2-methylpentyl 2-hydroxybenzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-02-2020 to 23-04-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-methylpentyl 2-hydroxybenzoate

EC Number:

850-366-8

Cas Number:

98969-19-8

Molecular formula:

C13H18O3

IUPAC Name:

2-methylpentyl 2-hydroxybenzoate

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Dry, refrigerated (2 to 8°C), protected from light and using an airtight container with silica gel.
- Other: colourless liquid

Method

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphoblastoid cells (TK6) ; from laboratory in-house cell stock originally obtained from European Collection of Authenticated Cell Cultures (ECACC; part of Public Health England, United Kingdom), were used in this study. The ECACC states that its stocks of the TK6 cell line are obtained from the American Type Culture Collection (ATCC).
- Suitability of cells: TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)
- Normal cell cycle time (negative control): See below for further information ; TK6 cell stocks are checked for mycoplasma and confirmed to be mycoplasma free. The doubling time for TK6 cells is between 13 and 15 hours (Lorge et al. (2016), Standardised cell sources and recommendations for good cell culture practices in genotoxicity testing. Mutation Research ; 809 : 1-15).

For cell lines:
- Absence of Mycoplasma contamination: TK6 cell stocks are checked for mycoplasma and confirmed to be mycoplasma free.
- Number of passages if applicable: Experiment 01: passage number 24 and Experiment 02: passage number 13 ; Experiment 03: passage number 17 and Experiment 04: passage number 23 ; (cell initiation dates indicated in the full study report).
- Methods for maintenance in cell culture: Vial-stock TK6 cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air.
- Cell cycle length, doubling time or proliferation index : The doubling time for TK6 cells is between 13 and 15 hours from Lorge et al. (2016).
- Modal number of chromosomes: Based on literature sources: Lorge et al. (2016), TK6 cells are expected to have a modal chromosome number of 47. Reported as confirmed by the laboratory.
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes [cleansing of working-cell stocks prior to ultra-cold storage is routine handling practice, according to Lorge et al. (2016)]
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored during the study. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on historical (and control) data these temporary deviations are not considered to affect the integrity of the study.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: from Recognised supplier (origin and dates within full study report). S9 Microsomal fraction: Lot No. 4189
- method of preparation of S9 mix: Documented in the full study report. Stored at -80ºC. Following thawing protein content was adjusted to 30 mg/mL by dilution with RPMI 1640 before incorporation into the S9 mix. S9-mix was prepared immediately before use. Excess was discarded.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.0% v/v S9 (with Nicotinamide adenine dinucleotide phosphate 0.25 mM and Glucose-6-phosphate 1.25 mM)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including Activity is presented in the full study report. Additionally, prior to testing each batch was subjected to in-house testing to assess batch-to-batch variation using bacterial promutagens: cyclophosphamide and benzo[a]pyrene (TA1535 and TA98, respectively) and was found to be acceptable. Furthermore, concurrent positive control substances all produced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.

Test concentrations with justification for top dose:

- Experiment 1:
3 hours exposure time
(+S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 4.0 μg/mL CP (PC)
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 2 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.00, 43.81, 53.01, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3 μg/mL plus 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 3 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
- Experiment 4 :
3 hours exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.54, 32.07, 33.67, 35.35, 37.12, 39.98, 40.93, 42.97, 45.17, 47.38, 49.75, 52.24, 54.85, 57.59, 60.47, 63.49, 66.67, 70.00 μg/mL and and 50 ng/mL MMC
The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:
Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL
Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL
Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in DMSO at all tested concentrations and up to 200.00 mg/mL, within solubility checks were performed in-house. Test item concentrations were used within 1 hour after preparation. The final concentration of DMSO in the culture medium was 1.0% (v/v).
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content (if applicable). See 'Test Material Information' for further details (if applicable). A test item-DMSO formulation solubility test was performed in R10P (RPMI 1640 media containing 10% heat inactivated horse serum, antibiotics and Pluronic F68) to assess the suitability of the solvent and to help with determining suitable test concentrations for the micronucleus assay. In the solubility test, precipitate was visible at a concentration of 2000 μg/mL after addition to R10P media. Following overnight incubation (humidified atmosphere of 5% CO2 at a temperature of 37°C) of the tubes (containing R10P media with test concentrations or solvent control), precipitate was visible at a concentration of 2000 μg/mL. In order to meet guideline criteria, 2000 μg/mL was therefore selected as the top concentration for all three treatment schedules. On the day of testing a stock solution of test item in DMSO was prepared at 200.0 mg/mL and further diluted in DMSO to prepare the required test solutions. In Experiment 2, 3 and 4, narrower dose ranges and lower top concentrations were prepared and dosed. Further details on test item concentrations are provided above and below.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

Remarks:

without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1 (single)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Vial-stock TK6 cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. TK6 cells were harvested by centrifugation and re-suspended at 1.5 x 10^5 cells per mL in tissue culture medium, then dispensed into treatment tubes (3 hour treatments) or culture flasks (continuous treatments) in 4.95 mL aliquots.
- Test substance added in medium : Based on findings in the preliminary assessment of solubility, the test item was prepared in DMSO at 200.0 mg/mL. It was then prepared in a dilution series, typically with a 1.5-fold dilution factor and finally diluted for treatment by adding 50 μL to 4.95 mL tissue culture medium containing 1.5 x 10^5 cells per mL. For tests performed in the presence of metabolic activation, S9 mix was incorporated into the culture medium. Formulated test item (10 μL) was added to each tube/flask and gently mixed to ensure homogeneity. Cultures following the 3 hour treatment schedules, either in the presence or absence of S9 mix, were incubated (humidified atmosphere of 5% CO2 at a temperature of 37°C) for 3 hours in culture medium (±S9 mix), after which cells were washed and the medium replaced with fresh culture medium and then dispensed into culture flasks. Cultures were then incubated until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control. Cultures following the continuous exposure treatment schedule (without S9 mix) were incubated (humidified atmosphere of 5% CO2 at a temperature of 37°C) until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control. Appropriate positive controls were also included in each assay.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: See above.
- Exposure duration/duration of treatment: Experiment 1 and 2: 3 hours exposure time and/or 24 hours exposure time ; Experiment 3 and 4: 3 hours exposure time
- Harvest time after the end of treatment (sampling/recovery times): See above. Until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Not applicable, see below.
- If cytokinesis blocked method was used for micronucleus assay: TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Following the treatment and recovery periods (where applicable), cells were counted using an automated cell counter. Cells in the solvent treated controls were required to have achieved between 1.5 and 2.0 population doublings. If cells in the solvent treated controls achieved a population doubling (PD) of <1.5, cultures were incubated further until sufficient proliferation had been achieved. From the six solvent treated cultures, a minimum of two were required to achieve a PD of >1.5. If a PD of >2.0 was observed, the experiment was repeated. Relative Population Doubling (RPD) was calculated for each culture. After RPDs were calculated from cell counts, these data were used to select the concentrations from which to prepare slides for micronucleus analysis. From the cytotoxicity profile (inferred from RPDs), a minimum of 3 and a maximum of 6 concentrations per test item per treatment schedule were selected for microscopic analysis (according to appropriate criteria as specified in the guideline OECD TG 487). Cultures treated with concentrations selected for slide preparation and analysis (including solvent treated and positive controls) were harvested by centrifugation and resuspended in fresh medium (RPMI supplemented with Pluronic F68 alone), with cell densities adjusted to 2.5 x 10^5 cells per mL. Subsequently, thin monolayer cell preparations were made using a cytology centrifuge. The monolayers were allowed to air dry before fixation with methanol. Following fixation, slides were stained with 0.12 mg/mL acridine orange in Dulbecco’s phosphate buffered saline (pH 7.4).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): All slides, including those for solvent and positive controls, were coded prior to analysis by someone other than the scorer.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Micronucleus analysis was performed on 2000 mononuclear cells per culture per test item concentration and control sample (i.e. where possible 1000 mononuclear cells per culture). Micronucleus scoring was performed by manual counting under a fluorescence microscope with typical magnification of x400. The number of mononuclear and micronucleated mononuclear cells scored were recorded. Micronucleus frequencies were recorded per 1000 mononuclear cells per culture. Mononucleated cells were selected if they were not overlapping and cytoplasm was clearly visible around the nuclei. Micronuclei were selected if they were less than one-third of the diameter of the main nucleus and located within the intact cytoplasm, evenly stained, spherical with a well-defined outline and not connected to the main nucleus. The number of micronuclei analysed from 2000 mononuclear cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran Armitage) in order to confirm a dose-response (p < 0.05).
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Not applicable.
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Population Doubling (PD) and/or Relative Population Doubling (RPD)
- Any supplementary information relevant to cytotoxicity: Cells in the solvent treated controls were required to have achieved between 1.5 and 2.0 population doublings. If cells in the solvent treated controls achieved a population doubling (PD) of <1.5, cultures were incubated further until sufficient proliferation had been achieved. From the six solvent treated cultures, a minimum of two were required to achieve a PD of >1.5. If a PD of >2.0 was observed, the experiment was repeated. Relative Population Doubling (RPD) was calculated for each culture. After RPDs were calculated from cell counts, these data were used to select the concentrations from which to prepare slides for micronucleus analysis. From the cytotoxicity profile (inferred from RPDs), a minimum of 3 and a maximum of 6 concentrations per test item per treatment schedule were selected for microscopic analysis (according to appropriate criteria as specified in the guideline OECD TG 487). Micronucleus analysis was performed on 2000 mononuclear cells per culture per test item concentration and control sample (i.e. where possible 1000 mononuclear cells per culture).

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
Statistical analysis is performed and/or provided the acceptability criteria are met :
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
(a) at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
(c) any of the results were outside the historical solvent control range (Poisson-based 95% control limits)
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
(a) none of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) there was no concentration-related increase when evaluated using an appropriate trend test
(c) all results were inside the distribution of the historical solvent control data (Poisson-based 95% control limits)
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item Such equivocal responses may occur regardless of the number of times the experiment is repeated. Expert judgement in accordance with the OECD TG 487 and related mutagenicity guidelines is therefore utilised in the assessment.

OTHER:
ACTIVATION: The S9-mix was prepared prior to the dosing of the test cultures. Metabolic activation was achieved by adding 1.0% v/v S9 in the exposure medium (with Nicotinamide adenine dinucleotide phosphate 0.25 mM and Glucose-6-phosphate 1.25 mM).

Rationale for test conditions:

In accordance with the OECD TG 487 guidelines.

Evaluation criteria:

Statistical analysis is performed and/or provided the acceptability criteria are met :
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
(a) at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
(c) any of the results were outside the historical solvent control range (Poisson-based 95% control limits)
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
(a) none of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) there was no concentration-related increase when evaluated using an appropriate trend test
(c) all results were inside the distribution of the historical solvent control data (Poisson-based 95% control limits)
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item Such equivocal responses may occur regardless of the number of times the experiment is repeated. Expert judgement in accordance with the OECD TG 487 and related mutagenicity guidelines is therefore utilised in the assessment.

An in vitro micronucleus test is considered acceptable if it meets the following Acceptability Criteria:
(a) Solvent (DMSO) controls : the frequency of mononucleate cells with micronuclei for the solvent cultures must approximate those of the acceptable ranges from the Test Facility’s historical control database and/or published values.
(b) Positive controls : the positive control chemicals must induce a statistically significant (p <0.05) increase in the frequency of micronuclei in mononucleate cells compared with the concurrent solvent controls.

Statistics:

Statistical analysis was performed using software package(s). The number of micronuclei analysed from 2000 mononuclear cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran Armitage) in order to confirm a dose-response (p < 0.05).
See above for further information on the statistical analysis and acceptability criteria.

Results and discussion

Test results

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no reported significant changes in pH, when the test item was dosed into media.
- Effects of osmolality: There was no reported significant changes in osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media. Specifically: at the end of each treatment period the osmolality and pH of the solvent control and highest test concentration in treatment medium were determined for each treatment schedule. There were no relevant changes observed between the solvent controls and test concentrations evaluated.
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation and time of the determination:
3 hours exposure
Experiment 1: (+S9 and -S9): upon addition of the test item to the culture medium, precipitate was observed at concentrations of 888.9 μg/mL and above. At the end of the treatment period precipitate was observed at a concentration of 175.6 μg/mL and above. In Experiment 2 and 3 : (+S9 and -S9): no precipitate was reported, up to 125.0 μg/mL dose level. In Experiment 4: (+S9 and -S9): no precipitate was reported, up to 70.0 μg/mL dose level.
24 hours (continuous) exposure
Experiment 1: (-S9): Upon addition of the test item to the culture medium, precipitate was observed at concentrations of 888.9 μg/mL and above. At the end of the treatment period precipitate was observed at a concentration of 263.4 μg/mL and above. In Experiment 2: (-S9): no precipitate was reported, up to 103.3 μg/mL dose level.
- Definition of acceptable cells for analysis: The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity (or as technically possible). The lowest dose level had little or no cytotoxicity (little or no effect on RPD, or approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined. See above and below for more information. For test items that do not yield apparent cytotoxicity, where solubility is a limiting factor, the lowest concentration at which minimal precipitate was visible in cultures was selected as the highest concentration for slide preparation and micronucleus analysis. For test items that do not yield apparent cytotoxicity or solubility issues, the three highest test concentrations were selected for further microscopic analysis.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES (if applicable): Not applicable.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables.
For all test methods and criteria for data analysis and interpretation: See tables. Also see above “Evaluation” and “Statistics” fields.
- Concentration-response relationship where possible: See tables.
- Statistical analysis: See tables.
- Other: Concentrations tested were as follows
- Experiment 1:
3 hours exposure time
(+S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 4.0 μg/mL CP (PC)
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 2 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.00, 43.81, 53.01, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3 μg/mL plus 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 3 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
- Experiment 4 :
3 hours exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.54, 32.07, 33.67, 35.35, 37.12, 39.98, 40.93, 42.97, 45.17, 47.38, 49.75, 52.24, 54.85, 57.59, 60.47, 63.49, 66.67, 70.00 μg/mL and and 50 ng/mL MMC
The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:
Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL
Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL
Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See tables.
o In the case of the cytokinesis-block method: CBPI and/or ; distribution of mono-, bi- and multi-nucleated cells : see tables.
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells): see tables.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: see tables.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- All the positive control items produced a statistically significant increase in the number of binucleated cells with micronuclei (statistically significant p ≤ 0.01 or were within the 95% Historic Control Data limit range presented in the full study report).
- Negative (solvent/vehicle) historical control data: All vehicle (DMSO) controls had frequencies of mono- and binucleated cells with micronuclei within the range expected for normal human lymphocytes. (Within the 95% Historic Control Data limit range presented in the full study report).

Any other information on results incl. tables

The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:

Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL

Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL

Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

 

Table 1. Experiment 1 : Cytotoxicity From 3 hour Exposure Testing in the Presence of S9 Mix and Absence of S9 Mix

Short Treatment (3 hour) +S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	ND	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	-0.34	100.34	ND	ND	ND	N
52.02	9.21	90.79	ND	ND	ND	N
78.04	12.33	87.67	ND	ND	ND	N
117.1	100.0	-62.38	ND	ND	ND	N
175.6	100.0	-146.24	ND	ND	ND	Y
263.4	ND	ND	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
CPA 4 µg/mL	17.45	82.55	ND	ND	ND	N
Short Treatment (3 hour) -S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	ND	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	12.21	87.79	ND	ND	ND	N
52.02	19.25	80.75	ND	ND	ND	N
78.04	76.15	23.85	ND	ND	ND	N
117.1	100.0	-166.63	ND	ND	ND	N
175.6	100.0	-182.87	ND	ND	ND	Y
263.4	ND	ND	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
MMC 50 ng/mL	23.31	76.69	ND	ND	ND	N

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; CPA = cyclophosphamide; ND = Not determined; Precipitate observed at the end of treatment period = N = no / Y = yes.

 

Table 2. Experiment 3: Cytotoxicity and/or Cytotoxicity and Slide-Scored Data From 3 hour Exposure Testing in the Presence of S9 Mix and Absence of S9 Mix

Short Treatment (3 hour) +S9 mix Experiment 3 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture		MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	12/1000		24/2000	N/A	N
			12/1000
7.500	ND	ND	ND		ND	ND	N
15.00	12.27	87.73	ND		ND	ND	N
30.00 #	12.27	87.73	13/1000		26/2000	ns	N
			13/1000
53.01 #	18.70	81.30	7/1000		14/2000	ns	N
			7/1000
58.31 #	30.84	69.16	10/1000		19/2000	ns	N
			9/1000
64.14 #	29.81	70.19	12/1000		23/2000	ns	N
			11/1000
70.56 #	56.10	43.90	7/1000		15/2000	ns	N
			8/1000
77.62	55.19	44.81	ND		ND	ND	N
85.38	53.96	46.04	ND		ND	ND	N
93.91	100.0	-165.58	ND		ND	ND	N
103.3	100.0	-155.06	ND		ND	ND	N
113.6	100.0	-166.10	ND		ND	ND	N
125.0	100.0	-162.92	ND		ND	ND	N
CPA 4 µg/mL #	36.88	63.12	60/1000		112/2000	1.602x10^-15	N
			52/1000
Short Treatment (3 hour) -S9 mix Experiment 3 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %		MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0		ND	ND	ND	N
7.500	3.95	96.05		ND	ND	ND	N
15.00	9.47	90.53		ND	ND	ND	N
30.00	11.99	88.01		ND	ND	ND	N
53.01	100.0	-5.38		ND	ND	ND	N
58.31	52.24	47.76		ND	ND	ND	N
64.14	100.0	-73.46		ND	ND	ND	N
70.56	100.0	-92.20		ND	ND	ND	N
77.62	100.0	-137.76		ND	ND	ND	N
85.38	100.0	-123.08		ND	ND	ND	N
93.91	100.0	-188.71		ND	ND	ND	N
103.3	100.0	-167.72		ND	ND	ND	N
113.6	100.0	-194.89		ND	ND	ND	N
125.0	100.0	-171.57		ND	ND	ND	N
MMC 50 ng/mL	41.02	58.98		ND	ND	ND	N

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; CPA = cyclophosphamide; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no, ns = not significant (by Fisher’s exact test); # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 1.899x10^-01 = not significant.

 

Table 3. Experiment 4: Cytotoxicity and Slide-Scored Data From 3 hour Exposure Testing in the Absence of S9 Mix

Short Treatment (3 hour) -S9 mix Experiment 4 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	8/1000	18/2000	N/A	N
			10/1000
7.500	ND	ND	ND	ND	ND	N
15.00	ND	ND	ND	ND	ND	N
30.54	ND	ND	ND	ND	ND	N
32.07	ND	ND	ND	ND	ND	N
33.67	ND	ND	ND	ND	ND	N
35.35	ND	ND	ND	ND	ND	N
37.12	ND	ND	ND	ND	ND	N
39.98	ND	ND	ND	ND	ND	N
40.93	ND	ND	ND	ND	ND	N
42.97	6.90	93.10	ND	ND	ND	N
45.12 #	1.39	98.61	19/1000+	19/2000+	ns	N
47.38	16.71	83.29	ND	ND	ND	N
49.75	21.62	78.38	ND	ND	ND	N
52.24	16.84	83.16	ND	ND	ND	N
54.85	18.63	81.37	ND	ND	ND	N
57.79 #	23.21	76.79	9/1000	17/2000	ns	N
			8/1000
60.47 #	48.61	51.39	7/1000	15/2000	ns	N
			8/1000
63.49 #	55.84	44.16	7/1000	15/2000	ns	N
			8/1000
66.67 #	51.13	48.87	ND	ND	ND	N
70.00	100.0	-145.56	ND	ND	ND	N
MMC 50 ng/mL #	24.87	75.13	60/1000	124/2000	<2.2x10^-16	N
			64/1000

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no, ns = not significant (by Fisher’s exact test); + = All 2000 cells scored from one culture as the duplicate culture was unsuitable for analysis when examined under the microscope; # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 4.385x10^-01 = not significant.

 

Table 4. Experiment 1 & 2 Cytotoxicity and/or Experiment 2: Cytotoxicity and Slide-Scored Data From Continuous (24 hour) Exposure Testing in the Absence of S9 Mix

Continuous Treatment (24 hour) -S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	N/A	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	8.33	91.67	ND	ND	ND	N
52.02	14.07	85.93	ND	ND	ND	N
78.04	91.35	8.65	ND	ND	ND	N
117.1	100.0	-162.17	ND	ND	ND	N
175.6	100.0	-21.17	ND	ND	ND	N
263.4	100.0	-234.34	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
MMC 30 ng/mL	45.51	54.49	ND	ND	ND	N
COL 7.5 ng/mL	39.57	60.43	ND	ND	ND	N
Continuous Treatment (24 hour) -S9 mix Experiment 2 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	7/1000	19/2000	N/A	N
			12/1000
7.500	ND	ND	ND	ND	ND	N
15.00	ND	ND	ND	ND	ND	N
30.00 #	-3.46	103.46	7/1000	16/2000	ns	N
			9/1000
43.81	20.97	79.03	ND	ND	ND	N
48.81 #	20.51	79.49	7/1000	15/2000	ns	N
			8/1000
53.01 #	22.70	77.30	9/1000	16/2000	ns	N
			7/1000
58.31 #	31.69	68.31	7/1000	21/2000	ns	N
			14/1000
64.14 #	59.85	40.15	7/1000	15/2000	ns	N
			8/1000
70.56	92.41	7.59	ND	ND	ND	N
77.62	87.62	12.38	ND	ND	ND	N
85.38	100.0	-146.07	ND	ND	ND	N
93.91	100.0	-109.05	ND	ND	ND	N
103.3	100.0	-115.11	ND	ND	ND	N
MMC 30 ng/mL #	44.67	55.33	65/1000	113/2000	<2.2x10^-16	N
			48/1000
COL 7.5 ng/mL #	38.35	61.65	63/1000	122/2000	<2.2x10^-16	N
			59/1000

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; COL = Colchicine; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no; ns = not significant (by Fisher’s exact test); # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 9.073x10^-01 = not significant.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to TK6 human lymphoblastoid cells.

Executive summary:

The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in TK6 human lymphoblastoid cells using the in vitro micronucleus test method with manual scoring of microscope slides, either in the presence or absence of a metabolic activation system (1.0% v/v S9-mix). The test as applied to the test system under three treatment schedules 3 hours in both the absence and presence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix), and a continuous (24 hour) treatment in the absence of S9 mix. Cells were harvested for micronucleus analysis when cells in the solvent control treated cultures achieved between 1.5 and 2.0 normal cell cycles in the relevant exposure condition. In all treatments, the solvent used was dimethyl sulphoxide (DMSO). TK6 human lymphoblastoid cells were not cytokinesis blocked and cell division was determined using a measure of relative population doubling (RPD). Duplicate cultures were used for each test concentration and micronuclei were scored in a minimum of 1000 mononuclear cells per culture (where possible), using two cultures per treatment concentration (2000 mononuclear cells in total). Initially, a test item-DMSO formulation solubility test was performed in R10P (RPMI 1640 media containing 10% heat inactivated horse serum, antibiotics and Pluronic F68) to assess the suitability of the solvent and to help with determining suitable test concentrations for the micronucleus assay. In the solubility test, precipitate was visible at a concentration of 2000μg/mL after addition to R10P media. Following overnight incubation (humidified atmosphere of 5% CO2 at a temperature of 37°C) of the tubes (containing R10P media with test concentrations or solvent control), precipitate was visible at a concentration of 2000μg/mL.In order tomeet guideline criteria, 2000μg/mL was therefore selected as the top concentration for all three treatment schedules for Experiment 1. On the day of testing a stock solution of test item in DMSO was prepared at 200.0 mg/mL and further diluted in DMSO to prepare the required test solutions. Upon addition of test item to the culture medium in Experiment 1 precipitate was observed at concentrations of 888.9 μg/mL and above in all treatment schedules. At the end of the treatment period precipitate was observed at concentrations of 175.6 μg/mL and above in the three hour treatment schedules and 263.4 μg/mL and above in the continuous treatment schedule. In Experiment 2, 3 and 4, narrower dose ranges and lower top concentrations were prepared and dosed due to non achievement of required cytotoxicity (55% ±5%) dose levels. The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were: Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL, Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL and Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL. In Experiment 3, cytotoxicity of 56.10% was observed at a concentration of 70.56 μg/mL in the 3 hour +S9 mix treatment. In Experiment 4 cytotoxicity of 55.84% was observed at a concentration of 63.49 μg/mL in the 3 hour -S9 mix treatment. In Experiment 2 cytotoxicity of 59.85% was observed at a concentration of 64.14 μg/mL in the continuous treatment. In further experiments, no precipitate was observed at any concentrations tested during any stage of the experiment. In final tests, where slides were selected for analysis, for all treatment schedules, data for background micronucleus induction in the solvent controls were consistent with the historical control database (based on 95% Poisson confidence limits) for TK6 cells. The concurrent positive controls produced statistically significant (p <0.05) increases in micronuclei compared with the concurrent negative controls and were within the expected range based on the historical control database. No statistically significant increases in micronucleus formation were observed at any test item concentration analysed, all micronucleus values fell within the 95% Poisson confidence limits of the solvent control and there were no concentration related increases when evaluated with a Cochran-Armitage trend tests. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to TK6 human lymphoblastoid cells, in vitro in the presence or absence of S9 mix.