Registration Dossier
Registration Dossier
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EC number: 832-827-5 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
SynNova Base Oil has no mutagenic activity on the growth of the bacterial strains.
SynNova Base Oil was considered as not clastogenic in Chinese hamster V79 cells.
No mutagenic effect of SynNova Base Oil was observed in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay.
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:
- 09 April 2019 to 09 May 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- histidine (his) and tryptophan (trp)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The composition of solution refers to 1000 mL.
Induction of Liver Enzymes
Male Wistar rats (349-395 g, animals were 9-10 weeks old at initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 27 November 2018 (Citoxlab code: E13013).
Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 30 November 2018 (Citoxlab code: E13013, Expiry date: 30 November 2020).
The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 26.7 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately. - Test concentrations with justification for top dose:
- Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In Assay 1 and Assay 2 the same concentrations were used based on the absence of positive effect of the test item.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1, 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. - Vehicle / solvent:
- Propylene Glycol (PG) + 2% Polysorbate 80 was used as solvent to prepare the stock solution of the test material. The test item was freshly formulated at a concentration of 100 mg/mL in the vehicle on the day of administration in the Pharmacy of Citoxlab Hungary Ltd. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent. The formulation container was magnetic stirred continuously up to the end of dose administration procedures.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Dimethyl sulfoxide (DMSO), Distilled water & Propylene glycol with 2% (w/v) Polysorbate 80
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene (2AA) & 4-nitro-1,2-phenylene-diamine (NPD)
- Details on test system and experimental conditions:
- DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 and an Assay 2. In the Preliminary Range Finding Test as well as in the Assay 1, the plate incorporation method was used. In Assay 2 the plate incorporation method or the pre-incubation method was used.
Preliminary Compatibility Test
Based on the available information (trial formulations of the test item performed at the Test Facility), the test item was insoluble in the generally used vehicles (distilled water, DMSO, Acetone, Ethanol, PG + 1% Polysorbate 80). At the concentration level of 200 mg/mL settling emulsion was detected in Propylene Glycol + 2% Polysorbate 80. This emulsion with continuously stirring was suitable for the test. Therefore, PG + 2% Polysorbate 80 was selected for vehicle (solvent) of the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.
Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, a 100 mg/mL stock solution was prepared in PG + 2% Polysorbate 80. Six test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.
Test Item Concentrations in the Mutagenicity Tests (Assay 1 and Assay 2)
Based on the results of the preliminary test and main tests, in the Assay 1, 2 a 100 mg/mL stock solution was prepared in PG + 2% Polysorbate 80, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.
Procedure for Exposure in the Assay 1
Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
Procedure for Exposure in the Assay 2
Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours. - Rationale for test conditions:
- The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of the testing laboratory. .
- Evaluation criteria:
- The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in all Salmonella typhimurium and Escherichia coli WP2 uvrA bacterial strains
According to the guidelines [5][6][7][8], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation. - Statistics:
- The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- 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:
- cytotoxicity
- Remarks:
- Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was detected on the plates in both Salmonella typhimurium strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations in the preliminary experiment.
No inhibitory or toxic effects of the test item were detected in the preliminary experiment.
MUTAGENICITY TESTS (ASSAY 1 AND ASSAY 2)
In the Assay 1, the plate incorporation method was used. In the Assay 2, in case of all examined Salmonella typhimurium strains without metabolic activation the plate incorporation method was used. In case of all examined Salmonella typhimurium strains with metabolic activation and Escherichia coli WP2 uvrA with and without metabolic activation the pre-incubation method was used. The Assay 1, 2 were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1, 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
Precipitate/slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and/or 1581 μg/plate concentrations in Assays 1 and 2.
Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.19). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In Assay 2 (plate incorporation method/pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.38). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.
VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid. - Conclusions:
- The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, Assay 1 (Plate Incorporation Method) and Assay 2 (Plate Incorporation Method or Pre-Incubation Method).
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item SynNova Base Oil (Batch Number: TS20371) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study. - Executive summary:
The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), Assay 1 (Plate Incorporation Method) and Assay 2 (Plate Incorporation Method or Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in Propylene Glycol + 2% Polysorbate 80 at a maximum concentration of 100 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the absence and presence of metabolic activation were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate in Assays 1 and 2.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
Precipitate/slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and/or 1581 μg/plate concentrations in Assays 1-2.
Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item SynNova Base Oil (Batch Number: TS20371) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 April 2019 to 24 April 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Version / remarks:
- OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Commission Regulation (EU) 2017/735 of 14 February 2017, B.10. "Mutagenicity – In Vitro Mammalian Chromosome Aberration Test" amending Commission Regulation (EC) No 440/2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- somatic and/or germ cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- V79: Chinese hamster lung, male
ECACC Cat. No.: 86041102
Lot No.: 10H016
Date of working lot: 24 January 2019
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10°C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5). - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
Induction of Rat Liver Enzymes
Male Wistar rats (292-387 g animals were 8 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation dates of the induction of liver enzymes used for preparation S9 used in this study was 05 January 2018.
Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 08 January 2018 (Citoxlab Hungary Ltd. code: E12790, Expiry date: 08 January 2020).
The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used in the study was determined to be 30.45 g/L. The sterility of the preparation was confirmed.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately. - Test concentrations with justification for top dose:
- In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation). - Vehicle / solvent:
- Based on the result of the trial formulations of the test item performed at the Test Facility, at 200 mg/mL concentration insolubility was detected using the generally used vehicles (distilled water, DMSO, Acetone, Ethanol, PG + 1% Polysorbate 80). At the concentration level of 200 mg/mL settling emulsion was detected in Propylene Glycol + 2% Polysorbate 80, but this emulsion with continuously stirring was suitable for the test. Therefore, PG + 2% Polysorbate 80 was selected for vehicle of the study. The vehicle was compatible with the survival of the cells and the S9 activity.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Propylene glycol with 2% (w/v) Polysorbate 80
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Toxicity and Concentration Selection
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
In this Preliminary Toxicity Test, the cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time and for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC% (Relative Increase in Cell Counts).
Chromosome Aberration Assays
The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.
In Assay 2, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.
Treatment of the Cells
For the cytogenetic experiments, 1-3 day old cultures (more than 50% confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 105 cells/dish concentration and incubated for approximately 24 hours at 37°C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
Cells were treated with different concentration test item solutions, untreated, negative (vehicle) or positive control solution (treatment volume was 100 μL/dish ) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in the preliminary and both main tests.
For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC%(Relative Increase in Cell Counts).
Preparation of Chromosomes
2-2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells* was dropped onto clean microscope slides and air-dried. The slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.
*Note: Fixed cells were stored frozen in case if any additional action was required (as documented in the raw data and reported). After the finalization of the report, the remaining frozen cell suspension samples will be discarded.
Examination of Slides
The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
The coded slides were sent for evaluation to the attention of Principal Investigator:
Natalie Danford, B.Sc., MPH, PhD.
Principal Investigator
Microptic Cytogenetics
2 Langland Close
Mumbles
Swansea, SA3 4LY, United Kingdom
The metaphase analysis was conducted under the control of the Principal Investigator. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
At least 150* metaphases with 22±2 chromosomes (centromeres) from each culture (replicate) were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
The aberrations are defined in the following way:
Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
Chromosome-type:structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage. The examination of slides from a culture may be halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded.
The vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.
The metaphase analysis was conducted in compliance with Good Laboratory Practice as required by the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997). These Principles are in conformity with other international GLP regulations.
When the metaphase analysis has been performed, the original raw data (record sheets) for the metaphase analysis and the microscope slides are shipped back to the Test Facility (Citoxlab Hungary Ltd). After the slide reading phase of the study had ended, the Principal Investigator issued a Work Phase Report. - Rationale for test conditions:
- In accordance with the test guidelines.
- Evaluation criteria:
- The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.
Evidence of a dose-response relationship (if any) was considered to support the conclusion.
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- VEHICLE AND CONCENTRATION SELECTION
Based on the available solubility information (trial formulations of the test item performed at the Test Facility), PG + 2% Polysorbate 80 was selected for vehicle of the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
Concentration Selection Cytotoxicity Assays (3-hour treatment with and without metabolic activation, 20-hour harvesting time; and 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays.
A total of ten test concentrations between 2000 and 3.906 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD No. 473 guideline instructions (up to the solubility limit).
CHROMOSOME ABERRATION ASSAYS
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 1, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-31.25 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 2, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-62.5 μg/mL concentration range with metabolic activation and in the 250-31.25 μg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in the experiment with and without metabolic activation. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 62.5, 31.25 and 15.625 μg/mL (a total of three) were evaluated in the experiment without metabolic activation due to the observed insolubility of the test item.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
VALIDITY OF THE STUDY
The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and without metabolic activation; while cytotoxicity was not detected in Assay 1 and Assay 2 with and without metabolic activation. The evaluated concentration ranges of the Assays were considered to be adequate, as they covered the range from insolubility to no or little insolubility.
Three test item concentrations were evaluated in each experiment.
The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the acceptable historical control range.
In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations (Tables 9-12 of Appendix 5) demonstrating the sensitivity of the test system in each assay.
The study was considered to be valid. - Conclusions:
- The test item SynNova Base Oil was tested for potential clastogenic activity using the Chromosome Aberration Assays. The study included a Concentration Selection Cytotoxicity Assay and two Chromosome Aberration Assays.
The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.
Treatment with the test item did not result in a statistically and biologically significant, reproducible, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, SynNova Base Oil did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, SynNova Base Oil was considered as not clastogenic in this test system. - Executive summary:
SynNova Base Oil was tested in vitro in a Chromosome Aberration Assays using Chinese hamster V79 lung cells. The test item was formulated in PG (propylene glycol) + 2% Polysorbate 80 and it was examined up to solubility limit concentration according to the OECD No. 473 guideline recommendations. In the two independent Chromosome Aberration Assays using duplicate cultures, at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each evaluated test item treated, negative (vehicle) and positive control sample.
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 1, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-31.25 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 2, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-62.5 μg/mL concentration range with metabolic activation and in the 250-31.25 μg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in the experiment with and without metabolic activation. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 62.5, 31.25 and 15.625 μg/mL (a total of three) were evaluated in the experiment without metabolic activation due to the observed insolubility.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases and/or endoreduplicated metaphases were found in some cases in the negative (vehicle) control or positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with SynNova Base Oil.
The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.
In conclusion, SynNova Base Oil did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, SynNova Base Oil was considered as not clastogenic in this test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 April 2019 to 06 June 2019
- 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)
- Version / remarks:
- OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 29 July 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008)
- Deviations:
- no
- 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:
- No further details specified in the study report.
- Target gene:
- thymidine kinase (tk) locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 60797977
Supplier: American Type Culture Collection (Manassas, Virginia, USA)
Date of receipt: 27 February 2014
Date of working lot: 17 May 2017 (MP14)
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. 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 assays. - Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- Not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- The post-mitochondrial fraction (S9 fraction) was prepared from rat liver [3, 4] by the Microbiological Laboratory of Citoxlab Hungary Ltd. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived annually.
Induction of Liver Enzymes:
Male Wistar rats (292-387 g, animals were 8 weeks old at initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage (for both inducers) for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 05 January 2018.
Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. Sterility of the preparation was confirmed.
The protein concentration was determined by colorimetric test by chemical analyser at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 30.45 g/L. The date of preparation of S9 fraction for this study was 08 January 2018 (Citoxlab code: E12790, Expiry date: 08 January 2020).
The biological activity of each batch of S9 was characterized in the Salmonella assay using 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study was found active under the test conditions.
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath. - Test concentrations with justification for top dose:
- Based on the preliminary toxicity test and in agreement with the Sponsor, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 1, 3-hour treatment without metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 2, 3-hour treatment with metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 2, 24-hour treatment without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
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 concentrations were selected for the main assays according to the OECD No. 490 guideline instructions. A total of seven concentrations with and without metabolic activation were selected for Assays 1-2. - Vehicle / solvent:
- PG + 2% Polysorbate 80 was used for vehicle of the test item. In each case, sterile vehicle was prepared and used for formulation. Based on the available information (trial formulations of the test item performed in another in vitro study at the Test Facility [19/045-020C]), due to its chemical nature the test item was insoluble in generally used vehicles (distilled water, DMSO, Acetone, Ethanol, PG + 1% Polysorbate 80), only a settling emulsion could be achieved up to 200 mg/mL concentration with continuously stirring detected in Propylene Glycol + 2% Polysorbate 80. Therefore, PG + 2% Polysorbate 80 was selected for vehicle of the study.
Data of the chemicals used for vehicle are shown below:
Name: Propylene glycol with 2% (w/v) Polysorbate 80
Dispense code: S55001 / S55002*
Manufacturer: Citoxlab Hungary Ltd.
Expiry Date: 02 May 2019 / 26 May 2019
Storage condition: Room temperature
The Propylene glycol with 2% (w/v) Polysorbate 80 solution was prepared from Polysorbate 80 produced by Sigma-Aldrich (Batch number: BCBV5152, Expiry date: 30 June 2022) and Propylene glycol produced by Merck (Batch number: K50526178, Expiry date: 31 August 2023 and Batch number: K50917678, Expiry date: 31 December 2023).
The vehicle was filtered sterile using a 0.22 μm syringe filter before use (Supplier: Millipore, Lot No.: MP171520SA, Expiry date: April 2020). - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Propylene glycol with 2% (w/v) Polysorbate 80 & Dimethyl sulfoxide (DMSO)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- Main Mutation Assays
In Assay 1, cells were treated for 3 hours in the presence and absence of S9-mix. In Assay 2, cells were treated for 3 hours in the presence of S9-mix and for 24 hours in the absence of S9-mix.
A suitable volume (0.2 mL for a final volume of 20 mL (10 μL/mL) of RPMI-5 medium, vehicles (solvents), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6E+6 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37°C ± 1°C (approximately 5% CO2 in air). Gentle shaking was used during the treatments. Measurement of pH and osmolality was also performed after the treatment period.
Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer. Where sufficient cells survived, cell density was adjusted to a concentration of 2x105 cells/mL (if possible). Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.
Plating for Survival
Cultures of cell density 2E+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 37 ºC ± 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 2x105 cells/mL (whenever possible) and transferred to flasks for further growth.
On completion of the expression period, seven 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 1E+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 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days). Wells containing viable clones were identified by eye using background illumination and counted.
Plating for -trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1x104 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 2x103 cells per well.
Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (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. - Rationale for test conditions:
- In accordance with the test guidelines.
- 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. - 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:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- 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 PG + 2% Polysorbate 80 as vehicle was 2000 μg/mL (the recommended maximum concentration).
In the preliminary experiment no cytotoxicity was detected. Insolubility / minimal amount of insolubility was observed with and without metabolic activation at concentration range of 2000-125 μg/mL.
Concentrations were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline (based on the limited solubility of the test item). Seven concentrations were selected for the main experiments in each assay.
MUTATION ASSAYS
In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel subcultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks (12 days) in culturing plates with and without selective agent (TFT) for determination of mutations and viability.
Assay 1
In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL with and without metabolic activation.
In Assay 1, there were no large changes in pH or osmolality after treatment. In Assay 1, insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment with metabolic activation at 250 and 125 μg/mL concentrations and without metabolic activation at 250 μg/mL concentration.
Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observered. Thus, an evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
Absence of S9-mix (3-hour treatment)
In the absence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
Assay 2
In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL with metabolic activation and 1000, 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL without metabolic activation.
In Assay 2, there were no large changes in pH or osmolality after treatment. In Assay 2, insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment with metabolic activation at 250 μg/mL concentration and without metabolic activation at concentration range of 1000-250 μg/mL.
Presence of S9-mix (3-hour treatment)
In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative and confirmed the clearly negative result of the first test with metabolic activation.
Absence of S9-mix (24-hour treatment)
In the absence of S9-mix (24-hour treatment), no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 1000-15.625 μg/mL).
No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly 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 (50-170E-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 assays (for historical control data see Appendix 18). 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) controls of the test item and positive control item as well as the untreated control samples at the end of the expression period (PEviability) were acceptable in all assays.
The number of test concentrations evaluated was seven 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 concentrations up to the highest practical concentration (based on the limited solubility of the test item) were examined in the study and lower test concentrations were spaced by a factor of two. The examined concentration range covered the range from insolubility to no insolubility.
Suspension growth value of the untreated and negative (vehicle) controls were lower than the recommended value in Assay 2, however as all of the observed spontaneous mutation frequency values of the untreated and negative (vehicle) control samples were in the recommended range, this fact was considered to be acceptable and not to adversely affect the results of the study.
The overall study was considered to be valid. - Conclusions:
- The Mouse Lymphoma Assay on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.
In this Mouse Lymphoma Assay, treatment with the test item did not result in a statistically significant and/or biologically relevant increase in the mutation frequency in the presence or absence of a rat metabolic activation system (S9 fraction) in Assay 1 and Assay 2. Therefore, no mutagenic activity of the test item was observed in the performed experiments.
In conclusion, no mutagenic effect of SynNova Base Oil was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. - Executive summary:
An 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 SynNova Base Oil test item 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). The design of this study was based on the OECD No. 490 guideline, and the study was performed in compliance with Citoxlab Hungary Ltd. standard operating procedures and with the OECD Principles of Good Laboratory Practice.
Propylene glycol (PG) + 2% Polysorbate 80 was used as vehicle of the test item in this study. Based on the preliminary toxicity test and in agreement with the Sponsor, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 1, 3-hour treatment without metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 2, 3-hour treatment with metabolic activation: 250, 125, 62.5, 31.25, 15.625, 7.813 and 3.906 μg/mL,
Assay 2, 24-hour treatment without metabolic activation: 1000, 500, 250, 125, 62.5, 31.25 and 15.625 μg/mL.
In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility / minimal amount of insolubility was observed in the final treatment medium at the end of the treatment in Assays 1-2 with and without metabolic activation at higher concentrations.
In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. Thus, an evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
In Assay 2, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative and confirmed the clearly negative result of the first test with metabolic activation.
In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 1000-15.625 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly 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 SynNova Base Oil was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Referenceopen allclose all
Summary Tables of the Results
Summary Table of the Range Finding Test
Concentrations (µg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimurium tester strains |
|||
TA98 |
|
TA100 |
|
||
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
14.7 |
23.3 |
92.0 |
96.7 |
MF |
0.88 |
1.27 |
1.03 |
0.97 |
|
DMSO control |
Mean |
14.0 |
22.7 |
-- |
94.3 |
MF |
0.84 |
1.24 |
-- |
0.95 |
|
Distilled water control |
Mean |
-- |
-- |
88.3 |
-- |
MF |
-- |
-- |
0.99 |
-- |
|
PG + 2% Polysorbate 80 control |
Mean |
16.7 |
18.3 |
89.3 |
99.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
13.7 |
23.3 |
98.3 |
94.0 |
MF |
0.82 |
1.27 |
1.10 |
0.94 |
|
2500 |
Mean |
16.0 |
19.0 |
96.3 |
92.3 |
MF |
0.96 |
1.04 |
1.08 |
0.93 |
|
1000 |
Mean |
14.0 |
14.7 |
91.0 |
95.7 |
MF |
0.84 |
0.80 |
1.02 |
0.96 |
|
316 |
Mean |
12.0 |
23.0 |
100.7 |
98.3 |
MF |
0.72 |
1.25 |
1.13 |
0.99 |
|
100 |
Mean |
20.7 |
22.3 |
94.0 |
101.0 |
MF |
1.24 |
1.22 |
1.05 |
1.01 |
|
31.6 |
Mean |
18.3 |
20.3 |
95.7 |
101.7 |
MF |
1.10 |
1.11 |
1.07 |
1.02 |
|
10 |
Mean |
17.7 |
18.7 |
92.0 |
96.3 |
MF |
1.06 |
1.02 |
1.03 |
0.97 |
|
NDP (4µg) |
Mean |
400.0 |
-- |
-- |
-- |
MF |
28.57 |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2470.7 |
-- |
2469.3 |
MF |
|
109.00 |
-- |
26.18 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1060.0 |
-- |
MF |
-- |
-- |
12.00 |
-- |
|
Summary Table of the Assay 1
Concentrations (µg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
18.3 |
21.0 |
94.7 |
106.0 |
12.7 |
11.0 |
8.0 |
7.3 |
43.3 |
47.3 |
MF |
0.98 |
1.02 |
1.03 |
1.14 |
0.79 |
1.00 |
1.09 |
1.05 |
0.98 |
0.97 |
|
DMSO control |
Mean |
18.3 |
22.3 |
-- |
105.7 |
-- |
12.7 |
7.7 |
7.0 |
-- |
47.7 |
MF |
0.98 |
1.08 |
-- |
1.13 |
-- |
1.15 |
1.05 |
1.00 |
-- |
0.98 |
|
Distilled water control |
Mean |
-- |
-- |
92.0 |
-- |
13.7 |
-- |
-- |
-- |
42.0 |
-- |
MF |
-- |
-- |
1.00 |
-- |
0.85 |
-- |
-- |
-- |
0.95 |
-- |
|
PG + 2% Polysorbate 80 control |
Mean |
18.7 |
20.7 |
92.0 |
93.3 |
16.0 |
11.0 |
7.3 |
7.0 |
44.0 |
48.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
17.0 |
21.0 |
84.0 |
87.3 |
11.0 |
12.7 |
7.3 |
7.3 |
40.0 |
45.7 |
MF |
0.91 |
1.02 |
0.91 |
0.94 |
0.69 |
1.15 |
1.00 |
1.05 |
0.91 |
0.94 |
|
1581 |
Mean |
17.3 |
20.3 |
93.3 |
99.3 |
11.3 |
9.7 |
7.3 |
7.7 |
42.0 |
49.3 |
MF |
0.93 |
0.98 |
1.01 |
1.06 |
0.71 |
0.88 |
1.00 |
1.10 |
0.95 |
1.01 |
|
500 |
Mean |
17.0 |
22.3 |
77.3 |
93.0 |
12.3 |
10.7 |
6.3 |
7.7 |
41.3 |
46.7 |
MF |
0.91 |
1.08 |
0.84 |
1.00 |
0.77 |
0.97 |
0.86 |
1.10 |
0.94 |
0.96 |
|
158.1 |
Mean |
19.0 |
19.7 |
80.7 |
93.0 |
10.0 |
12.3 |
7.0 |
7.7 |
39.3 |
46.3 |
MF |
1.02 |
0.95 |
0.88 |
1.00 |
0.63 |
1.12 |
0.95 |
1.10 |
0.89 |
0.95 |
|
50 |
Mean |
17.3 |
19.3 |
78.7 |
90.3 |
10.7 |
10.0 |
7.7 |
7.3 |
41.3 |
49.0 |
MF |
0.93 |
0.94 |
0.86 |
1.00 |
0.67 |
0.91 |
1.05 |
1.05 |
0.94 |
1.01 |
|
15.81 |
Mean |
17.7 |
18.0 |
85.3 |
87.0 |
13.0 |
10.0 |
8.0 |
8.3 |
41.7 |
47.3 |
MF |
0.95 |
0.87 |
0.93 |
0.93 |
0.81 |
0.91 |
1.09 |
1.19 |
0.95 |
0.97 |
|
NPD (4µg) |
Mean |
410.7 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
22.40 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2408.0 |
-- |
2452.0 |
-- |
257.0 |
-- |
233.7 |
-- |
-- |
MF |
-- |
107.82 |
-- |
23.21 |
-- |
20.29 |
-- |
33.38 |
-- |
-- |
|
2AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
274.3 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
5.76 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1253.3 |
-- |
1290.7 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
13.62 |
-- |
94.44 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
417.3 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
54.43 |
-- |
-- |
-- |
|
MMS (2µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1084.0 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
25.81 |
-- |
|
Summary Table of the Assay 2
Concentrations (µg/plate) |
Mean values of revertants / Mutation factor (MF) |
Salmonella typhimuriumtester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
17.3 |
23.3 |
95.3 |
104.3 |
15.3 |
15.3 |
7.7 |
9.0 |
54.0 |
57.7 |
MF |
0.90 |
1.00 |
0.96 |
1.05 |
1.05 |
1.05 |
0.96 |
1.29 |
1.09 |
1.01 |
|
DMSO control |
Mean |
18.0 |
21.7 |
-- |
98.7 |
-- |
13.3 |
8.7 |
8.0 |
-- |
57.0 |
MF |
0.93 |
0.93 |
-- |
0.99 |
-- |
0.91 |
1.08 |
1.14 |
-- |
0.99 |
|
Distilled water control |
Mean |
-- |
-- |
96.3 |
-- |
14.7 |
-- |
-- |
-- |
49.7 |
-- |
MF |
-- |
-- |
0.97 |
-- |
1.00 |
-- |
-- |
-- |
1.00 |
-- |
|
PG + 2% Polysorbate 80 control |
Mean |
19.3 |
23.3 |
99.0 |
99.7 |
14.7 |
14.7 |
8.0 |
7.10 |
49.7 |
57.3 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
19.3 |
20.3 |
95.3 |
108.3 |
16.0 |
15.0 |
9.3 |
6.7 |
53.3 |
57.7 |
MF |
1.00 |
0.87 |
0.96 |
1.09 |
1.09 |
1.02 |
1.17 |
0.95 |
1.07 |
1.01 |
|
1581 |
Mean |
21.7 |
24.3 |
95.3 |
105.0 |
16.0 |
15.3 |
6.7 |
8.0 |
56.7 |
55.3 |
MF |
1.12 |
1.04 |
0.96 |
1.05 |
1.09 |
1.05 |
0.83 |
1.14 |
1.14 |
0.97 |
|
500 |
Mean |
19.7 |
19.7 |
92.7 |
94.0 |
17.3 |
14.7 |
10.3 |
6.0 |
53.7 |
56.3 |
MF |
1.02 |
0.84 |
0.94 |
.094 |
1.18 |
1.00 |
1.29 |
0.86 |
1.08 |
0.98 |
|
158.1 |
Mean |
19.0 |
25.0 |
99.0 |
96.7 |
16.7 |
16.0 |
9.7 |
6.7 |
48.7 |
59.0 |
MF |
0.98 |
1.07 |
1.00 |
0.97 |
1.14 |
1.09 |
1.21 |
0.95 |
0.98 |
1.03 |
|
50 |
Mean |
19.3 |
27.3 |
104.7 |
96.0 |
17.0 |
15.7 |
7.0 |
7.0 |
53.7 |
58.0 |
MF |
1.00 |
1.17 |
1.06 |
0.96 |
1.16 |
1.07 |
0.88 |
1.00 |
1.08 |
1.01 |
|
15.81 |
Mean |
19.0 |
22.0 |
95.3 |
102.0 |
14.7 |
16.0 |
9.0 |
9.7 |
48.7 |
57.7 |
MF |
0.98 |
0.94 |
0.96 |
1.02 |
1.00 |
1.09 |
1.13 |
1.38 |
0.98 |
1.01 |
|
NPD (4µg) |
Mean |
449.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
24.96 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2466.7 |
-- |
2432.0 |
-- |
233.7 |
-- |
210.0 |
-- |
-- |
MF |
-- |
113.85 |
-- |
24.65 |
-- |
17.53 |
-- |
26.25 |
-- |
-- |
|
2AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
258.3 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.53 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1201.3 |
-- |
1210.7 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
12.47 |
-- |
82.55 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
448.0 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
51.69 |
-- |
-- |
-- |
|
MMS (2µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1108.0 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
22.31 |
-- |
|
Historical Control Data
(Period of 2011-2018)
Untreated control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
T1535 |
TA1537 |
E. coli |
|
Mean |
22.3 |
101.5 |
12.1 |
7.9 |
36.3 |
27.4 |
108.9 |
11.6 |
9.4 |
41.3 |
St. dev. |
5.4 |
19.5 |
4.6 |
3.5 |
10.8 |
6.7 |
18.4 |
3.6 |
3.8 |
10.3 |
Range |
9-50 |
54-210 |
1-46 |
1-26 |
11-82 |
10-56 |
65-204 |
1-39 |
1-29 |
16-89 |
n |
1860 |
1846 |
1857 |
1866 |
1875 |
1878 |
1869 |
1877 |
1881 |
1875 |
DMSO control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
21.4 |
97.5 |
12.1 |
7.7 |
35.3 |
27.6 |
106.4 |
11.4 |
9.1 |
40.4 |
St. Dev. |
5.3 |
18.8 |
4.5 |
3.4 |
10.7 |
6.7 |
19.3 |
3.9 |
3.7 |
10.2 |
Range |
6-55 |
40-217 |
1-43 |
1-27 |
7-81 |
11-67 |
53-229 |
2-33 |
1-29 |
9-85 |
n |
2007 |
1992 |
2004 |
2016 |
2019 |
2024 |
2013 |
2027 |
2028 |
2022 |
Distilled water control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
23.0 |
101.0 |
12.1 |
8.7 |
37.4 |
29.0 |
108.9 |
11.4 |
10.0 |
42.3 |
St. Dev. |
5.5 |
20.3 |
4.4 |
3.5 |
10.7 |
6.7 |
20.3 |
3.4 |
3.7 |
10.1 |
Range |
11-45 |
45-215 |
2-47 |
2-24 |
12-84 |
10-53 |
64-222 |
3-39 |
1-24 |
13-91 |
n |
399 |
1863 |
1869 |
405 |
1905 |
402 |
1875 |
1887 |
402 |
1893 |
DMF control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.4 |
90.2 |
11.4 |
7.7 |
37.3 |
27.1 |
98.9 |
11.1 |
8.9 |
40.0 |
St. Dev. |
5.2 |
16.8 |
4.3 |
3.4 |
12.8 |
6.8 |
18.0 |
3.3 |
3.5 |
11.0 |
Range |
8-38 |
54-152 |
1-34 |
1-19 |
16-99 |
11-49 |
60-156 |
3-21 |
1-23 |
17-76 |
n |
276 |
276 |
276 |
276 |
267 |
276 |
276 |
276 |
273 |
267 |
Acetone control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
22.4 |
97.7 |
12.1 |
7.5 |
36.2 |
28.5 |
106.8 |
11.1 |
8.8 |
41.3 |
St. Dev. |
5.0 |
14.7 |
5.6 |
2.9 |
9.6 |
6.6 |
14.2 |
3.4 |
3.3 |
9.1 |
Range |
11-39 |
62-160 |
4-49 |
1-17 |
17-63 |
15-52 |
66-177 |
4-22 |
1-19 |
17-70 |
n |
314 |
315 |
315 |
318 |
312 |
315 |
315 |
318 |
318 |
315 |
Positive reference control data |
||||||||||
|
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
368.8 |
1208.5 |
1165.0 |
444.3 |
1034.2 |
2410.8 |
2425.1 |
229.2 |
219.0 |
255.4 |
St. Dev. |
100.9 |
185.0 |
179.4 |
147.1 |
140.2 |
274.5 |
252.0 |
117.0 |
49.0 |
98.2 |
Range |
152-2336 |
536-2120 |
208-2440 |
149-2104 |
488-2496 |
312-4918 |
1192-5240 |
101-2216 |
117-838 |
125-2512 |
n |
1860 |
1848 |
1857 |
1866 |
1878 |
1878 |
1869 |
1881 |
1881 |
1875 |
TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E. coli: Escherichia coli WPR uvrA, n: number of cases
SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAYS
Summarized results of the Concentration Selection Cytotoxicity without metabolic activation (3h/20h)
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
- |
3/20 |
7.50E+06 |
108 |
normal / normal (pH: 7.7; osm 345 mmol/kg) |
Negative (vehicle) control |
- |
- |
3/20 |
7.10E+06 |
100 |
normal / normal (pH: 7.7; osm 465 mmol/kg) |
SynNova Base Oil |
2000 |
- |
3/20 |
7.15E+06 |
101 |
precipitate / precipitate (pH: 7.7; osm: 431 mmol/kg) |
1000 |
- |
3/20 |
7.10E+06 |
100 |
precipitate / precipitate (pH: 7.7; osm: 441 mmol/kg) |
|
500 |
- |
3/20 |
6.70E+06 |
92 |
precipitate / precipitate (pH: 7.7; osm: 459 mmol/kg) |
|
250 |
- |
3/20 |
7.10E+06 |
100 |
precipitate / precipitate (pH: 7.7; osm: 466 mmol/kg) |
|
125 |
- |
3/20 |
6.85E+06 |
95 |
precipitate / precipitate (pH: 7.7; osm: 469 mmol/kg) |
|
62.5 |
- |
3/20 |
6.65E+06 |
91 |
precipitate# / precipitate (pH: 7.7; osm: 466 mmol/kg) |
|
31.25 |
- |
3/20 |
6.50E+06 |
88 |
normal / precipitate# (pH: 7.7; osm: 477 mmol/kg) |
|
15.625 |
- |
3/20 |
6.55E+06 |
89 |
normal / precipitate# (pH: 7.7; osm: 478 mmol/kg) |
|
7.813 |
- |
3/20 |
7.00E+06 |
98 |
normal / normal (pH: 7.7; osm 478 mmol/kg) |
|
3.906 |
- |
3/20 |
7.35E+06 |
105 |
normal / normal (pH: 7.7; osm 484 mmol/kg) |
*: compared to the negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
Summarized results of the Concentration Selection Cytotoxicity with metabolic activation (3h/20h)
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
+ |
3/20 |
7.80E+06 |
131 |
normal / normal (pH: 7.7; osm 377 mmol/kg) |
Negative (vehicle) control |
- |
+ |
3/20 |
6.45E+06 |
100 |
normal / normal (pH: 7.7; osm 464 mmol/kg) |
SynNova Base Oil |
2000 |
+ |
3/20 |
6.80E+06 |
108 |
precipitate / precipitate (pH: 7.7; osm: 423 mmol/kg) |
1000 |
+ |
3/20 |
6.30E+06 |
97 |
precipitate / precipitate (pH: 8.0; osm: 477 mmol/kg) |
|
500 |
+ |
3/20 |
6.85E+06 |
109 |
precipitate / precipitate (pH: 7.7; osm: 451 mmol/kg) |
|
250 |
+ |
3/20 |
6.45E+06 |
100 |
precipitate / precipitate (pH: 7.7; osm: 459 mmol/kg) |
|
125 |
+ |
3/20 |
6.15E+06 |
93 |
precipitate / precipitate# (pH: 7.7; osm: 469 mmol/kg) |
|
62.5 |
+ |
3/20 |
6.35E+06 |
98 |
precipitate# / precipitate# (pH: 7.7; osm: 461 mmol/kg) |
|
31.25 |
+ |
3/20 |
6.20E+06 |
94 |
normal / normal (pH: 7.7; osm: 461 mmol/kg) |
|
15.625 |
+ |
3/20 |
6.30E+06 |
97 |
normal / normal (pH: 7.7; osm: 462 mmol/kg) |
|
7.813 |
+ |
3/20 |
6.55E+06 |
102 |
normal / normal (pH: 7.7; osm 469 mmol/kg) |
|
3.906 |
+ |
3/20 |
6.70E+06 |
106 |
normal / normal (pH: 8.0; osm 471 mmol/kg) |
*: compared to the negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
Summarized results of the Concentration Selection Cytotoxicity without metabolic activation (20h/20h)
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
- |
20/20 |
7.35E+06 |
113 |
normal / normal (pH: 7.5; osm 342 mmol/kg) |
Negative (vehicle) control |
- |
- |
20/20 |
6.75E+06 |
100 |
normal / normal (pH: 7.7; osm 472 mmol/kg) |
SynNova Base Oil |
2000 |
- |
20/20 |
6.25E+06 |
89 |
precipitate / precipitate (pH: 7.7; osm: 445 mmol/kg) |
1000 |
- |
20/20 |
6.40E+06 |
93 |
precipitate / precipitate (pH: 7.7; osm: 456 mmol/kg) |
|
500 |
- |
20/20 |
6.95E+06 |
104 |
precipitate / precipitate (pH: 7.7; osm: 469 mmol/kg) |
|
250 |
- |
20/20 |
6.40E+06 |
93 |
precipitate / precipitate (pH: 7.7; osm: 481 mmol/kg) |
|
125 |
- |
20/20 |
6.40E+06 |
93 |
precipitate / precipitate (pH: 7.7; osm: 479 mmol/kg) |
|
62.5 |
- |
20/20 |
6.30E+06 |
90 |
precipitate# / precipitate (pH: 7.7; osm: 485 mmol/kg) |
|
31.25 |
- |
20/20 |
6.25E+06 |
89 |
normal / precipitate# (pH: 7.7; osm: 481 mmol/kg) |
|
15.625 |
- |
20/20 |
6.55E+06 |
96 |
normal / precipitate# (pH: 7.4; osm: 496 mmol/kg) |
|
7.813 |
- |
20/20 |
7.05E+06 |
106 |
normal / normal (pH: 7.4; osm 487 mmol/kg) |
|
3.906 |
- |
20/20 |
6.40E+06 |
93 |
normal / normal (pH: 7.4; osm 502 mmol/kg) |
*: compared to the negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
CYTOTOXICITY RESULTS OF THE MAIN EXPERIMENTS
Cytotoxicity results of Chromosome Aberration Assay 1 experiment without metabolic activation
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
- |
3/20 |
5.55E+06 |
89 |
normal / normal (pH: 7.4; osm 342 mmol/kg) |
Negative (vehicle) control |
- |
- |
3/20 |
6.00E+06 |
100 |
normal / normal (pH: 7.4; osm 476 mmol/kg) |
SynNova Base Oil |
250 |
- |
3/20 |
5.55E+06 |
89 |
precipitate / precipitate (pH: 7.4; osm: 471 mmol/kg) |
125 |
- |
3/20 |
5.35E+06 |
84 |
precipitate / precipitate (pH: 7.4; osm: 481 mmol/kg) |
|
62.5 |
- |
3/20 |
5.55E+06 |
89 |
precipitate / precipitate (pH: 7.4; osm: 468 mmol/kg) |
|
31.25 |
- |
3/20 |
5.05E+06 |
77 |
precipitate# / precipitate# (pH: 7.4; osm: 480 mmol/kg) |
|
15.625 |
- |
3/20 |
6.40E+06 |
84 |
normal / normal (pH: 7.4; osm: 481 mmol/kg) |
|
Positive control (1 µL/mL EMS) |
- |
- |
3/20 |
7.75E+06 |
46 |
normal / normal (pH: 7.4; osm: 350 mmol/kg) |
*: compared to thenegative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
Cytotoxicity results of Chromosome Aberration Assay 1 experiment with metabolic activation
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
+ |
3/20 |
4.70E+06 |
112 |
normal / normal (pH: 7.4; osm 339 mmol/kg) |
Negative (vehicle) control |
- |
+ |
3/20 |
4.40E+06 |
100 |
normal / normal (pH: 7.4; osm 466 mmol/kg) |
SynNova Base Oil |
250 |
+ |
3/20 |
4.65E+06 |
110 |
precipitate / precipitate (pH: 7.4; osm: 459 mmol/kg) |
125 |
+ |
3/20 |
4.40E+06 |
100 |
precipitate / precipitate (pH: 7.4; osm: 477 mmol/kg) |
|
62.5 |
+ |
3/20 |
4.35E+06 |
98 |
precipitate / precipitate (pH: 7.4; osm: 451 mmol/kg) |
|
31.25 |
+ |
3/20 |
4.40E+06 |
100 |
precipitate# / precipitate# (pH: 7.4; osm: 442 mmol/kg) |
|
15.625 |
+ |
3/20 |
4.30E+06 |
96 |
normal / normal (pH: 7.4; osm: 459 mmol/kg) |
|
Positive control (6 µg/mL CP) |
- |
+ |
3/20 |
3.30E+06 |
57 |
normal / normal (pH: 7.4; osm: 353 mmol/kg) |
*: compared to the negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
Cytotoxicity results of Chromosome Aberration Assay 2 experiment without metabolic activation
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
- |
20/20 |
9.25E+06 |
103 |
normal / normal (pH: 7.7; osm 344 mmol/kg) |
Negative (vehicle) control |
- |
- |
20/20 |
9.05E+06 |
100 |
normal / normal (pH: 7.7; osm 477 mmol/kg) |
SynNova Base Oil |
250 |
- |
20/20 |
6.85E+06 |
67 |
precipitate / precipitate (pH: 7.7; osm: 478 mmol/kg) |
125 |
- |
20/20 |
7.20E+06 |
72 |
precipitate / precipitate (pH: 7.7; osm: 484 mmol/kg) |
|
62.5 |
- |
20/20 |
7.60E+06 |
78 |
precipitate / precipitate (pH: 7.7; osm: 473 mmol/kg) |
|
31.25 |
- |
20/20 |
8.20E+06 |
87 |
precipitate# / precipitate# (pH: 7.7; osm: 480 mmol/kg) |
|
15.625 |
- |
20/20 |
8.05E+06 |
85 |
normal / normal (pH: 7.7; osm: 478 mmol/kg) |
|
Positive control (0.4 µL/mL EMS) |
- |
- |
20/20 |
5.00E+06 |
39 |
normal / normal (pH: 7.7; osm: 372 mmol/kg) |
*: compared to the negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
Cytotoxicity results of Chromosome Aberration Assay 2 experiment with metabolic activation
Test group |
Dose (µg/mL) |
S9-mix |
Treatment / sampling time |
Cell number (total) |
RICC (%)* |
Observations beginning / end of treatment |
Untreated control |
- |
+ |
3/20 |
8.35E+06 |
108 |
normal / normal (pH: 7.4; osm 338 mmol/kg) |
Negative (vehicle) control |
- |
+ |
3/20 |
7.90E+06 |
100 |
normal / normal (pH: 7.4; osm 477 mmol/kg) |
SynNova Base Oil |
250 |
+ |
3/20 |
7.90E+06 |
100 |
precipitate / precipitate (pH: 7.4; osm: 472 mmol/kg) |
125 |
+ |
3/20 |
7.80E+06 |
98 |
precipitate / precipitate (pH: 7.4; osm: 467 mmol/kg) |
|
62.5 |
+ |
3/20 |
7.65E+06 |
95 |
normal / precipitate# (pH: 7.4; osm: 476 mmol/kg) |
|
31.25 |
+ |
3/20 |
7.95E+06 |
101 |
normal / normal (pH: 7.4; osm: 492 mmol/kg) |
|
15.625 |
+ |
3/20 |
7.45E+06 |
92 |
normal / normal (pH: 7.4; osm: 464 mmol/kg) |
|
Positive control (6 µg/mL CP) |
- |
+ |
3/20 |
4.70E+06 |
42 |
normal / normal (pH: 7.4; osm: 350 mmol/kg) |
*: compared to the negative (vehicle) control (PG +2% Polysorbate 80) #: minimal amount
RICC: Relative Increase in Cell Counts osm: osmolaity
Note: Duplicate counts were performed at each counting.
SUMMARY TABLES OF THE MAIN EXPERIMENTS
Summary table of Chromosome Aberration Assay 1 without metabolic activation
Concentration (µg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling |
RICC* (%) |
Insolubility## |
Mean % aberrant cells### |
SynNova Base Oil without metabolic activation (-S9) |
||||
Untreated control |
3h / 20h |
89 |
- |
NE |
Negative (vehicle) control [300] |
3h / 20h |
100 |
- |
2.7 |
250 µg/mL [300] |
3h / 20h |
89 |
+ |
2.7 |
125 µg/mL [300] |
3h / 20h |
84 |
+ |
2.0 |
62.5 µg/mL [300] |
3h / 20h |
89 |
+ |
2.7 |
31.25 µg/mL |
3h / 20h |
77 |
+a |
NE |
15.625 µg/Ml |
3h / 20h |
84 |
- |
NE |
Positive control [300] |
3h / 20h |
46 |
- |
11.0*** |
Negative (vehicle) control: PG + 2% Polysorbate 80
Positive control (-S9): Ethyl methanesulfonate, 1 µL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Summary table of Chromosome Aberration Assay 1 with metabolic activation
Concentration (µg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling |
RICC* (%) |
Insolubility## |
Mean % aberrant cells### |
SynNova Base Oil without metabolic activation (+S9) |
||||
Untreated control |
3h / 20h |
112 |
- |
NE |
Negative (vehicle) control [300] |
3h / 20h |
100 |
- |
3.0 |
250 µg/mL [300] |
3h / 20h |
110 |
+ |
2.7 |
125 µg/mL [300] |
3h / 20h |
100 |
+ |
3.3 |
62.5 µg/mL [300] |
3h / 20h |
98 |
+ |
5.0 |
31.25 µg/mL |
3h / 20h |
100 |
+a |
NE |
15.625 µg/Ml |
3h / 20h |
96 |
- |
NE |
Positive control [126] |
3h / 20h |
57 |
- |
39.7*** |
Negative (vehicle) control: PG + 2% Polysorbate 80
Positive control (-S9): Cyclophosphamide 6 µg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Summary table of Chromosome Aberration Assay 2 without metabolic activation
Concentration (µg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling |
RICC* (%) |
Insolubility## |
Mean % aberrant cells### |
SynNova Base Oil without metabolic activation (-S9) |
||||
Untreated control |
20h / 20h |
103 |
- |
NE |
Negative (vehicle) control [300] |
20h / 20h |
100 |
- |
1.7 |
250 µg/mL |
20h / 20h |
67 |
+ |
NE |
125 µg/mL |
20h / 20h |
72 |
+ |
NE |
62.5 µg/mL [300] |
20h / 20h |
78 |
+ |
3.3 |
31.25 µg/mL [300] |
20h / 20h |
87 |
+a |
2.3 |
15.625 µg/mL [300] |
20h / 20h |
85 |
- |
3.0 |
Positive control [136] |
20h / 20h |
39 |
- |
38.2.*** |
Negative (vehicle) control: PG + 2% Polysorbate 80
Positive control (-S9): Ethyl methanesulfonate, 0.4 µL/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Summary table of Chromosome Aberration Assay 2 with metabolic activation
Concentration (µg/mL) [Number of analyzed cells] |
Time of Treatment / Sampling |
RICC* (%) |
Insolubility## |
Mean % aberrant cells### |
SynNova Base Oil without metabolic activation (+S9) |
||||
Untreated control |
3h / 20h |
108 |
- |
NE |
Negative (vehicle) control [300] |
3h / 20h |
100 |
- |
1.7 |
250 µg/mL [300] |
3h / 20h |
100 |
+ |
1.3 |
125 µg/mL [300] |
3h / 20h |
98 |
+ |
3.7 |
62.5 µg/mL [300] |
3h / 20h |
95 |
+a |
2.3 |
31.25 µg/mL |
3h / 20h |
101 |
- |
NE |
15.625 µg/mL |
3h / 20h |
92 |
- |
NE |
Positive control [56] |
3h / 20h |
42 |
- |
92.9*** |
Negative (vehicle) control: PG + 2% Polysorbate 80
Positive control (-S9): Cyclophosphamide 6 µg/mL
NE: not evaluated
RICC: Relative Increase in Cell Counts
#: compared to the negative (vehicle) control
##: in the final treatment medium at the end of the treatment
###: excluding gaps
a: minimal amount
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
HISTORICAL CONTROL DATA
3h/20h treatment/sampling time without S9-mix
|
Aberration rate (phases with aberration in %) |
|||
Negative control |
Positive control (EMS) |
|||
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
Mean |
2.71 |
1.20 |
22.67 |
18.02 |
SD |
1.65 |
0.82 |
12.43 |
8.16 |
Range |
0-7 |
0-3 |
4-63 |
4-40 |
n |
46 |
46 |
40 |
40 |
3h/20h treatment/sampling time with S9-mix
|
Aberration rate (phases with aberration in %) |
|||
Negative control |
Positive control (CP) |
|||
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
Mean |
3.24 |
1.45 |
73.00 |
69.50 |
SD |
1.57 |
0.95 |
23.51 |
25.64 |
Range |
0-8 |
0-4 |
21-100 |
21-100 |
n |
43 |
43 |
21 |
21 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Notes:
1. In the period 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.
2. In studies performed before the update OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for positive control sample was halted when 15 aberrant cells were counted.
20h/28h treatment/sampling time without S9-mix
|
Aberration rate (phases with aberration in %) |
|||
Negative control |
Positive control (EMS) |
|||
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
Mean |
2.58 |
1.13 |
34.97 |
30.96 |
SD |
1.81 |
0.86 |
10.94 |
9.80 |
Range |
0-8 |
0-4 |
5-76 |
5-68.2 |
n |
43 |
43 |
43 |
43 |
3h/28h treatment/sampling time with S9-mix
|
Aberration rate (phases with aberration in %) |
|||
Negative control |
Positive control (CP) |
|||
Incl. gaps |
Excl. gaps |
Incl. gaps |
Excl. gaps |
|
Mean |
3.07 |
1.47 |
52.63 |
48.80 |
SD |
1.64 |
1.07 |
23.98 |
24.49 |
Range |
0-8 |
0-5 |
7-93.8 |
6-93.8 |
n |
43 |
43 |
21 |
21 |
SD = standard deviation
Range = min.-max. values
n = number of experiments
EMS = Ethyl methanesulfonate
CP = Cyclophosphamide
Notes:
1. In the period 2008-2009, NNDA (N-Nitrosodimethylamine) was used as positive control substance in the experiments with metabolic activation. Mean aberration frequency for NNDA was 22.91 (including gaps) and 18.07 (excluding gaps) in 22 experiments.
2. In studies performed before the update OECD guideline (2014) 200 metaphases were scored for chromosomal aberration per samples. Minimum and maximum values reflect the total number of aberrant cells in 200 metaphases. Furthermore, in those studies counting for positive control sample was halted when 15 aberrant cells were counted.
3. These historical controls were used for this study due to the lack of the 20h/20h treatment/sampling time without S9-mix historical control.
Results of the Preliminary Toxicity Test
(3-hour treatment in the presence of metabolic activation)
Test item (or solvent) concentration |
Total cell number, (relative Survival*, %) after treatment Day 0 |
Total cell number, (Relative Survival*, %) on Day 1 |
Total cell number, (Relative Survival*, %) on Day 2 |
Observations at the beginning / after treatment** |
Untreated control |
7.85E+06 (101) |
2.09E+07 (117) |
2.34E+07 (104) |
B: normal A: normal (pH: 7.0, osmolality: 299 mmol/kg) |
Negative (vehicle) control |
7.75E+06 (100) |
1.79E+07 (100) |
2.24E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 412 mmol/kg) |
2000 µg/mL |
5.40E+06 (70) |
1.91E+07 (107) |
2.06E+07 (92) |
B: discoloured medium#, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 395 mmol/kg) |
1000 µg/mL |
6.30E+06 (81) |
1.75E+07 (98) |
2.14E+07 (96) |
B: discoloured medium#, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 407 mmol/kg) |
500 µg/mL |
6.50E+06 (84) |
1.74E+07 (97) |
2.36E+07 (105) |
B: discoloured medium#, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 407 mmol/kg) |
250 µg/mL |
3.75E+06 (48) |
5.48E+07 (31) |
2.20E+07 (98) |
B: precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 432 mmol/kg) |
125 µg/mL |
4.40E+06 (57) |
1.71E+07 (96) |
2.24E+07 (100) |
B: precipitate# A: precipitate#(pH: 7.0, osmolality: 424 mmol/kg) |
62.5 µg/mL |
5.30E+06 (68) |
1.68E+07 (94) |
2.32E+07 (104) |
B: normal A: normal (pH: 7.0, osmolality: 427 mmol/kg) |
31.25 µg/mL |
5.60E+06 (72) |
1.44E+07 (81) |
2.18E+07 (97) |
B: normal A: normal (pH: 7.0, osmolality: 441 mmol/kg) |
15.625 µg/mL |
4.55E+06 (59) |
1.31E+07 (73) |
2.26E+07 (101) |
B: normal A: normal (pH: 7.0, osmolality: 435 mmol/kg) |
7.813 µg/mL |
3.80E+06 (49) |
1.59E+07 (89) |
1.92E+07 (86) |
B: normal A: normal (pH: 7.0, osmolality: 429 mmol/kg) |
3.906 µg/mL |
4.30E+06 (55) |
1.50E+07 (84) |
2.00E+07 (89) |
B: normal A: normal (pH: 7.0, osmolality: 422 mmol/kg) |
*: compared to the relevant negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Results of the Preliminary Toxicity Test
(3-hour treatment in the absence of metabolic activation)
Test item (or solvent) concentration |
Total cell number, (relative Survival*, %) after treatment Day 0 |
Total cell number, (Relative Survival*, %) on Day 1 |
Total cell number, (Relative Survival*, %) on Day 2 |
Observations at the beginning / after treatment** |
Untreated control |
1.05E+07 (114) |
2.10E+07 (105) |
2.20E+07 (99) |
B: normal A: normal (pH: 7.0, osmolality: 295 mmol/kg) |
Negative (vehicle) control |
9.20E+06 (100) |
2.00E+07 (100) |
2.22E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 421 mmol/kg) |
2000 µg/mL |
9.80E+06 (107) |
1.89E+07 (95) |
2.02E+07 (91) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 399 mmol/kg) |
1000 µg/mL |
8.85E+06 (96) |
1.68E+07 (84) |
2.24E+07 (101) |
B: discoloured medium, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 412 mmol/kg) |
500 µg/mL |
9.40E+06 (102) |
1.76E+07 (88) |
2.46E+07 (11) |
B: discoloured medium#, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 406 mmol/kg) |
250 µg/mL |
9.10E+06 (99) |
1.86E+07 (93) |
2.12E+07 (95) |
B: discoloured medium#, precipitate A: discoloured medium#, precipitate (pH: 7.0, osmolality: 427 mmol/kg) |
125 µg/mL |
8.95E+06 (97) |
1.79E+07 (89) |
2.12E+07 (95) |
B: discoloured medium#, precipitate# A: discoloured medium#, precipitate#(pH: 7.0, osmolality: 417 mmol/kg) |
62.5 µg/mL |
9.30E+06 (101) |
1.79E+07 (89) |
2.30E+07 (104) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 421 mmol/kg) |
31.25 µg/mL |
9.30E+06 (101) |
1.70E+07 (85) |
2.16E+07 (97) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 433 mmol/kg) |
15.625 µg/mL |
1.02E+07 (110) |
1.71E+07 (86) |
2.26E+07 (102) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 412 mmol/kg) |
7.813 µg/mL |
9.20E+06 (100) |
1.67E+07 (83) |
2.32E+07 (105) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 417 mmol/kg) |
3.906 µg/mL |
9.20E+06 (100) |
1.83E+07 (92) |
2.76E+07 (124) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 418 mmol/kg) |
*: compared to the relevant negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Results of the Preliminary Toxicity Test
(24-hour treatment in the absence of metabolic activation)
Test item (or solvent) concentration |
Total cell number, (relative Survival*, %) after treatment Day 0 |
Total cell number, (Relative Survival*, %) on Day 1 |
Total cell number, (Relative Survival*, %) on Day 2 |
Observations at the beginning / after treatment** |
Untreated control |
1.87E+07 (136) |
1.86E+07 (100) |
2.30E+07 (111) |
B: normal A: normal (pH: 7.0, osmolality: 294 mmol/kg) |
Negative (vehicle) control |
1.38E+07 (100) |
1.86E+07 (100) |
2.08E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 413 mmol/kg) |
2000 µg/mL |
1.78E+07 (129) |
1.88E+07 (101) |
2.16E+07 (104) |
B: discoloured medium#, precipitate A: precipitate (pH: 7.0, osmolality: 355 mmol/kg) |
1000 µg/mL |
1.09E+07 (79) |
1.88E+07 (101) |
2.18E+07 (105) |
B: discoloured medium#, precipitate A: precipitate (pH: 7.0, osmolality: 421 mmol/kg) |
500 µg/mL |
1.26E+07 (91) |
1.79E+07 (96) |
2.34E+07 (113) |
B: discoloured medium#, precipitate A: precipitate#(pH: 7.0, osmolality: 409 mmol/kg) |
250 µg/mL |
1.11E+07 (80) |
1.73E+07 (93) |
2.08E+07 (100) |
B: discoloured medium#, precipitate A: precipitate#(pH: 7.0, osmolality: 419 mmol/kg) |
125 µg/mL |
1.08E+07 (78) |
1.77E+07 (95) |
2.20E+07 (106) |
B: discoloured medium#, precipitate# A: normal(pH: 7.0, osmolality: 423 mmol/kg) |
62.5 µg/mL |
1.22E+07 (99) |
1.76E+07 (94) |
2.06E+07 (99) |
B: discoloured medium# A: normal (pH: 7.0, osmolality: 428 mmol/kg) |
31.25 µg/mL |
8.05E+06 (58) |
1.68E+07 (90) |
2.08E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 447 mmol/kg) |
15.625 µg/mL |
1.30E+07 (94) |
1.67E+07 (90) |
2.24E+07 (108) |
B: normal A: normal (pH: 7.0, osmolality: 413 mmol/kg) |
7.813 µg/mL |
1.23E+07 (89) |
1.85E+07 (99) |
2.08E+07 (100) |
B: normal A: normal (pH: 7.0, osmolality: 412 mmol/kg) |
3.906 µg/mL |
1.18E+07 (85) |
1.86E+07 (100) |
2.06E+07 (99) |
B: normal A: normal (pH: 7.0, osmolality: 417 mmol/kg) |
*: compared to the relevant negative (vehicle) control (PG + 2% Polysorbate 80) #: minimal amount
**: B: at the beginning of the treatment, A: at the end of the treatment
Summary Tables on Survival Data
Survival Results of Assay 1 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
Relative Survival§(%RS) |
Relative Total Growth (RTG) |
+ |
3 |
A1 |
250 µg/mL |
75/384 |
1.021 |
116 |
106 |
125 µg/mL |
79/384 |
0.988 |
107 |
95 |
|||
62.5 µg/mL |
96/384 |
0.866 |
82 |
85 |
|||
31.25 µg/mL |
63/384 |
1.130 |
112 |
103 |
|||
15.625 µg/mL |
86/384 |
0.935 |
87 |
98 |
|||
7.813 µg/mL |
82/384 |
0.965 |
99 |
111 |
|||
3.906 µg/mL |
84/384 |
0.950 |
96 |
111 |
|||
Vehicle control |
91/384 |
0.900 |
100 |
100 |
|||
Vehicle control for CP |
70/384 |
1.064 |
144 |
123 |
|||
Untreated control |
68/384 |
1.082 |
138 |
124 |
|||
Positive control (CP) |
205/384 |
0.392 |
38 |
26 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
§= Relative survival values (%) corrected with the post treatment cell concentrations
Survival Results of Assay 1 (II)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
Relative Survival§(%RS) |
Relative Total Growth (RTG) |
- |
3 |
A1 |
250 µg/mL |
118/384 |
0.737 |
81 |
96 |
125 µg/mL |
79/384 |
0.988 |
107 |
90 |
|||
62.5 µg/mL |
98/384 |
0.854 |
90 |
98 |
|||
31.25 µg/mL |
93/384 |
0.886 |
93 |
106 |
|||
15.625 µg/mL |
89/384 |
0.914 |
92 |
104 |
|||
7.813 µg/mL |
93/384 |
0.886 |
95 |
102 |
|||
3.906 µg/mL |
147/384 |
0.6000 |
62 |
83 |
|||
Vehicle control |
89/384 |
0.914 |
100 |
100 |
|||
Vehicle control for NQO |
80/384 |
0.980 |
110 |
113 |
|||
Untreated control |
78/384 |
0.996 |
116 |
135 |
|||
Positive control (NQO) |
161/384 |
0.543 |
49 |
47 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Niroquinoline-N-oxide (0.15 µg/mL)
§= Relative survival values (%) corrected with the post treatment cell concentrations
Survival Results of Assay 2 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
Relative Survival§(%RS) |
Relative Total Growth (RTG) |
+ |
3 |
A2 |
250 µg/mL |
94/384 |
0.880 |
73 |
79 |
125 µg/mL |
80/384 |
0.980 |
86 |
81 |
|||
62.5 µg/mL |
82/384 |
0.965 |
83 |
108 |
|||
31.25 µg/mL |
70/384 |
1.064 |
89 |
95 |
|||
15.625 µg/mL |
98/384 |
0.854 |
73 |
80 |
|||
7.813 µg/mL |
120/384 |
0.727 |
61 |
70 |
|||
3.906 µg/mL |
114/384 |
0.759 |
62 |
76 |
|||
Vehicle control |
67/384 |
1.091 |
100 |
100 |
|||
Vehicle control for CP |
80/384 |
0.980 |
103 |
99 |
|||
Untreated control |
90/384 |
0.907 |
88 |
165 |
|||
Positive control (CP) |
282/384 |
0.193 |
17 |
17 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
§= Relative survival values (%) corrected with the post treatment cell concentrations
Survival Results of Assay 2 (II)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
Relative Survival§(%RS) |
Relative Total Growth (RTG) |
- |
24 |
A2 |
1000 µg/mL |
90/384 |
0.907 |
104 |
121 |
500 µg/mL |
74/384 |
1.029 |
114 |
116 |
|||
250 µg/mL |
97/384 |
0.860 |
97 |
118 |
|||
125 µg/mL |
110/384 |
0.781 |
87 |
92 |
|||
62.5 µg/mL |
98/384 |
0.854 |
95 |
93 |
|||
31.25 µg/mL |
112/384 |
0.770 |
90 |
99 |
|||
15.625 µg/mL |
75/384 |
1.021 |
116 |
120 |
|||
Vehicle control |
97/384 |
0.860 |
100 |
100 |
|||
Vehicle control for NQO |
97/384 |
0.860 |
148 |
144 |
|||
Untreated control |
118/384 |
0.737 |
124 |
165 |
|||
Positive control (NQO) |
182/384 |
0.467 |
57 |
57 |
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
§= Relative survival values (%) corrected with the post treatment cell concentrations
Summary Tables of Viability Data
Viability Results of Assay 1 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
+ |
3 |
A1 |
250 µg/mL |
103/384 |
0.822 |
125 µg/mL |
99/384 |
0.847 |
|||
62.5 µg/mL |
104/384 |
0.816 |
|||
31.25 µg/mL |
100/384 |
0.841 |
|||
15.625 µg/mL |
98/384 |
0.854 |
|||
7.813 µg/mL |
93/384 |
0.886 |
|||
3.906 µg/mL |
79/384 |
0.988 |
|||
Vehicle control |
96/384 |
0.866 |
|||
Vehicle control for CP |
90/384 |
0.907 |
|||
Untreated control |
90/384 |
0.907 |
|||
Positive control (CP) |
193/384 |
0.430 |
A1 = Assay 1
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
Viability Results of Assay 1 (II)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
- |
3 |
A1 |
250 µg/mL |
82/384 |
0.965 |
125 µg/mL |
87/384 |
0.928 |
|||
62.5 µg/mL |
78/384 |
0.996 |
|||
31.25 µg/mL |
63/384 |
1.130 |
|||
15.625 µg/mL |
80/384 |
0.980 |
|||
7.813 µg/mL |
78/384 |
0.996 |
|||
3.906 µg/mL |
89/384 |
0.914 |
|||
Vehicle control |
75/384 |
1.021 |
|||
Vehicle control for NQO |
73/384 |
1.038 |
|||
Untreated control |
68/384 |
1.082 |
|||
Positive control (NQO) |
91/384 |
0.900 |
A1 = Assay 1
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.15 µg/mL)
Viability Results of Assay 2 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
+ |
3 |
A2 |
250 µg/mL |
103/384 |
0.822 |
125 µg/mL |
92/384 |
0.893 |
|||
62.5 µg/mL |
60/384 |
1.160 |
|||
31.25 µg/mL |
76/384 |
1.012 |
|||
15.625 µg/mL |
77/384 |
1.004 |
|||
7.813 µg/mL |
61/384 |
1.150 |
|||
3.906 µg/mL |
77/384 |
1.004 |
|||
Vehicle control |
73/384 |
1.038 |
|||
Vehicle control for CP |
74/384 |
1.029 |
|||
Untreated control |
75/384 |
1.021 |
|||
Positive control (CP) |
209/384 |
0.380 |
A2 = Assay 2
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide (4 µg/mL)
Viability Results of Assay 2 (II)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Study phase |
Test item or control concentration |
Number of empty wells/total number of wells |
Plating Efficiency (PE) |
- |
24 |
A2 |
1000 µg/mL |
66/384 |
1.101 |
500 µg/mL |
76/384 |
1.012 |
|||
250 µg/mL |
75/384 |
1.021 |
|||
125 µg/mL |
75/384 |
1.021 |
|||
62.5 µg/mL |
81/384 |
0.973 |
|||
31.25 µg/mL |
80/384 |
0.980 |
|||
15.625 µg/mL |
71/384 |
1.055 |
|||
Vehicle control |
79/384 |
0.988 |
|||
Vehicle control for NQO |
82/384 |
0.965 |
|||
Untreated control |
76/384 |
1.012 |
|||
Positive control (NQO) |
107/384 |
0.799 |
A2 = Assay 2
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = 4-Nitroquinoline-N-oxide (0.1 µg/mL)
Summary Tables of Mutagenicity Data
Mutagenicity Results of Assay 1 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item of control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
+ |
3 |
250 µg/mL |
6374/768 |
52/768 |
79/768 |
0.030 |
113.7 |
125 µg/mL |
640/768 |
56/768 |
72/768 |
0.002 |
107.6 |
||
62.5 µg/mL |
643/768 |
48/768 |
77/768 |
0.000002 |
108.8 |
||
31.25 µg/mL |
649/768 |
60/768 |
59/768 |
0.105 |
100.1 |
||
15.625 µg/mL |
631/768 |
54/768 |
83/768 |
0.031 |
104.1 |
||
7.813 µg/mL |
638/768 |
59/768 |
71/768 |
0.024 |
104.6 |
||
3.906 µg/mL |
651/768 |
54/768 |
63/768 |
1.041 |
83.6 |
||
Vehicle control |
636/768 |
62/768 |
70/768 |
-- |
108.8 |
||
Vehicle control for CP |
647/768 |
43/768 |
78/768 |
-- |
94.5 |
||
Untreated control |
604/768 |
64/768 |
100/768 |
-- |
132.5 |
||
Positive control (CP: 4 µg/mL) |
161/768 |
127/768 |
480/768 |
♦♦ 3.22E-11 |
1816.9* |
In linear trend analysisß2/var (ß) = 0.61, not significant
* = Statistically significant
♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.48(at p<0.05)
♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn ß = slope of the curve var(ß) = variance of the slope
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 1 (II)
(3-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item of control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
- |
3 |
250 µg/mL |
644/768 |
56/768 |
68/768 |
0.059 |
91.2 |
125 µg/mL |
610/768 |
59/768 |
99/768 |
1.031 |
124.1 |
||
62.5 µg/mL |
635/768 |
44/768 |
89/768 |
0.004 |
95.4 |
||
31.25 µg/mL |
626/768 |
65/768 |
77/768 |
0.080 |
90.5 |
||
15.625 µg/mL |
653/768 |
48/768 |
67/768 |
0.036 |
101.8 |
||
7.813 µg/mL |
648/768 |
52/768 |
68/768 |
0.256 |
85.3 |
||
3.906 µg/mL |
662/768 |
37/768 |
69/768 |
0.458 |
81.3 |
||
Vehicle control |
630/768 |
51/768 |
87/768 |
-- |
97.0 |
||
Vehicle control for NQO |
624/768 |
60/768 |
84/768 |
-- |
100.1 |
||
Untreated control |
608/768 |
74/768 |
86/768 |
-- |
108.0 |
||
Positive control (NQO: 0.15 µg/mL) |
154/768 |
173/768 |
441/768 |
♦♦ 1.52E-14 |
892.8* |
In linear trend analysisß2/var (ß) = 0.21, not significant
* = Statistically significant
♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.48(at p<0.05)
♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn ß = slope of the curve var(ß) = variance of the slope
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (I)
(3-hour treatment in the presence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item of control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
+ |
3 |
250 µg/mL |
590/768 |
98/768 |
80/768 |
1.613 |
160.3 |
125 µg/mL |
633/768 |
66/768 |
69/768 |
0.176 |
108.2 |
||
62.5 µg/mL |
630/768 |
82/768 |
56/768 |
1.987 |
85.4 |
||
31.25 µg/mL |
596/768 |
94/768 |
78/768 |
0.037 |
125.2 |
||
15.625 µg/mL |
611/768 |
93/768 |
64/768 |
0.077 |
112.2 |
||
7.813 µg/mL |
607/768 |
84/768 |
77/768 |
0.456 |
102.3 |
||
3.906 µg/mL |
631/768 |
76/768 |
61/768 |
0.709 |
97.8 |
||
Vehicle control |
599/768 |
94/768 |
75/768 |
-- |
119.8 |
||
Vehicle control for CP |
629/768 |
80/768 |
59/768 |
-- |
97.0 |
||
Untreated control |
618/768 |
77/768 |
73/768 |
-- |
106.4 |
||
Positive control (CP: 4 µg/mL) |
210/768 |
128/768 |
430/768 |
♦♦ 1.51E-14 |
1705.3* |
In linear trend analysisß2/var (ß) = 2.01, not significant
* = Statistically significant
♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.48(at p<0.05)
♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn ß = slope of the curve var(ß) = variance of the slope
+ = in the presence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for CP = DMSO
DMSO = Dimethyl sulfoxide
CP = Cyclophosphamide
Note: Mutation frequency refers to 106viable cells
Mutagenicity Results of Assay 2 (II)
(24-hour treatment in the absence of S9-mix)
S9-mix |
Treatment period (hours) |
Test item of control concentration |
Number of empty wells/total number of wells |
Number of large colonies/total number of wells |
Number of small colonies/total number of wells |
Dn2/var(Dn) ♦ |
Mutation frequency |
- |
24 |
1000 µg/mL |
618/768 |
64/768 |
86/768 |
0.142 |
98.7 |
500 µg/mL |
638/768 |
60/768 |
70/768 |
0.005 |
91.6 |
||
250 µg/mL |
648/768 |
45/768 |
75/768 |
0.089 |
83.2 |
||
125 µg/mL |
622/768 |
66/768 |
80/768 |
0.310 |
103.3 |
||
62.5 µg/mL |
619/768 |
71/768 |
78/768 |
0.630 |
110.0 |
||
31.25 µg/mL |
639/768 |
57/768 |
72/768 |
0.028 |
93.8 |
||
15.65 µg/mL |
653/768 |
58/768 |
57/768 |
0.359 |
76.9 |
||
Vehicle control |
643/768 |
57/768 |
68/768 |
-- |
89.9 |
||
Vehicle control for NQO |
613/768 |
98/768 |
57/768 |
-- |
116.8 |
||
Untreated control |
615/768 |
76/768 |
79/768 |
-- |
109.7 |
||
Positive control (NQO: 0.1 µg/mL) |
168/768 |
275/768 |
325/768 |
♦♦ 4.38E-14 |
951.5* |
In linear trend analysisß2/var (ß) = 0.16, not significant
* = Statistically significant
♦ = Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn)> 5.48(at p<0.05)
♦♦ = Evaluated by T-test for independent samples (compared to the DMSO vehicle control).
Dn= Difference of log mutant frequency of dose “n” and that of the vehicle control
var(Dn) = variance of Dn ß = slope of the curve var(ß) = variance of the slope
- = in the absence of S9-mix
Negative (vehicle) control = PG + 2% Polysorbate 80
Negative (vehicle) control for NQO = DMSO
DMSO = Dimethyl sulfoxide
NQO = Nitroquinoline-N-oxide
Note: Mutation frequency refers to 106viable cells
Historical Control Data
(update on 11 January 2017)
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 SD Min. Max. n |
94.3 26.9 39.3 198.5 84 |
103.6 35.3 52.6 235.6 43 |
106.4 27.4 41.7 179.1 44 |
90.4 22.7 33.4 121.8 26 |
99.6 19.0 55.1 125.0 13 |
96.3 24.6 43.2 141.1 13 |
|
Dimethyl sulfoxide (DMSO) |
|
|
|
||
Treatments |
3h,S9+ |
3h,S9- |
24h.S9- |
|
|
|
Average SD Min. Max. n |
97.3 33.7 44.2 269.9 101 |
97.3 38.5 33.7 261.6 57 |
98.9 26.8 47.1 159.4 50 |
|
|
|
Mutation Frequency of the Positive Controls (2006-2016) |
||||||
|
Cyclophosphamide |
4-Nitroquinoline-N-oxide |
||||
Treatments |
3h,S9+ |
|
|
|
3h,S9- |
24h,S9- |
Average SD Min. Max. n |
1178.7 524.7 196.1 2642.5 106 |
|
|
|
722.2 330.0 223.5 1687.3 58 |
831.9 337.2 245.0 1577.6 52 |
h = hour
SD = Standard Deviation
S9+ = experiment with metabolic activation
S9- = experiment without metabolic activation
n = number of cases
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial Reverse Mutation Assay
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), Assay 1 (Plate Incorporation Method) and Assay 2 (Plate Incorporation Method or Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in Propylene Glycol + 2% Polysorbate 80 at a maximum concentration of 100 mg/mL. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the absence and presence of metabolic activation were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate in Assays 1 and 2.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
Precipitate/slight precipitate was detected on the plates in all examined bacterial strains with and without metabolic activation at 5000 and/or 1581 μg/plate concentrations in Assays 1-2.
Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item SynNova Base Oil (Batch Number: TS20371) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
Chromosome Aberration Assays using Chinese hamster V79 lung cells
In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 1, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-31.25 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic.
In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 250, 125, 62.5, 31.25 and 15.625 μg/mL (experiment with and without metabolic activation).
In Assay 2, precipitate/minimal amount of precipitate was detected at the end of the treatment period in the final treatment medium in the 250-62.5 μg/mL concentration range with metabolic activation and in the 250-31.25 μg/mL concentrations without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in the experiment with and without metabolic activation. Therefore, concentrations of 250, 125 and 62.5 μg/mL (a total of three) were evaluated in the experiment with metabolic activation, and concentrations of 62.5, 31.25 and 15.625 μg/mL (a total of three) were evaluated in the experiment without metabolic activation due to the observed insolubility.
None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.
Polyploid metaphases and/or endoreduplicated metaphases were found in some cases in the negative (vehicle) control or positive control or test item treated samples in the performed experiments, but their incidence was not related to treatment with SynNova Base Oil.
In conclusion, SynNova Base Oil did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, SynNova Base Oil was considered as not clastogenic in this test system.
Mammalian cell assay
An 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 SynNova Base Oil test item 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).
Propylene glycol (PG) + 2% Polysorbate 80 was used as vehicle of the test item in this study.
In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. Thus, an evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was noted at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
In Assay 2, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 250-3.906 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative and confirmed the clearly negative result of the first test with metabolic activation.
In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all seven concentrations (concentration range of 1000-15.625 μg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any of the evaluated concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered as being clearly negative.
In conclusion, no mutagenic effect of SynNova Base Oil was observed in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Justification for classification or non-classification
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