Registration Dossier
Registration Dossier
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EC number: 933-779-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
OECD 471 (in vitro Bacterial reverse mutation test): Negative (±S9)
OECD 487 (in vitro Micronucleus Test): Negative (±S9)
OECD 490 (in vitro mouse lymphoma assay): Negative (±S9)
BlueScreen (in vitro human lymphoblastoid TK6 cells (GLuc-T01) assay): Negative (±S9)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 April 2012 - 22 June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- The major Japanese Regulatory Authorities including METI, MHLW and MAFF
- Qualifier:
- according to guideline
- Guideline:
- other: The USA EPA (TSCA) OPPTS harmonised guidelines
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Sponsor's identification: Ocimene PQ
- Description: colourless liquid
- Composition: 73.1% Cis-beta-Ocimene 73.1%; Dipentene 21%
- Batch No.of test material: A120304A
- Expiration date of the lot/batch: 04 March 2014
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: all formulations were used within four hours of preparation and were assumed to be stable fo rthis period
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- the test material was accurately weighed and approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer on the day of each experiment
- formulated concentrations were adjusted to allow for the stated water/impurity content (26.9%) of the test substance
- prior to use, the solvent was dried to remove water, using molecular sieves (2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x10^-4 microns) - Target gene:
- Histidine or tryptophan locus
- 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-mix
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Mutation test 1:
- Salmonella strains TA98 and TA1537 (absence and presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
- Salmonella strains TA1535 and TA100 (absence and presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 50, 150, 500, 1500, 5000 µg/plate.
Mutation test 2:
- Salmonella strains TA1537 and TA98 (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- Salmonella strains TA1535 and TA100 (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate
- Salmonella strain TA1537 (presence of S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
- Salmonella strain TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test substance was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL, but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. - Untreated negative controls:
- yes
- Remarks:
- 2-Aminoanthracene; Benzo(a)pyrene
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Vehicle control
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. The cultures were monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
- Exposure duration: approximately 48-hour incubation at 37°C
MEASUREMENTS
- After the incubation period, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.
PRELIMINARY TOXICITY TEST
- A preliminary test was conducted in order to select appropriate dose levels for use in the main test.
- 0.1 mL bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented top agar, 0.1 mL of test substance formulation and 0.5 mL S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30mL/plate).
- 10 concentrations of the test substance formulation and a vehicle control (acetone) were tested.
- 0.1 mL of the maximum concentration of the test substance and 2 mL molten trace histidine or tryptophan supplemented top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test substance.
MUTATION TEST 1
- 0.1 mL of one of the bacterial cultures were dispensed into sets of test tubes with 2mL molten, trace histidine or tryptophane supplemented top agar, 0.1 mL vehicle, test material formulation or postiive control and 0.5 mL of S9-mix or phosphate buffer.
- The test tubes were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate)
- The procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.
MUTATION TEST 2
- Performed using fresh bacterial cultures, test substance and control solutions.
- Based on results from Experiment 1 and the change in test methodology led to a change in the test substance dose range.
CONTROLS
1) Vehicle control - solvent treatment group
2) Positive controls - used in a series of plates without S9-mix:
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 2µg/plate for WP2uvrA
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 3µg/plate for TA100
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 5µg/plate for TA1535
- 9-Aminoacridine (9AA): 80 µg/plate for TA1537
3) Negative controls - non-mutagenic substances in the absence of metabolising enzymes were used in the series of plates with S9-mix:
- 2-Aminoanthracene (2AA): 2 µg/plate for TA100
- 2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
- 2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
- Benzo(a)pyrene (BP): 5µg/plate for TA98 - Evaluation criteria:
- Any one or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Results will be reported as equivocal when data generated will prohibit making a definite judgement about test substance activity. - Statistics:
- Not specified
- Key result
- 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
- Key result
- 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
- Key result
- Species / strain:
- S. typhimurium TA 98
- 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 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:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary test:
- The test substance was toxic to TA100 at 5000 µg/plate and non-toxic to WP2uvrA.
- The test substance formulation and S9-mix used in this experiment were both shown to be sterile.
Controls:
- The results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
- All positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S0-mix and the sensitivity of the bacterial strains.
Mutation test 1:
- The test substance (from the 15 µg/plate) caused a visible reduction in the growth of the Salmonella bacterial background lawns (except for TA98 in the presence of the S9-mix)
- No toxicity was seen to E.coli WP2uvrA
Mutation test 2:
- The test substance caused a slightly stronger toxic response with weakened bacterial background lawns initially from 5µg/plate in the absence of S9-mix and 15 µg/plate in the presence of the S9-mix.
- Weakened lawns in the E.coli WP2uvrA strain were noted at 1500 µg/plate only in the absence of metabolic activation.
Acceptance criteria met: yes - Conclusions:
- Under the conditions of the study and based on the results, the test substance, Ocimene PQ, was considered to be non-mutagenic to Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).
- Executive summary:
This study was conducted to evaluate the ability of the test substance, Ocimene PQ, to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptopjhan locus in the genome of five strains of bacteria. The study was performed in accordance with OECD 471, EU Method B.13/14, the major Japanese Regulatory Authorities including METI, MHLW and MAFF, and the USA EPA (TSCA) OPPTS harmonised guidelines.
The bacterial strains employed in this study were Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. The bacterial strains wree treated with the test substance using the Ames plate incorporation (Mutation test 1) and pre-incubation (Mutation test 2) methods at up to seven dose levels, tested in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). A preliminary toxicity assay was initially performed in order to select appropriate dose levels for the main experiment. The test ubstance was found to be toxic to S. typhimuriumTA100 at the 5000 µg/plate and non-toxic to E.coli WP2uvrA.
The dose range for Mutation test 1 was determined by the preliminary test and ranged from 0.15 -5000 µg/plate, depending on the bacterial strain and the presence or absence of the S9 -mix. Mutation test 2 was repeated on a separate dat (pre-incubation), using an amended dose range of 0.05 -5000 µg/plate, fresh cultures of bacterial strains and fresh test substance preparations. Additional dose levels and an expanded dose range were selected where applicable, in order to achieve the four non-toxic dose levels and the toxic limit of the test substance.
In Mutation test 1, the test substance caused a visible reduction in the growth of the Salmonella bacterial background lawns from the 15 µg/plate (except for TA98 in the presence of the S9-mix), but no toxicity was seen to E.coli WP2uvrA. Mutation test 2, the test substance caused a slightly stronger toxic response with weakened bacterial background lawns initially from 5µg/plate in the absence of S9-mix and 15 µg/plate in the presence of the S9-mix. Weakened lawns in the E.coli WP2uvrA strain were noted at 1500 µg/plate only in the absence of metabolic activation. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology. The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains with any dose of the test material, either in the presence or absence of the S9 -mix or in either exposure method employed.
There was a test substance precipitate (oily in appearance) seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies.The vehicle (acetone) control plates gave revertant colony counts within the normal range and all positive chemicals induced marked increases in the frequency of revertant colonies, both in the presence and absence of metabolic activation. The acceptance criteria were met and therefore the sensitivity of the assay and effiicacy of the S9 -mix were validated.
Under the conditions of the study and based on the results, it can be conluded that the test substance, Ocimene PQ, was considered to be non-mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-07-13 to 2019-03-27
- 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
- GLP compliance:
- yes (incl. QA statement)
- 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: Emerald Kalama Chemical Ltd (Dans Road, WA8 0RF Widnes, Cheshire, United Kingdom): Lot# A170524D
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, Protected from humidity, Under nitrogen atmosphere
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: The test item was diluted in the vehicle (ethanol) at concentrations of: 272.46 mg/mL for the preliminary cytotoxicity test, 163.476 mg/mL for the first experiment, 54.492 mg/mL for the second experiment
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Colourless liquid - Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- L5178Y TK+/- cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France)
- Suitability of cells: L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei.
- Cell cycle length, doubling time or proliferation index: average cell cycle time is approximately 10-12 hours
- Number of passages if applicable: Not applicable
- Methods for maintenance in cell culture if applicable: The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma and for the modal chromosome number.
- Modal number of chromosomes: The modal chromosome number is 40
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL). This medium was supplemented by heat inactivated horse serum at 10% (v/v); Humidified atmosphere of 5% CO2/95% air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: Not specified
- Periodically 'cleansed' against high spontaneous background: Not specified - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver post-mitochondrial fraction (S9 fraction)
- Test concentrations with justification for top dose:
- Preliminary Cytotoxicity Test: To assess the cytotoxicity of the test item, eight dose levels (one culture/dose level) were tested both with and without metabolic activation: 0.016, 0.041, 0.10, 0.26, 0.64, 1.6, 4 and 10 mM.
Main Experiments:
Short treatment without S9 mix: 3 h treatment + 24 h recovery: were 0.031, 0.063, 0.125, 0.25, 0.375, 0.5, 0.75 and 1 mM.
Continuous treatment without S9 mix: 24 h treatment + 0 h recovery: were 0.031, 0.063, 0.125, 0.25, 0.375, 0.5, 0.75 and 1 mM
Experiments with S9 mix: were 0.188, 0.375, 0.75, 1.5, 2, 3, 4, and 6 mM - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was ethanol.
Note: Positive controls were dissolved in water. - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicine (0.5 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 3 x 10^5 cells/mL
DURATION
- Exposure duration: See Table 1 and Table 2.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 27 hours depending on the treatment as shown in Tables 1 and 2.
SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giesma (5%)
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the final cell counting, the cells were washed with culture medium containing 10% inactivated horse serum and 1% pluronic acid. The cells were suspended in 49.5% culture medium containing 10% inactivated horse serum, 50% PBS and 0.5% pluronic acid, before being fixed.
Following the fixation, the cells were kept at 5°C for at least an overnight period. Depending on the observation at the end of the recovery period (presence or absence of precipitate and/or cytotoxicity), three dose levels of the test item-treated cultures were selected for spreading on slides. Cells were dropped onto clean glass slides. The slides were air dried before being stained for approximately 15 min in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above.
NUMBER OF CELLS EVALUATED: For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 2000 mononucleated cells per dose
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Analysis was performed by microscopic evaluation, on the basis of the recommendations of Miller et al. (1995) (e), according to the following criteria:
• micronuclei should be located within the cytoplasma of the cell,
• micronuclei should be clearly surrounded by a nuclear membrane,
• micronuclei should be round or oval in shape,
• the micronucleus area should be less than one-third of the area of the main nucleus,
• micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
• micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
• micronuclei should have similar staining intensity to that of the main nuclei,
• micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
• only mononucleated cells with a number of micronuclei ≥ 5 will be scored to exclude apoptosis and nuclear fragmentation.
DETERMINATION OF CYTOTOXICITY
- Method: Population Doubling (PD).
- Any supplementary information relevant to cytotoxicity: population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2. The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%). - Rationale for test conditions:
- The micronuclei observed in the cytoplasm of interphase cells may originate from acentric fragments (chromosome fragments lacking a centromere) or whole chromosomes that are unable to migrate with the rest of the chromosomes during the anaphase of cell division. The assay thus has the potential to detect the activity of both clastogenic and aneugenic chemicals
- Evaluation criteria:
- Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
• a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
• for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
• a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.
Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met. - Statistics:
- For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the X2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
Preliminary Cytotoxicity Test: Using a test item concentration of 272.46 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose level of 10 mM (corresponding to 1362.3 µg/mL) was achievable. Thus, the dose levels selected for the treatment of the preliminary test were 0.016, 0.041, 0.10, 0.26, 0.64, 1.6, 4 and 10 mM.
At the highest dose level of 10 mM, the pH of the culture medium was approximately 7.4 as for the vehicle control and the osmolality was 363 mOsm/kg H2O (397 mOsm/kg H2O for the vehicle control). Therefore, this dose level could be selected as the highest dose level for the main experiments, and none of the tested dose levels was considered to produce extreme culture conditions.
RANGE-FINDING/SCREENING STUDIES:
An emulsion was observed in the culture medium at the end of the 3-hour treatment periods, at dose levels ≥ 4 mM.
A precipitate was observed in the culture medium at the end of the 24-hour treatment period, at dose levels ≥ 4 mM.
Following the 3 and 24-hour treatments without S9 mix, a severe cytotoxicity was observed at dose levels ≥ 0.64 mM, as shown by a 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a slight to severe cytotoxicity was observed at dose levels ≥ 0.64 mM, as shown by a 30 to 100% decrease in the PD.
NUMBER OF CELLS WITH MICRONUCLEI
Short treatment without S9 mix: 3 h treatment + 24 h recovery:
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. No dose response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
Continuous treatment without S9 mix: 24 h treatment + 0 h recovery:
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. No dose response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
Experiments with S9 mix:
A statistically significant increase in the frequency of micronucleated cells was observed at the dose level of 0.75 mM relative to the vehicle control. At this dose level, the frequency of micronucleated cells of both replicate cultures was above the corresponding vehicle reference data range (i.e. 11 and 4‰ versus [0.0-3.5‰] for reference data). Since no dose-response relationship was demonstrated by the linear regression test, these results did not meet either the criteria for a positive or a negative response.
In order to check the reproducibility of these increases, a second experiment was undertaken under the same experimental conditions but using a narrower range of dose levels, as follows: 0.125, 0.25, 0.375, 0.5, 0.75, 1, 1.5, and 2 mM.
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. No dose response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment, despite the use of a narrower range of dose levels, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See Table 12 and 13
- Negative (solvent/vehicle) historical control data: See Table 12 and 13
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Population Doubling (PD) (please see Tables 3, 4, 6, 8, & 10) - Conclusions:
- Under the experimental conditions of the study, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene), did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
- Executive summary:
A Key OECD Guideline 487 study was conducted to evaluate the potential of the test material, (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene), to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.
After a preliminary cytotoxicity test, the test material, diluted in ethanol, was tested in two independent experiments with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:
First experiment
Second experiment
Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery
With S9 mix
3 h treatment + 24 h recovery
3 h treatment + 24 h recovery
Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.
Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations. The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
A precipitate was observed in the culture medium at the end of the 3h-treatment periods at dose levels ≥ 0.5 mM. No precipitate or emulsion was observed in the culture medium at the end of the 24h treatment period, at any of the tested dose levels.
Without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the main experiment were 0.031, 0.063, 0.125, 0.25, 0.375, 0.5, 0.75 and 1 mM.
Cytotoxicity
A slight to severe cytotoxicity was induced at dose levels ≥ 0.25 mM.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
- 0.125, 0.25 and 0.375 mM for the 3h-treatment, the latter inducing the recommended level of cytotoxicity,
- 0.063, 0.125 and 0.25 mM for the 24h-treatment, the latter inducing the recommended level of cytotoxicity.
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the corresponding vehicle controls. No dose response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
These results met the criteria for a negative response.
With S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected were as follows:
- 0.188, 0.375, 0.75, 1.5, 2, 3, 4, and 6 mM for the first experiment,
- 0.125, 0.25, 0.375, 0.5, 0.75, 1, 1.5, and 2 mM for the second experiment.
Cytotoxicity
In the first experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 1.5 mM.
In the second experiment, no noteworthy cytotoxicity was induced at any of the tested dose levels.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
. 0.188, 0.375 and 0.75 mM, the latter being the lowest precipitating dose level,
. 0.25, 0.375 and 0.5 mM, the latter being the lowest precipitating dose level.
In the first experiment, a statistically significant increase in the frequency of micronucleated cells was observed at the dose level of0.75 mMrelative to the vehicle control. At this dose level,the frequency of micronucleated cells of both replicate cultures was above the corresponding vehicle reference data range. Since no dose-response relationship was demonstrated by the linear regression test, these results did not meet either the criteria for a positive or a negative response.
In order to check the reproducibility of the results obtained, a second experiment was undertaken under the same experimental conditions.
In the second experiment, nostatistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. No dose-response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.
Under the experimental conditions of the study, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene), did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-06-12 to 2018-12-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- yes
- Remarks:
- Deviations had no impact on the study on the results or integrity of the study.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Limited (Dans Road WA8 0RF Widnes Cheshire, United Kingdom); Lot# A170524D
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤ 70 % relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Based on the results of the preliminary solubility test and the available information, at 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) or Distilled water as vehicles they were not suitable for this study. However, the 400 mg/mL concentration was achievable using Acetone as vehicle (solvent). Thus, Acetone was selected for vehicle (solvent) of this study. This vehicle (solvent) was compatible with the survival of the cells and the S9 activity.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : Colourless liquid - Target gene:
- tk+/- (thymidine kinase) locus in L5178Y cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, USA); 60797977
- Suitability of cells: While many mammalian cell gene mutation systems are available, however the mouse lymphoma assay (MLA), employing the tk+/- (thymidine kinase) locus in L5178Y cells, has the advantage of detecting both gene mutations and chromosome aberrations. The principle of this assay is based on placing cells under selective pressure so that only mutant cells are able to survive. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-) for the gene that produces the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products but if a toxic base analogue (5-trifluorothymidine) is present in the medium, the enzyme will incorporate the analogue into the cells. Thus, the cells will not survive unless the enzyme is rendered inactive, by mutation. Resistance to 5-trifluorothymidine (TFT) results in a lack of thymidine kinase (TK) activity so the mutants (tk-/-) are unable to incorporate the toxic analogue and therefore survive in its presence.
Two types of TFT-resistant mutant colonies occur and these are designated as large colonies (normal-growing) and small (slow-growing) colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this assay, gene mutations within the tk gene (11 to 13 kilobases) and chromosomal events involving the gene may be detected. The TK system has a high spontaneous mutant frequency and high numbers of cells can be treated and sampled, therefore it is statistically robust.
- Methods for maintenance in cell culture if applicable: Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than three times before used in the main study.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Three types of RPMI 1640 medium were prepared as shown in Table 1.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver post-mitochondrial fraction (S9 fraction)
- Test concentrations with justification for top dose:
- Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. Three-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments.
The highest concentration tested in the preliminary test was 2000 µg/mL (the recommended maximum concentration). Treatment of cell cultures was made as described in the next section for the main mutation assays. However, single cultures were only used and positive controls were not included. After the treatment period, cell concentrations were determined using a haemocytometer. Cells were transferred for the expression period for two extra days and repeated cell counting was performed. Visual examination for precipitation of test item in the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment period.
The highest selected concentration for the main tests was based on the observed cytotoxicity. A total of 8 concentrations with and without metabolic activation were selected for Assay 1 (Table 2) and Assay 2 (Table 3). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone (Source: VWR; LOT# 17J024003)
- Justification for choice of solvent/vehicle: Based on the results of the preliminary solubility test and the available information, at 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) or Distilled water as vehicles they were not suitable for this study. However, the 400 mg/mL concentration was achievable using Acetone as vehicle (solvent). Thus, Acetone was selected for vehicle (solvent) of this study. This vehicle (solvent) was compatible with the survival of the cells and the S9 activity.
Note: Dimethyl sulfoxide (DMSO) was used for vehicle (solvent) of the positive control chemicals. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 10^7 cells were placed in each of a series of 75 cm^2 sterile flasks
DURATION
- Exposure duration: 3 hours (±S9) and 24 hours (-S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Duplicate replicates
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Plating for Survival:
Cultures of cell density 2 x 10^5 cells/mL, were further diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for two weeks. Wells containing viable clones were identified by eye using background illumination and counted.
Expression Period:
To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2 x 10^5 cells/mL (whenever possible) and transferred to flasks for further growth.
On completion of the expression period, at least five test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.
Plating for Viability:
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1 x 10^4 cells/mL with RPMI-20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.
Plating for 5-trifluorothymidine (TFT) resistance:
At the end of the expression period, the cell concentration was adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2 x 10^3 cells per well.
Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for 12 days and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.
NUMBER OF CELLS EVALUATED: 10^6 viable cells
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: RTG was calculated as the percentage total growth of the treated cultures compared to the corresponding negative (vehicle/solvent) control value. - Evaluation criteria:
- The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG). - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.
The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In Assay 1 and Assay 2, there were no large changes in pH after treatment.
- Effects of osmolality: In Assay 1 and Assay 2, there were no large changes in osmolality after treatment.
- Water solubility: No insolubility was observed in the final treatment medium at the end of the treatment with and without metabolic activation in Assay 1 and Assay 2.
RANGE-FINDING/SCREENING STUDIES:
Preliminary Experiment:
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment using Acetone as vehicle was 2000 µg/mL (the recommended maximum concentration).
Insolubility and excessive cytotoxicity was detected in the preliminary experiment. Precipitate / minimal amount of precipitate was detected in the final treatment medium at the end of the treatment in the 2000-250 µg/mL concentration range with metabolic activation, in the 2000-125 µg/mL concentration range in case of short treatment and long treatment without metabolic activation.
Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-62.5 µg/mL concentration range using the short treatment with and without metabolic activation and in the long treatment without metabolic activation.
Concentrations up to the cytotoxic range were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline. Eight concentrations were selected for the main experiments in each assay.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See Table 5.
- Negative (solvent/vehicle) historical control data: See Table 5.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells] - Conclusions:
- No mutagenic effect of the test material [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
- Executive summary:
A Key OECD Guideline 490 in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).
Acetone was used as vehicle of the test material in this study. Based on the results of the preliminary experiment, the following test material concentrations were examined in the mutation assays:
Assay 1 (3-hour treatment with metabolic activation): 80, 60, 50, 40, 20, 10 and 5 and 2.5 µg/mL;
Assay 1 (3-hour treatment without metabolic activation): 80, 60, 50, 40, 20, 10, 5 and 2.5 µg/mL;
Assay 2 (3-hour treatment with metabolic activation): 60, 57.5, 55, 52.5, 50, 40, 20 and 10 µg/mL;
Assay 2 (24-hour treatment without metabolic activation): 45, 40, 35, 30, 20, 10, 5 and 2.5 µg/mL.
In Assay 1 and Assay 2, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assay 1 and 2 with and without metabolic activation.
In Assay 1 and Assay 2, following a 3-hour treatment with metabolic activation, marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 80 µg/mL concentration. In Assay 2 with metabolic activation no cells survived the expression period in the samples of 60 µg/mL concentration. An evaluation was made using data of six concentrations (concentration range of 50-2.5 µg/mL) in Assay 1 and seven concentrations (concentration range of 57.5-10 µg/mL) in Assay 2.
No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations, although a concentration related increase was indicated by the linear trend analysis. Reproducibility was seen between replicates. These experiments were considered to be negative.
In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 80, 60 and 50 µg/mL concentration. The relative total growth value of the next concentration (40 µg/mL) was slightly below <10%. No significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of five concentrations (concentration range of 40 - 2.5 µg/mL). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 1, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF.
Reproducibility was seen between replicates. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.
In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 45, 40 and 30 µg/mL concentrations. Only the cells of replicate ‘A’ survived the expression period in the samples of 35 µg/mL concentration. No significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of four concentrations (concentration range of 20 - 2.5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.
The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
In conclusion, no mutagenic effect of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- BlueScreen HC Assay
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2014
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- Genotoxicity and cytotoxicity of Ocimene PQ were assessed with the BlueScreen HC genotoxicity screening assay, using a protocol with and without metabolic activation in a 96-well microplate format. The assay evaluates genotoxicity by the induction in GLuc expression.
- GLP compliance:
- not specified
- Type of assay:
- other: Mammalian cell based genotoxicity screening assay
- Specific details on test material used for the study:
- TEST MATERIAL
Test item is the registered substance (EC 933-779-9) even though the test material CAS number indicates to its previous identity (used in pre-registration)
- CAS number: 3338-55-44
- Compound ID: GTX 110
- State: supplied as a colourless liquid
- Expiration date of the lot/batch: not specified
- Purity: not specified
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was combined with 100% DMSO to give a 1250 mM stock solution. The compound precipitated when added to water and therefore further dilution of the DMSO stock solution was required. - Target gene:
- GADD45a
- Species / strain / cell type:
- mammalian cell line, other: human TK6 (GLuc-T01)
- Details on mammalian cell type (if applicable):
- A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01) with a patented Gaussia luciferase (GLuc) reporter system that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to genotoxic stress.
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Highest concentration tested: 156 μg/ml
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: 96-well plate
(1) BlueScreen HC Assay - WITHOUT METABOLIC ACTIVATION
Overview
- A dilution series of the test material is generated in a 96-well, black microplate with an optically clear base.
- A standard genotoxic compound (4-nitroquinoline 1-oxide, 4-NQO) is added as an intra-plate quality control check.
- A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01), which contains a patented Gaussia luciferase (GLuc) reporter system that exploits the proper regulation of the GADD45a gene, which mediates the adaptive response to genotoxic stress.
- Exposure to a genotoxic compound increases expression of GLuc, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a coelenterazine substrate, added to the microplate wells just before measurement.
- Each dilution of the test compound is combined with an equal volume of a specialised growth medium containing BlueScreen HC cells. Duplicate dilution sreies of each compound are performed with a single microplate assay.
Duration
- The microplates were covered with a breathable membrane and incubated at 37°C with 5% CO2 and 95% humidity for 48 hours.
Measurements
- The assay plates are then analysed using a microplate reader that provides measurements of fluorescence and flash luminescence for cells and solutions in the microplate wells.
- A recent protocol enhancement has been added, that measures cytotoxicity by lysis of the cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.
- This estimation of relative cell density replaces the previous optical absorbance measure.
- Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminiscence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes.
- Luminescence is normalised to the fluorescence signal to correct for variation in cell yield caused by cytotoxicity.
Results
- Raw data collected from BlueScreen HC assay plates are saved to an MS Excel file.
- The luminescence and fluorescence data are automatically analysed using the BlueScreen HC software template to produce a result summary, with data presented both in tabulated and graphical formats, which provides a semi-quantitative assessment of cytotoxicity and genotoxicity.
- The overall assay outcome presented for a compound results from the average of the duplicate test series performed for the compound.
(2) BlueScreen HC S9 Metabolic Activation Assay - WITH METABOLIC ACTIVATION
Overview
- Exposure to a genotoxic compound increases GLuc expression, which is quantified at the assay endpoint by the detection of luminescence generated from the reaction of GLuc with a coelenterazine substrate, added to the microplate wells just prior to measurement.
- Cell density is determined by subsequent lysis of cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.
Cells
- This protocol uses the same cultured TK6 cell strain (GLuc-T01) as the assay without metabolic activation and is also performed in 96-well plates.
Duration: - The test substance is incubated with test cells n the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours.
Washing and recovery
- Cells were washed in phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery medium for 45 hours at 37ºC with 5% CO2 and 95% humidity.
Positive control:
- Cyclophosphamide - a commonly used standard compound in genotoxicity studies using metabolic activation. - Evaluation criteria:
- GENOTOXICIITY Without metabolic activation:
- The statistically defined threshold for a positive genotoxicity result is 1.8, i.e. 80% induction over and above the baseline for vehicle-treated cells.
GENOTOXOCITY With metabolic activation:
- The statistically defined threshold for a positive result is 1.5, i.e 50% induction over and above the baseline for vehicle-treated control cells.
CYTOTOXICITY
- The defined condition for a positive cytotoxicity result in the BlueScreen HC assay without S9 metabolic activation is a reduced relative cell density to less than 80%, compared to the vehicle-treated control. - Statistics:
- Not specified
- Key result
- Species / strain:
- mammalian cell line, other: TK6 (GLuc-T01)
- Remarks:
- Screening test
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mammalian cell line, other: TK6 (GLuc-T01)
- Remarks:
- Screening test
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The positive control substances (4-NQO and CPA) passed the test criteria in each case following the standard procedure for the BlueScreen HC and BlueScreen HC S9 metabolic activation assays, respectively.
- Remarks on result:
- other:
- Remarks:
- Highest conc tested 156 μg/ml
- Conclusions:
- Based on the results of the study and the evaluation criteria, the test substance Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene, was negative for genotoxicity with and without metabolic activation in the BlueScreen HC S9 assay.
- Executive summary:
The aim of this study was to assess the genotoxicity and cytotoxicity of the test substance, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene, with the BlueScreen HC genotoxicity screening assay, using a protocol with and without metabolic activation. The assay evaluates genotoxicity by the induction in GLuc expression.
The test substance was combined with 100% DMSO to give a 1250 mM colourless stock solution. It precipitated when added to water therefore further dilution of the DMSO stock solution was needed. The highest concentration of the test substance tested was at 156 μg/ml
The BlueScreen HC assay was performed in a 96 -well microtitre plate without metabolic activation (-S9). A dilution series of the test susbtance was generated in the microtitre plate. A standard genotoxic compound (4 -nitroquinoline 1 -oxide) was employed as a positive control and was added as an intra-plate quality control check to the microtitre plate at concentrations of 0.5 µg/mL and 0.125 µg/mL. The test system used was a genetically modified strain of cultured human lymphoblastoid TK6 cells (GLuc-T01) with a patented reported system, which explots the proper regulation of the GADD45a gene. This target gene mediates the adaptive response to genotoxic stress. The BlueScreen HC S9 assay was performed in the same cultured TK6 cell strain in a 96 -well microtitre plate as the first assay, but instead in the presence of metabolic activation (+S9). The test material was incubated with the test cells in the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours. Following this incubation period, the cells were washed in a phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C (5% CO2, 95% humidity). Cyclophosphate was employed as positive control, tested at concentrations of 25 µg/mL and 5µg/mL. A coelenterazine substrate was added to the microplate wells in both assays just prior to measurement, which reacts with GLuc resulting in luminescence. Cell exposure to a genotoxic compound increases GLuC expression that can be quantified by luminscence detection. Cytotoxicity was measured by lysis of cells and the addition of a fluorescent DNA binding stain, followed by the assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes. The quanity of luminescence is normalised to the fluorescence signal to correct for the variation in cell yield caused by cytotoxicity.
Based on the results of the study and the evaluation criteria, the test substance Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene, was negative for genotoxicity with and without metabolic activation in the BlueScreen HC S9 assay.
Referenceopen allclose all
Main experiment
- The master strains were checked prior to use for characteristics, viability and spontaneous reversion rate, which were all found to be satisfactory.
- The amino acid supplemented top agar and S9 -mix were shown to be sterile.
- The culture density for each bacterial strain was checked and considered to be acceptable.
- The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology.
- The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology.
- A test substance precipitate (oily in appearance) was seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies.
Acceptance criteria - the reverse mutation assay may be considered valid if the following criteria are met:
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.
- All tester strain cultures should show a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- All tester strain cultures should be in the range of 0.9-9 x10^9 bacterial per mL.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control cehmicals used in the study should induce marked increases in the frequency of revertant colonise, both in the presence and absence of metabolic activation.
- There should be a minimum of four non-toxic test substance dose levels.
- There should be no evidence of excessive contamination.
Table 3. Preliminary Cytotoxicity Test |
||||||
Treatment condition |
Treatment |
Cell concentration used for treatment (x 104 cells/mL) |
Post-treatment cell count (x 104 cells/mL) |
PD |
PD as % of Control |
Decrease in PD (%) |
without S9 mix: 3-h treatment + 24-h recovery |
Vehicle Control |
30 |
86.0 |
1.5 |
100 |
|
Test Item (mM) |
|
|
|
|
|
|
0.016 |
30 |
84.0 |
1.5 |
98 |
2 |
|
0.041 |
30 |
78.5 |
1.4 |
91 |
9 |
|
0.10 |
30 |
71.5 |
1.3 |
82 |
18 |
|
0.26 |
30 |
69.5 |
1.2 |
80 |
20 |
|
0.64 |
30 |
28.8 |
# |
# |
# |
|
1.6 |
30 |
1.6 |
# |
# |
# |
|
4 E |
30 |
0.3 |
# |
# |
# |
|
10 E |
30 |
0.3 |
# |
# |
# |
|
|
||||||
without S9 mix: 24-h treatment + 0-h recovery |
Vehicle Control |
30 |
100.0 |
1.7 |
100 |
|
Test Item (mM) |
|
|
|
|
|
|
0.016 |
30 |
90.0 |
1.6 |
91 |
9 |
|
0.041 |
30 |
105.0 |
1.8 |
104 |
None |
|
0.10 |
30 |
104.0 |
1.8 |
103 |
None |
|
0.26 |
30 |
135.0 |
2.2 |
125 |
None |
|
0.64 |
30 |
21.4 |
# |
# |
# |
|
1.6 |
30 |
0.9 |
# |
# |
# |
|
4 P |
30 |
0.0 |
# |
# |
# |
|
10 P |
30 |
0.0 |
# |
# |
# |
|
|
||||||
with S9 mix: 3-h treatment + 24-h recovery |
Vehicle Control |
30 |
116.0 |
2.0 |
100 |
|
Test Item (mM) |
|
|
|
|
|
|
0.016 |
30 |
111.0 |
1.9 |
97 |
3 |
|
0.041 |
30 |
107.0 |
1.8 |
94 |
6 |
|
0.10 |
30 |
90.0 |
1.6 |
81 |
19 |
|
0.26 |
30 |
88.0 |
1.6 |
80 |
20 |
|
0.64 |
30 |
77.5 |
1.4 |
70 |
30 |
|
1.6 |
30 |
63.0 |
1.1 |
55 |
45 |
|
4 E |
30 |
41.0 |
0.5 |
23 |
77 (too cytotoxic) |
|
10 E |
30 |
1.8 |
# |
# |
# |
|
PD: population doubling
Vehicle control: Ethanol
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
P: precipitate was noted in the culture medium at the end of treatment
E: emulsion was noted in the culture medium at the end of treatment
Table 4. First experiment without S9 mix, 3-h treatment + 24-h recovery: cytotoxicity |
||||||
Treatment |
Cell concentration used for treatment (x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of Control |
Decrease in PD (%) |
Vehicle Control |
30 |
C1 |
124.0 |
1.9 |
100 |
|
C2 |
104.0 |
|||||
Test item (mM) |
||||||
0.031 |
30 |
C1 |
143.0 |
2.1 |
108 |
None |
C2 |
112.0 |
|||||
0.063 |
30 |
C1 |
125.0 |
2.1 |
109 |
None |
C2 |
131.0 |
|||||
0.125 |
30 |
C1 |
123.0 |
2.0 |
105 |
None |
C2 |
122.0 |
|||||
0.250 |
30 |
C1 |
73.5 |
1.3 |
66 |
34 |
C2 |
71.5 |
|||||
0.375 |
30 |
C1 |
44.3 |
0.9 |
47 |
53 |
C2 |
67.5 |
|||||
0.50 P |
30 |
C1 |
15.2 |
# |
# |
# |
C2 |
25.2 |
|||||
0.75 P |
30 |
C1 |
3.7 |
# |
# |
# |
C2 |
1.4 |
|||||
1 P |
30 |
C1 |
5.1 |
# |
# |
# |
C2 |
13.6 |
|||||
Positive controls |
||||||
MMC (1 µg/mL ) |
30 |
C1 |
42.3 |
0.6 |
33 |
67 |
C2 |
50.7 |
|||||
COL (0.5 µg/mL) |
30 |
C1 |
21.4 |
# |
# |
# |
C2 |
19.5 |
|||||
PD: population doubling
Vehicle control: ethanol
MMC: Mitomycin C
COL: Colchicine
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
P: precipitate was noted in the culture medium at the end of treatment
Table 5. First experiment without S9 mix, 3-h treatment + 24-h recovery: cytogenetic results |
||||||||||||
Treatment |
Mean PD as % of Control |
Culture |
No. of cells Analysed |
Number of cells with n micronuclei |
Total micronucleated cells |
Frequency of micronucleated cells (‰) |
||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
Per culture |
Per dose |
Per culture |
Per dose |
||||
Vehicle Control |
100 |
C1 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
2 |
1.0 |
1.0 |
C2 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
1.0 |
||||
Test item (mM) |
||||||||||||
0.031 |
108 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.063 |
109 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.125 |
105 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
7 |
2.0 |
3.5 |
C2 |
1000 |
5 |
0 |
0 |
0 |
0 |
5 |
5.0 |
||||
0.250 |
66 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
3 |
2.0 |
1.5 |
C2 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
1.0 |
||||
0.375 |
47 |
C1 |
1000 |
5 |
0 |
0 |
0 |
0 |
5 |
8 |
5.0 |
4.0 |
C2 |
1000 |
3 |
0 |
0 |
0 |
0 |
3 |
3.0 |
||||
0.50 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.75 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
1 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
Positive controls |
||||||||||||
MMC (1 µg/mL ) |
33 |
C1 |
1000 |
148 |
16 |
2 |
0 |
0 |
166 |
387 |
166.0 |
193.5*** |
C2 |
1000 |
185 |
29 |
7 |
0 |
0 |
221 |
221.0 |
||||
COL (0.5 µg/mL) |
# |
C1 |
1000 |
43 |
3 |
0 |
0 |
0 |
45 |
90 |
45.0 |
45.0 *** |
C2 |
1000 |
44 |
1 |
0 |
0 |
0 |
45 |
45.0 |
||||
PD: population doubling Statistics: 2 x 2 contingency table:
Vehicle control: ethanol ***: p < 0.001
MMC: Mitomycin C
COL: Colchicine
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
P: precipitate was noted in the culture medium at the end of treatment
Table 6. First experiment without S9 mix, 24-h treatment + 0-h recovery: cytotoxicity |
||||||
Treatment |
Cell concentration used for treatment (x 104 cells/mL) |
Culture |
Post-treatment cell count (x 104 cells/mL) |
Mean PD |
Mean PD as % of Control |
Decrease in PD (%) |
Vehicle Control |
30 |
C1 |
69.0 |
1.2 |
100 |
|
C2 |
70.0 |
|||||
Test item (mM) |
||||||
0.031 |
30 |
C1 |
75.0 |
1.3 |
106 |
None |
C2 |
71.0 |
|||||
0.063 |
30 |
C1 |
54.5 |
1.0 |
86 |
14 |
C2 |
69.5 |
|||||
0.125 |
30 |
C1 |
61.0 |
1.1 |
93 |
None |
C2 |
70.5 |
|||||
0.250 |
30 |
C1 |
41.0 |
0.6 |
46 |
54 |
C2 |
47.3 |
|||||
0.375 |
30 |
C1 |
29.0 |
0.2 |
13 |
87 (too cytotoxic) |
C2 |
37.7 |
|||||
0.50 |
30 |
C1 |
8.5 |
# |
# |
# |
C2 |
7.3 |
|||||
0.75 |
30 |
C1 |
0.6 |
# |
# |
# |
C2 |
0.3 |
|||||
1 |
30 |
C1 |
0.8 |
# |
# |
# |
C2 |
1.1 |
|||||
Positive controls |
||||||
MMC (1 µg/mL ) |
30 |
C1 |
29.5 |
# |
# |
#
|
C2 |
28.3 |
|||||
COL (0.5 µg/mL) |
30 |
C1 |
16.1 |
# |
# |
# |
C2 |
25.5 |
|||||
PD: population doubling
Vehicle control: ethanol
MMC: Mitomycin C
COL: Colchicine
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
Table7. First experiment without S9 mix, 24-h treatment + 0-h recovery: cytogenetic results |
||||||||||||
Treatment |
Mean PD as % of Control |
Culture |
No. of cells Analysed |
Number of cells with n micronuclei |
Total micronucleated cells |
Frequency of micronucleated cells (‰) |
||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
Per culture |
Per dose |
Per culture |
Per dose |
||||
Vehicle Control |
100 |
C1 |
1000 |
5 |
0 |
0 |
0 |
0 |
5 |
6 |
5.0 |
3.0 |
C2 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
1.0 |
||||
Test item (mM) |
||||||||||||
0.031 |
106 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.063 |
86 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
9 |
2.0 |
4.5 |
C2 |
1000 |
6 |
1 |
0 |
0 |
0 |
7 |
7.0 |
||||
0.125 |
93 |
C1 |
1000 |
4 |
0 |
0 |
0 |
0 |
4 |
9 |
4.0 |
4.5 |
C2 |
1000 |
5 |
0 |
0 |
0 |
0 |
5 |
5.0 |
||||
0.250 |
46 |
C1 |
1000 |
2 |
1 |
0 |
0 |
0 |
3 |
6 |
3.0 |
3.0 |
C2 |
1000 |
3 |
0 |
0 |
0 |
0 |
3 |
3.0 |
||||
0.375 |
13 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.50 |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.75 |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
1 |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
Positive controls |
||||||||||||
MMC (1 µg/mL ) |
33 |
C1 |
1000 |
52 |
14 |
3 |
0 |
0 |
69 |
132 |
69.0 |
66.0 *** |
C2 |
1000 |
43 |
18 |
2 |
0 |
0 |
63 |
63.0 |
||||
COL (0.5 µg/mL) |
# |
C1 |
1000 |
99 |
16 |
1 |
0 |
0 |
116 |
155 |
116.0 |
77.5 *** |
C2 |
1000 |
31 |
8 |
0 |
0 |
0 |
39 |
39.0 |
||||
PD: population doubling Statistics: 2 x 2 contingency table:
Vehicle control: ethanol ***: p < 0.001
MMC: Mitomycin C
COL: Colchicine
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
Table 8. First experiment with S9 mix, 3-h treatment + 24-h recovery: cytotoxicity |
||||||
Treatment |
Cell concentration used for treatment (x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of Control |
Decrease in PD (%) |
Vehicle Control |
30 |
C1 |
84.0 |
1.9 |
100 |
|
C2 |
135.0 |
|||||
Test item (mM) |
||||||
0.188 |
30 |
C1 |
148.0 |
2.2 |
120 |
None |
C2 |
135.0 |
|||||
0.375 |
30 |
C1 |
133.0 |
2.2 |
117 |
None |
C2 |
139.0 |
|||||
0.75 P |
30 |
C1 |
100.0 |
1.8 |
99 |
1 |
C2 |
116.0 |
|||||
1.5 P |
30 |
C1 |
11.0 |
0.7 |
39 |
61 (too cytotoxic) |
C2 |
88.0 |
|||||
2 P |
30 |
C1 |
24.4 |
# |
# |
# |
C2 |
4.7 |
|||||
3 P |
30 |
C1 |
36.7 |
# |
# |
# |
C2 |
0.6 |
|||||
4 P |
30 |
C1 |
60.5 |
1.0 |
52 |
48 |
C2 |
57.5 |
|||||
6 P |
30 |
C1 |
0.0 |
# |
# |
# |
C2 |
0.4 |
|||||
Positive controls |
||||||
CPA (6 µg/mL) |
30 |
C1 |
58.0 |
0.9 |
50 |
50 |
C2 |
56.5 |
|||||
PD: population doubling
Vehicle control: ethanol
CPA: Cyclophosphamide
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
P: precipitate was noted in the culture medium at the end of treatment
Table 9. First experiment with S9 mix, 3-h treatment + 24-h recovery: cytogenetic results |
||||||||||||
Treatment |
Mean PD as % of Control |
Culture |
No. of cells Analysed |
Number of cells with n micronuclei |
Total micronucleated cells |
Frequency of micronucleated cells (‰) |
||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
Per culture |
Per dose |
Per culture |
Per dose |
||||
Vehicle Control |
100 |
C1 |
1000 |
3 |
0 |
0 |
0 |
0 |
1 |
4 |
3.0 |
2.0 |
C2 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
1.0 |
||||
Test item (mM) |
||||||||||||
0.188 |
120 |
C1 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
6 |
1.0 |
3.0 |
C2 |
1000 |
5 |
0 |
0 |
0 |
0 |
5 |
5.0 |
||||
0.375 |
117 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
3 |
2.0 |
1.5 |
C2 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
1.0 |
||||
0.75 P |
99 |
C1 |
1000 |
11 |
0 |
0 |
0 |
0 |
11 |
15 |
11.0 |
7.5 * |
C2 |
1000 |
14 |
0 |
0 |
0 |
0 |
4 |
4.0 |
||||
1.5 P |
39 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
2 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
3 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
4 P |
52 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
6 P |
# |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
Positive controls |
||||||||||||
CPA (6 µg/mL) |
50 |
C1 |
1000 |
114 |
15 |
3 |
0 |
0 |
132 |
247 |
132.0 |
123.5 *** |
C2 |
1000 |
104 |
10 |
1 |
0 |
0 |
115 |
115.0 |
||||
PD: population doubling Statistics: 2 x 2 contingency table:
Vehicle control: ethanol *: p < 0.05
CPA: Cyclophosphamide ***: p < 0.001
C1: Culture 1
C2: Culture 2
#: cell concentration at the end of treatment was lower than the cell concentration at the beginning of treatment
P: precipitate was noted in the culture medium at the end of treatment
Table 10. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytotoxicity |
||||||
Treatment |
Cell concentration used for treatment (x 104cells/mL) |
Culture |
Post-treatment cell count (x 104cells/mL) |
Mean PD |
Mean PD as % of Control |
Decrease in PD (%) |
Vehicle Control |
30 |
C1 |
201.0 |
2.7 |
100 |
|
C2 |
178.0 |
|||||
Test item (mM) |
||||||
0.125 |
30 |
C1 |
193.0 |
2.6 |
98 |
2 |
C2 |
172.0 |
|||||
0.25 |
30 |
C1 |
163.0 |
2.5 |
95 |
5 |
C2 |
181.0 |
|||||
0.375 |
30 |
C1 |
166.0 |
2.5 |
95 |
5 |
C2 |
178.0 |
|||||
0.5 P |
30 |
C1 |
176.0 |
2.6 |
98 |
2 |
C2 |
187.0 |
|||||
0.75 P |
30 |
C1 |
212.0 |
2.7 |
101 |
None |
C2 |
174.0 |
|||||
1 P |
30 |
C1 |
194.0 |
2.6 |
98 |
2 |
C2 |
173.0 |
|||||
1.5 P |
30 |
C1 |
136.0 |
2.3 |
86 |
14 |
C2 |
156.0 |
|||||
2 P |
30 |
C1 |
148.0 |
2.2 |
84 |
16 |
C2 |
133.0 |
|||||
Positive controls |
||||||
CPA (6 µg/mL) |
30 |
C1 |
71.0 |
1.3 |
47 |
53 |
C2 |
73.0 |
|||||
PD: population doubling
Vehicle control: ethanol
CPA: Cyclophosphamide
C1: Culture 1
C2: Culture 2
P: precipitate was noted in the culture medium at the end of treatment
Table 11. Second experiment with S9 mix, 3-h treatment + 24-h recovery: cytogenetic results |
||||||||||||
Treatment |
Mean PD as % of Control |
Culture |
No. of cells Analysed |
Number of cells with n micronuclei |
Total micronucleated cells |
Frequency of micronucleated cells (‰) |
||||||
n = 1 |
n = 2 |
n = 3 |
n = 4 |
n = 5 |
Per culture |
Per dose |
Per culture |
Per dose |
||||
Vehicle Control |
100 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
4 |
2.0 |
2.0 |
C2 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
2.0 |
||||
Test item (mM) |
||||||||||||
0.125 |
96 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
0.25 |
95 |
C1 |
1000 |
1 |
0 |
0 |
0 |
0 |
1 |
4 |
1.0 |
2.0 |
C2 |
1000 |
3 |
0 |
0 |
0 |
0 |
3 |
3.0 |
||||
0.375 |
95 |
C1 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
6 |
2.0 |
3.0 |
C2 |
1000 |
4 |
0 |
0 |
0 |
0 |
4 |
4.0 |
||||
0.5 P |
98 |
C1 |
1000 |
3 |
0 |
0 |
0 |
0 |
3 |
5 |
3.0 |
2.5 |
C2 |
1000 |
2 |
0 |
0 |
0 |
0 |
2 |
2.0 |
||||
0.75 P |
101 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
1 P |
98 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
1.5 P |
86 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
2 P |
84 |
C1 |
|
|
|
|
|
|
|
|
|
|
C2 |
|
|
|
|
|
|
|
|
||||
Positive controls |
||||||||||||
CPA (6 µg/mL) |
47 |
C1 |
1000 |
26 |
3 |
0 |
0 |
0 |
29 |
80 |
29.0 |
40.0 *** |
C2 |
1000 |
49 |
2 |
0 |
0 |
0 |
51 |
51.0 |
||||
PD: population doubling Statistics: 2 x 2 contingency table:
Vehicle control: Ethanol ***: p < 0.001
CPA: Cyclophosphamide
C1: Culture 1
C2: Culture 2
P: precipitate was noted in the culture medium at the end of treatmen
Table 12. Historical Data – Experiments without S9 Mix |
||||||
Parameter |
Frequency of Micronucleated cells in 1000 cells |
|||||
Treatment Conditions |
3 hours treatment + 24 hours recovery |
24 hours treatment + 0 hours recovery |
||||
Control Items |
Vehicle Control |
MMC (1 µg/mL) |
COL (0.5 µg/mL) |
Vehicle Control |
MMC (1 µg/mL) |
COL (0.5 µg/mL) |
N |
50 |
50 |
50 |
28 |
28 |
28 |
Mean |
1.7 |
133.0 |
22.9 |
2.1 |
46.4 |
34.5 |
SD |
1.0 |
67.5 |
13.3 |
1.1 |
19.8 |
17.5 |
Lower CL 95% |
1.4 |
113.8 |
19.1 |
1.6 |
38.7 |
27.7 |
Upper CL 95% |
1.9 |
152.2 |
26.6 |
2.5 |
54.1 |
41.2 |
5th Percentile |
0.5 |
23.0 |
7.5 |
0.0 |
11.5 |
14.5 |
Median |
1.5 |
138.3 |
20.0 |
2.0 |
49.5 |
27.0 |
95th Percentile |
4.0 |
238.5 |
54.5 |
3.5 |
72.5 |
65.5 |
Min |
0.0 |
18.0 |
5.5 |
0.0 |
8.0 |
10.0 |
Max |
4.5 |
306.0 |
65.0 |
5.0 |
97.5 |
75.0 |
COL: Colchicine
MMC: Mitomycin C
CL: Confidence Limit
Max: Maximal Value
SD: Standard Deviation
Min: Minimal Value
N – number of values
Table 13. Historical Data – Experiments with S9 Mix |
||
Parameter |
Frequency of Micronucleated cells in 1000 cells |
|
Treatment Conditions |
3 hours treatment + 24 hours recovery |
|
Control Items |
Vehicle Control |
CPA (6 µg/mL) |
N |
67 |
67 |
Mean |
1.5 |
101.9 |
SD |
0.9 |
49.3 |
Lower CL 95% |
1.3 |
89.8 |
Upper CL 95% |
1.7 |
113.9 |
5th Percentile |
0.5 |
26.0 |
Median |
1.5 |
105.5 |
95th Percentile |
3.0 |
184.5 |
Min |
0.0 |
14.0 |
Max |
3.5 |
251.0 |
CPA: Cyclophosphamide
CL: Confidence Limit
Max: Maximal Value
SD: Standard Deviation
Min: Minimal Value
N – number of values
Table 4. Results of Mutation Test (Assays 1 and 2) |
|||||
Assay |
Concentration (RTG) and MF values |
Increase in MF compared to vehicle control (GEF) |
Concentration related increase was indicated by the linear trend analysis |
Reproducible between cultures |
Conclusion |
Assay 1 |
50 µg/mL (56%): 105.7 |
32.9 (<GEF) |
Yes (Note: |
Yes
|
Negative
|
40 µg/mL (92%): 79.4 |
6.6 (<GEF) |
||||
20 µg/mL (88%): 83.1 |
10.3 (<GEF) |
||||
10 µg/mL (91%): 72.0 |
-0.8 (<GEF) |
||||
5 µg/mL (100%): 67.0 |
-5.8 (<GEF) |
||||
Vehicle (100%): 72.8 |
32.9 (<GEF) |
||||
Assay 1 |
40 µg/mL (7%): 165.6* |
80.6 (<GEF) |
No (Note: statistically significant increase in the MF values was seen at the highest evaluated concentration) |
Yes |
Negative |
20 µg/mL (96%): 78.9 |
-6.1 (<GEF) |
||||
10 µg/mL (93%): 114.8 |
29.8 (<GEF) |
||||
5 µg/mL (96%): 87.6 |
2.6 (<GEF) |
||||
2.5 µg/mL (83%):114.3 |
29.3 (<GEF) |
||||
Vehicle (100%): 85.0 |
|
||||
|
|||||
Assay 2 |
57.5 µg/mL (18%): 186.7 |
74.1 (<GEF) |
Yes (Note: |
Yes
|
Negative
|
55 µg/mL (24%): 173.2 |
60.6 (<GEF) |
||||
52.5 µg/mL (62%): 138.2 |
25.6 (<GEF) |
||||
50 µg/mL (52%): 152.2 |
39.6 (<GEF) |
||||
40 µg/mL (100%): 161.1 |
48.5 (<GEF) |
||||
Vehicle (100%): 112.6 |
|
||||
Assay 2 |
20 µg/mL (71%): 115.8 |
6.1 (<GEF) |
No (Note: No statistically significant increase in the MF values was seen) |
Yes |
Negative |
10 µg/mL (74%): 109.9 |
0.2 (<GEF) |
||||
5 µg/mL (81%): 116.3 |
6.6 (<GEF) |
||||
2.5 µg/mL (104%): 123.1 |
13.4 (<GEF) |
||||
Vehicle (100%): 109.7 |
|
||||
Notes: Results of the five or four highest evaluated concentrations are shown in the table. Statistical significance labelled by *.
MF: Mutation Frequency (refers to 106 viable cells), GEF: Global Evaluation Factor (=126 per 106 viable cells)
Table 5. Historical Control Data |
||||||
Mutation Frequency of the Negative Controls (2006-2016) |
||||||
|
Culture medium |
Distilled water |
||||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
3h,S9+ |
3h,S9- |
24h,S9- |
Average |
94.3 |
103.6 |
106.4 |
90.4 |
96.6 |
96.3 |
SD |
26.9 |
35.3 |
27.4 |
22.7 |
19.0 |
24.6 |
Min. |
39.3 |
52.6 |
41.7 |
33.4 |
55.1 |
43.2 |
Max. |
198.5 |
235.6 |
179.1 |
121.8 |
125.0 |
141.1 |
n |
84 |
43 |
44 |
26 |
13 |
13 |
Dimethyl sulfoxide (DMSO) |
||||||
Treatments |
3h,S9+ |
3h,S9- |
24h,S9- |
|
|
|
Average |
97.3 |
97.3 |
98.9 |
|
|
|
SD |
33.7 |
38.5 |
26.8 |
|
|
|
Min. |
44.2 |
33.7 |
47.1 |
|
|
|
Max. |
269.9 |
261.6 |
159.4 |
|
|
|
n |
101 |
57 |
50 |
|
|
|
Mutation Frequency of the Positive Controls (2006-2016) |
||||||
|
Cyclophosphamide |
4-Nitroquinoline-N-oxide |
||||
Treatments |
3h,S9+ |
|
|
|
3h,S9- |
24h,S9- |
Average |
1178.7 |
|
|
|
722.2 |
831.9 |
SD |
524.7 |
|
|
|
330.0 |
337.2 |
Min. |
196.1 |
|
|
|
223.5 |
245.0 |
Max. |
2642.5 |
|
|
|
1687.3 |
1577.6 |
n |
106 |
|
|
|
58 |
52 |
h = hour
SD = Standard Deviation
S9+ = experiment with metabolic activation
S9- = experiment without metabolic activation
n = number of cases
Mutation Assay 1
Presence of S9-mix (3-hour treatment):
In Assay 1, following a 3-hour treatment with metabolic activation marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation, no cells survived the expression period in the samples of 80 µg/mL concentration. Relative total growth of the highest evaluated concentration (50 µg/mL) was 56%. An evaluation was made using data of six concentrations (concentration range of 50-2.5 µg/mL).
No statistically significant increase in the mutation frequency values was observed. Concentration related increase was indicated by the linear trend analysis, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. This experiment was considered to be negative.
Absence of S9-mix (3-hour treatment):
In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 80, 60 and 50 µg/mL concentration. The relative total growth value of the highest evaluated concentration (40 µg/mL) was slightly below <10% (RTG value of 7%). No significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of five concentrations (concentration range of 40-2.5 µg/mL).
There was statistically significant increase in the mutation frequency values in the highest evaluated concentration, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF. Reproducibility was seen between. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.
Mutation Assay 2
Presence of S9-mix (3-hour treatment):
In Assay 2, following a 3-hour treatment with metabolic activation, marked or excessive cytotoxicity was seen at higher concentrations. No cells survived the expression period in the samples of 60 µg/mL concentration. Relative total growth of the highest evaluated concentration (57.5 µg/mL) was within the target range (RTG value of 18%). An evaluation was made using data of seven concentrations (concentration range of 57.5-10 µg/mL).
Concentration related increase was indicated by the linear trend analysis, however the increase was not statistically significant and the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance) in each case. Reproducibility was seen between replicates. This experiment was considered to be negative.
Absence of S9-mix (24-hour treatment):
In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 45, 40 and 30 µg/mL concentrations. Only the cells of replicate ‘A’ survived the expression period in the samples of 35 µg/mL concentration. No significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of four concentrations (concentration range of 20-2.5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. Reproducibility was seen between replicates. Therefore, this experiment was considered to be negative.
Validity of the Mutation Assays
Untreated, negative (vehicle/solvent) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle/solvent) and untreated controls were in the recommended range (50-170 x 10-6) in all cases.
The positive controls (Cyclophosphamide in the presence of metabolic activation and
4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all. All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD No. 490 criteria.
The plating efficiencies for the negative (vehicle/solvent) control of the test item and the untreated control samples at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays.
The number of test concentrations evaluated was at least four in Assay 1 and Assay 2, which met the acceptance criteria about the minimum number of evaluated concentrations.
The tested concentration range in the study was considered to be adequate as the selected range properly covered concentrations from cytotoxicity to no or little cytotoxicity. More closely spaced test item concentrations were used in the expected cytotoxic range. Furthermore, the highest evaluated concentration showed proper degree of cytotoxicity (approximately 80-90%, i.e. approximately 10-20 relative total growth)*.
*Notes:
In Assay 1 the experiment of 3-hour with metabolic activation, the relative total growth of the highest evaluated concentration (50 µg/mL) was 56%, thus a lower degree of cytotoxicity was observed than the recommendation of the relevant OECD guideline. However, in Assay 2 the experiment of 3-hour with metabolic activation, the relative total growth of the highest evaluated concentration (57.5 µg/mL) was 18%, therefore the RTG value of this concentration was within the target range of 10-20%. Thus, the results were considered to cover appropriate concentrations, being acceptable to justify the study for the exposures with metabolic activation.
In Assay 1 the experiment of 3-hour without metabolic activation there was no evidence of mutagenicity and there were a series of data points between 100% to 25% RTG and there was also a negative data point slightly below 10% RTG (at 40 µg/mL, RTG value of 7%). In Assay 2 the experiment of 24-hour without metabolic activation the relative total growth of the highest evaluated concentration (20 µg/mL) was 71%, thus a lower degree of cytotoxicity was observed than the recommendation of the relevant OECD guideline, however no cells survived at higher concentrations. There was no evidence of mutagenicity and a closely spaced concentration range was used to properly cover concentrations from cytotoxicity, to no or little cytotoxicity, in short and long treatment without metabolic activation. Therefore, taking together the two experiments without metabolic activation, it is concluded that it is valid to state that the test item is negative under this condition.
Suspension growth value of the untreated and negative (vehicle/solvent) control samples were in line with the recommended range in all cases (i.e. 8-32 fold in case of short treatments and 32-180 fold in case of long treatment).
The overall study was considered to be valid.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
A Key OECD Guideline 471 study (Harlan Laboratories Ltd., 2012; K = 1) was conducted to evaluate the ability of the test material (Ocimene PQ), to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.
The bacterial strains employed in this study were Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. The bacterial strains were treated with the test substance using the Ames plate incorporation (Mutation test 1) and pre-incubation (Mutation test 2) methods at up to seven dose levels, tested in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). A preliminary toxicity assay was initially performed in order to select appropriate dose levels for the main experiment. The test substance was found to be toxic to S. typhimurium TA100 at the 5000 µg/plate and non-toxic to E.coli WP2uvrA.
The dose range for Mutation test 1 was determined by the preliminary test and ranged from 0.15 -5000 µg/plate, depending on the bacterial strain and the presence or absence of the S9 -mix. Mutation test 2 was repeated on a separate date (pre-incubation), using an amended dose range of 0.05 -5000 µg/plate, fresh cultures of bacterial strains and fresh test substance preparations. Additional dose levels and an expanded dose range were selected where applicable, in order to achieve the four non-toxic dose levels and the toxic limit of the test substance.
In Mutation test 1, the test substance caused a visible reduction in the growth of the Salmonella bacterial background lawns from the 15 µg/plate (except for TA98 in the presence of the S9-mix), but no toxicity was seen to E.coli WP2uvrA. Mutation test 2, the test substance caused a slightly stronger toxic response with weakened bacterial background lawns initially from 5µg/plate in the absence of S9-mix and 15 µg/plate in the presence of the S9-mix. Weakened lawns in the E.coli WP2uvrA strain were noted at 1500 µg/plate only in the absence of metabolic activation. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology. The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains with any dose of the test material, either in the presence or absence of the S9 -mix or in either exposure method employed.
There was a test substance precipitate (oily in appearance) seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies. The vehicle (acetone) control plates gave revertant colony counts within the normal range and all positive chemicals induced marked increases in the frequency of revertant colonies, both in the presence and absence of metabolic activation. The acceptance criteria were met and therefore the sensitivity of the assay and efficacy of the S9 -mix were validated.
Under the conditions of the study and based on the results, it was concluded that the test substance, (Ocimene PQ), was not mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).
A Key OECD Guideline 487 in vitro study (Citoxlab France, 2019; K = 1) was conducted to evaluate the potential of the test material, (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene), to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.
After a preliminary cytotoxicity test, the test material, diluted in ethanol, was tested in two independent experiments with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:
First experiment (Without S9 mix: 3 h treatment + 24 h recovery; 24 h treatment + 0 h recovery; With S9 mix: 3 h treatment + 24 h recovery)
Second experiment (With S9 mix: 3 h treatment + 24 h recovery)
Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.
Then, after the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.
Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations. The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.
A precipitate was observed in the culture medium at the end of the 3h-treatment periods at dose levels ≥ 0.5 mM. No precipitate or emulsion was observed in the culture medium at the end of the 24h treatment period, at any of the tested dose levels.
Without S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the main experiment were 0.031, 0.063, 0.125, 0.25, 0.375, 0.5, 0.75 and 1 mM.
Cytotoxicity
A slight to severe cytotoxicity was induced at dose levels ≥ 0.25 mM.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
- 0.125, 0.25 and 0.375 mM for the 3h-treatment, the latter inducing the recommended level of cytotoxicity,
- 0.063, 0.125 and 0.25 mM for the 24h-treatment, the latter inducing the recommended level of cytotoxicity.
No statistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the corresponding vehicle controls. No dose response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
These results met the criteria for a negative response.
With S9 mix
With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected were as follows:
- 0.188, 0.375, 0.75, 1.5, 2, 3, 4, and 6 mM for the first experiment,
- 0.125, 0.25, 0.375, 0.5, 0.75, 1, 1.5, and 2 mM for the second experiment.
Cytotoxicity
In the first experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 1.5 mM.
In the second experiment, no noteworthy cytotoxicity was induced at any of the tested dose levels.
Micronucleus analysis
The dose levels selected for micronucleus analysis were as follows:
. 0.188, 0.375 and 0.75 mM, the latter being the lowest precipitating dose level,
. 0.25, 0.375 and 0.5 mM, the latter being the lowest precipitating dose level.
In the first experiment, a statistically significant increase in the frequency of micronucleated cells was observed at the dose level of0.75 mMrelative to the vehicle control. At this dose level,the frequency of micronucleated cells of both replicate cultures was above the corresponding vehicle reference data range. Since no dose-response relationship was demonstrated by the linear regression test, these results did not meet either the criteria for a positive or a negative response.
In order to check the reproducibility of the results obtained, a second experiment was undertaken under the same experimental conditions.
In the second experiment, nostatistically significant increase in the frequency of micronucleated cells was observed at any of the analyzed dose levels relative to the vehicle control. No dose-response relationship was demonstrated by the linear regression test and none of the analyzed dose levels showed frequencies of both replicate culture above the vehicle control historical range.
Since the increase in the frequency of micronucleated cells observed in the first experiment was not reproduced in the second experiment, this increase is considered not to be biologically relevant, and the overall results in the presence of metabolic activation are considered to meet the criteria for a negative response.
Under the experimental conditions of the study, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene), did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.
A Key OECD Guideline 490 in vitro mammalian cell assay (Citoxlab Hungary Ltd., 2019; K = 1) was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).
Acetone was used as vehicle of the test material in this study. Based on the results of the preliminary experiment, the following test material concentrations were examined in the mutation assays:
Assay 1 (3-hour treatment with metabolic activation): 80, 60, 50, 40, 20, 10 and 5 and 2.5 µg/mL;
Assay 1 (3-hour treatment without metabolic activation): 80, 60, 50, 40, 20, 10, 5 and 2.5 µg/mL;
Assay 2 (3-hour treatment with metabolic activation): 60, 57.5, 55, 52.5, 50, 40, 20 and 10 µg/mL;
Assay 2 (24-hour treatment without metabolic activation): 45, 40, 35, 30, 20, 10, 5 and 2.5 µg/mL.
In Assay 1 and Assay 2, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in Assay 1 and 2 with and without metabolic activation.
In Assay 1 and Assay 2, following a 3-hour treatment with metabolic activation, marked or excessive cytotoxicity was seen at higher concentrations. In Assay 1 with metabolic activation no cells survived the expression period in the samples of 80 µg/mL concentration. In Assay 2 with metabolic activation no cells survived the expression period in the samples of 60 µg/mL concentration. An evaluation was made using data of six concentrations (concentration range of 50-2.5 µg/mL) in Assay 1 and seven concentrations (concentration range of 57.5-10 µg/mL) in Assay 2.
No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations, although a concentration related increase was indicated by the linear trend analysis. Reproducibility was seen between replicates. These experiments were considered to be negative.
In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 80, 60 and 50 µg/mL concentration. The relative total growth value of the next concentration (40 µg/mL) was slightly below <10%. No significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of five concentrations (concentration range of 40 - 2.5 µg/mL). There was statistically significant increase in the mutation frequency values in the highest evaluated concentration in Assay 1, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF.
Reproducibility was seen between replicates. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.
In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 45, 40 and 30 µg/mL concentrations. Only the cells of replicate ‘A’ survived the expression period in the samples of 35 µg/mL concentration. No significant cytotoxicity was observed at lower concentrations.
An evaluation was made using data of four concentrations (concentration range of 20 - 2.5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.
The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.
In conclusion, no mutagenic effect of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Note: The final reports of the above OECD 490 study is currently awaited from the CRO. A spontaneous update of this dossier will be undertaken by the registrant upon receipt of all pending study reports from the CRO/s.
BlueScreen
The aim of this study was to assess the genotoxicity and cytotoxicity of the test substance, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene, with the BlueScreen HC genotoxicity screening assay, using a protocol with and without metabolic activation. The assay evaluates genotoxicity by the induction in GLuc expression.
The test substance was combined with 100% DMSO to give a 1250 mM colourless stock solution. It precipitated when added to water therefore further dilution of the DMSO stock solution was needed. The highest concentration of the test substance tested was at 156 μg/ml
The BlueScreen HC assay was performed in a 96 -well microtitre plate without metabolic activation (-S9). A dilution series of the test susbtance was generated in the microtitre plate. A standard genotoxic compound (4 -nitroquinoline 1 -oxide) was employed as a positive control and was added as an intra-plate quality control check to the microtitre plate at concentrations of 0.5 µg/mL and 0.125 µg/mL. The test system used was a genetically modified strain of cultured human lymphoblastoid TK6 cells (GLuc-T01) with a patented reported system, which explots the proper regulation of the GADD45a gene. This target gene mediates the adaptive response to genotoxic stress. The BlueScreen HC S9 assay was performed in the same cultured TK6 cell strain in a 96 -well microtitre plate as the first assay, but instead in the presence of metabolic activation (+S9). The test material was incubated with the test cells in the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours. Following this incubation period, the cells were washed in a phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C (5% CO2, 95% humidity). Cyclophosphate was employed as positive control, tested at concentrations of 25 µg/mL and 5µg/mL. A coelenterazine substrate was added to the microplate wells in both assays just prior to measurement, which reacts with GLuc resulting in luminescence. Cell exposure to a genotoxic compound increases GLuC expression that can be quantified by luminscence detection. Cytotoxicity was measured by lysis of cells and the addition of a fluorescent DNA binding stain, followed by the assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes. The quanity of luminescence is normalised to the fluorescence signal to correct for the variation in cell yield caused by cytotoxicity.
Based on the results of the study and the evaluation criteria, the test substance Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene, was negative for genotoxicity with and without metabolic activation in the BlueScreen HC S9 assay.
Justification for classification or non-classification
Based on the results observed in the in vitro assays, the test material Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene does not meet the criteria to be classified for mutagenicity under EU Regulation (EC) No 1272/2008 (CLP).
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