of 11 μg/mL; At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and vehicle controls
Main test: Duplicate cultures for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
200 cells/flask were seeded for cloning efficiency and 2 x 10^5 cells/flask were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

- OTHER:
- Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
- The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Rationale for test conditions:

The test item was a UVCB, therefore, the maximum proposed concentration level in the solubility test was set at 5000 μg/mL initially, which was the maximum recommended concentration level in regulatory guidelines.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

other: on-going

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Precipitation:

PRELIMINARY CYTOTOXICITY TEST:


MAIN TEST:

HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mutant Frequencies per survivor (x 10^-6): 226.52 ± 77.63 (Ethyl methanesulphonate, -S9); 319.07 ± 157.07 (Dimethyl benzanthracene, +S9)
- Negative (solvent/vehicle) historical control data:
Mutant Frequencies per survivor (x 10^-6): 13.92 ± 7.04 (-S9); 20.62 ± 13.64 (+S9)

None

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item for 4 h, with and without metabolic activation (2% S9); S9 fraction prepared from male rats, dosed with phenobarbital and β-Naphthaflavone.

 

Preliminary cytotoxicity test: μg/mL, 4 h exposure without and with metabolic activation

 

Mutation tests:

4 h exposure with metabolic activation: μg/mL

4 h exposure with metabolic activation: μg/mL

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

28 October 2016

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Esseterre / FRC15.091117.H1
- Appearance: Pale yellow to yellow mobile liquid
- Expiration date of the lot/batch: 09 November 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8ºC), in the dark

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

At the end of the exposure period in both experiments, the cell cultures were incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (2% final concentration of S9): S9 fraction, prepared from male rats dosed with phenobarbital and β-Naphthaflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test: TBD ; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix

Main Test
4 h exposure to the test item formulations without S9-mix: μg/mL
4 h exposure to the test item formulations with S9-mix: μg/mL
24 h exposure to the test item without S9-mix: µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
- The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 μg/mL. Additionally, the test item was considered to be 100% so no correction for purity made.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

CELLS:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 25 years; Main Experiment: male, aged 25 years; Main Experiment Repeat male, aged 33 years

CULTURE OF LYMPHOCYTES:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

METHOD OF APPLICATION: in medium; Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS).

DURATION
- Exposure duration: 4 h (± S9) and 24 h exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h exposure (-S9) in main experiments
- Fixation time: 24 h incubation period in treatment-free media after exposure to the test item in all experiments

SPINDLE INHIBITOR (cytogenetic assays): At the end of the exposure period in both experiments, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.

STAIN (for cytogenetic assays): Slides were stained in 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture was prepared for test item and vehicle control
- Main tests: Duplicate cultures were prepared for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cell harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of microscope slides: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Then the slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
- A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.
- The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Due to excessive test item toxicity, only 284 binucleate cells could be analyzed for micronuclei in the B culture at 80 μg/mL in the 4 h exposure group without S9-mix. Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = vehicle control culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

- OTHER:
Qualitative slide assessment: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

other: on-going

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Precipitation:

PRELIMINARY TOXICITY TEST
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 312.5 μg/mL in all three exposure groups.
- Hemolysis was observed following exposure to the test item at all the dose levels tested in the 4 h exposure groups whereas hemolysis was observed at and above 19.53 μg/mL in the 24 h continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition to hemolysis, a reduced pellet was observed at and above 78.13 μg/mL in the 4 h exposure groups whereas a reduced pellet was observed at and above 156.25 μg/mL in the 24 h continuous exposure group. A reduced pellet is an indication of cytotoxicity and that maximum exposure is occurring. This type of toxic response to the lymphocytes is not reflected in the CBPI data because the surviving cells are still dividing.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 39.06 μg/mL in the exposure groups in the absence of metabolic activation (S9). The maximum dose with binucleate cells present in the presence of S9 was 78.13 μg/mL. The test item induced evidence of severe toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on toxicity for all three exposure groups.

MAIN EXPERIMENT
- In the Main Experiment, the qualitative assessment of the slides determined that the toxicity was less than that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three exposure groups which did not achieve optimum toxicity as defined in the OECD 487 guideline (55±5%). No precipitate of test item was noted at any of the dose levels in any exposure groups tested.
- Hemolysis was observed following exposure to the test item at all the dose levels tested in the 4 h exposure groups whereas hemolysis was observed at and above 20 μg/mL in the 24 h continuous exposure group. Also and in addition to the hemolysis, a reduced pellet was observed at and above 60 and 70 μg/mL in the 4 h exposure groups in the absence and presence of S9, respectively, whereas no reduced pellet was observed at any of the dose levels in the 24 h continuous exposure group. Therefore, the hemolysis and reduced pellet were similar to that observed in the preliminary toxicity test.
- The CBPI data for the short exposure group in the presence of S9 only and for the 24 h exposure group confirm the qualitative observations in that no or only a moderate dose-related inhibition of CBPI was observed. In the presence of S9, only a moderate inhibition of cell proliferation was observed where only 24% cytostasis was achieved at the maximum dose level (120 μg/mL). In the absence of S9, a dose-related inhibition of CBPI was observed in the 24 h continuous exposure group where 17%, 33% and 43% cytostasis was achieved at 60, 70 and 80 μg/mL, respectively. In the 4 h group, only a moderate inhibition of CBPI was observed. Therefore, the experiment was repeated in an effort to achieve optimum toxicity in all three exposure groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
% binucleate cells with micronuclei: 2.25-18.85 (6.31 ± 2.90) - Mitomycin C (4 h, -S9); 1.75-5.75 (3.73 ± 1.06) - Demecolcine (24 h, -S9); 1.65-7.25 (3.48 ± 1.51) - Cyclophosphamide (4 h, +S9)
- Negative (solvent/vehicle) historical control data:
% binucleate cells with micronuclei:
4 h, -S9: 0.10-1.25 (0.38 ± 0.27); 95% CI - ± 0.09 (0.29-0.47)
24 h, -S9: 0.05-0.90 (0.43 ± 0.20); 95% CI - ± 0.07 (0.36-0.50)
4 h, +S9: 0.10-1.20 (0.41 ± 0.24); 95% CI - ± 0.08 (0.33-0.49)

Table 7.6.1/1: CBPI and Micronucleus Data – Main Experiment

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 2% S9; S9 fraction was obtained from the liver homogenates of male rats induced with phenobarbital and β-Naphthaflavone.

 

Preliminary Toxicity Test: μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix

Main Test

4 h exposure to the test item formulations without S9-mix: μg/mL

4 h exposure to the test item formulations with S9-mix: μg/mL

24 h exposure to the test item without S9-mix: μg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Chevallier, 2017	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA-	-S9 +S9	Up to cytotoxic or highest recommended concentration	-S9 : non mutagenic +S9 : non mutagenic

 

 

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to test substance at the following concentrations:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate for TA 98, TA 100 and TA 102 strains with and without S9 mix(plate incorporation method)

Mutagenicity experiments

Experiments without S9 mix: 312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in both mutagenicity experiments (plate incorporation method)

Experiments with S9 mix:

312.5, 625, 1250, 2500 and 5000 μg/plate for the five strains in the first experiment (plate incorporation method), and for the TA 98, TA 100 and TA 102 strains in the second experiment (pre-incubation method),

62.5, 125, 250, 500, 1000 and 2000 μg/plate for the TA 1535 strain in the second experiment (pre-incubation method),

31.3, 62.5, 125, 250, 500 and 1000 μg/plate for the TA 1537 strain in the second experiment (pre-incubation method).

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 mix prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

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

Experiments without S9 mix:

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A moderate toxicity was noted at the highest dose-level of 5000 μg/plate in the TA 1535, TA 1537 and TA 100 strains, in both experiments. No noteworthy toxicity was noted at any of the tested dose-levels towards the other strains used. 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 met the criteria of a negative response.

Experiments with S9 mix:

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, no noteworthy toxicity was noted at any of the tested dose-levels in any of the tested strains. In the second experiment using the pre-incubation method, a moderate to strong toxicity was noted at 1000 μg/plate in the TA 1537 strain, at 2000 μg/plate in the TA 1535 strain, at dose-levels ≥ 2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 98 strain, whereas no noteworthy toxicity was noted 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 met the criteria of a negative response.

 

Under the test conditions, test substance Pepper black oil is not mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) strains. Therefore the registered substance Olibanum oil is not mutagenic in these bacterial strains.

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 classification is proposed.