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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 (in vitro Bacterial reverse mutation test): Negative (±S9)

OECD 487 (in vitro Micronucleus Test): Negative (±S9)

OECD 490: (in vitro mouse lymphoma assay): Negative in absence of metabolic activation (-S9) and positive in the presence of metabolic activation (+S9).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October - 6 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Sponsor's identification: 1,4-Dimethoxy-2-tert-butylbenzene (Vetimoss)
- CAS number: 21112-37-8
- Description: clear colourless liquid
- Batch No.of test material: 1000509491
- Purity: 99.6%
- Expiration date of the lot/batch: 14 January 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: all formulations were used within four hours of preparation and were assumed to be stable fo rthis period

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- the test material was accurately weighed and approximate half-log dilutions were prepared in acetone by mixing on a vortex mixer on the day of each experiment
- formulated concentrations were adjusted to allow for the stated water/impurity content (26.9%) of the test substance
- prior to use, the solvent was dried to remove water, using molecular sieves (2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x10^-4 microns)
Target gene:
Histidine or tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 50, 150, 500, 1500, 5000 µg/plate

Mutation test 1:
- Salmonella strains (absence of S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- Salmonella strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 50, 150, 500, 1500, 5000 µg/plate.

Mutation test 2:
- Salmonella strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- Salmonella strains (presence of S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
- Escherichia coli strain WP2uvrA (absence and presence of S9-mix): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test substance was immiscible in sterile distilled water at 50 mg/mL, but fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house.
Untreated negative controls:
yes
Remarks:
2-Aminoanthracene; Benzo(a)pyrene
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle control
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation; in agar (plate incorporation)

DURATION
- Preincubation period: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. The cultures were monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
- Exposure duration: approximately 48-hour incubation at 37°C

MEASUREMENTS
- After the 48-hour incubation period at 37°C, the plates were assessed for numbers of revertant colonies using an automated colony counter and examined for effects on the growth of the bacterial background lawn.

PRELIMINARY TOXICITY TEST
- A preliminary test was conducted in order to select appropriate dose levels for use in the main test.
- 0.1 mL bacterial culture (TA100 or WP2uvrA) was dispensed into sets of small volume containers with 0.5 mL S9-mix or phosphate buffer and 0.1 mL of test substance formulation.
- The containers were sealed incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented top agar.
- The tube contents were mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
- The plates were placed in anaerobic jars or bags (one jar/bag for each each concentration of test substance/vehicle) during incubation.
- 5 concentrations of the test substance formulation and a vehicle control (dimethyl sulphoxide) were tested.
- 0.1 mL of the maximum concentration of the test substance and 2 mL molten trace histidine or tryptophan supplemented top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test substance.

EXPERIMENT 1
- Up to 7 concentrations of test substance were assayed in triplicate against each tester strain.
- The same process was followed as in the preliminary assay, except 0.1 mL bacterial culture (TA100 or WP2uvrA) was dispensed into sets of small volume containers with 0.5 mL S9-mix or phosphate buffer and 0.1 mL of test substance formulation.
- The procedure was repeated in triplicate for each bacterial strain and for each concentration of test substance both with and withou the S9-mix.

EXPERIMENT 2
- Performed using fresh bacterial cultures, test substance and control solutions.
- Based on results from Experiment 1, the test substance dose range was slightly altered.

CONTROLS
1) Vehicle control - solvent treatment group
- The untreated controls were dosed using the standard plate incorporation method.
- 0.1 mL of one of the bacterial cultures were dispensed into sets of test tubes with 2 mL molten trace histidine or tryptophan supplemented top agar.
- The tube contents were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
- The procedure was repeated in triplicate for each bacterial strain.
- The plates were incubated at 37°C for 48-hours and then the frequency of revertant colonies was measured.

2) Positive controls - used in a series of plates without S9-mix:
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 2µg/plate for WP2uvrA
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 3µg/plate for TA100
- N-ethyl-N'-nitro-N-nitrosguanidine (ENNG): 5µg/plate for TA1535
- 9-Aminoacridine (9AA): 80 µg/plate for TA1537
- 4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate

3) Negative controls - non-mutagenic substances in the absence of metabolising enzymes were used in the series of plates with S9-mix:
- 2-Aminoanthracene (2AA): 1 µg/plate for TA100
- 2-Aminoanthracene (2AA): 2 µg/plate for TA1535 and TA1537
- 2-Aminoanthracene (2AA): 10 µg/plate for WP2uvrA
- Benzo(a)pyrene (BP): 5µg/plate for TA98
Evaluation criteria:
Any one or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested.
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Results will be reported as equivocal when data generated will prohibit making a definite judgement about test substance activity.
Statistics:
Not specified
Key result
Species / strain:
other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary test:
- The test substance was toxic to TA100 from 50 and 500 µg/plate in the absence and presence of the S9-mix respectively, and non-toxic to WP2uvrA.
- The test substance formulation and S9-mix used in this experiment were both shown to be sterile.

Controls:
- The results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
- All positive control chemicals induced marked increases in the frequency of revertant colonies thus confirming the activity of the S0-mix and the sensitivity of the bacterial strains.

Main experiment:
- The test substance caused a visible reduction in the growth of all the bacterial background lawns of the Salmonella strains in the absence of the S9-mix from 15 µg/plate and the 150 µg/plate in the presence of the S9-mix.
- No toxicity was seen to E.coli WP2uvrA at any test substance dose level tested either in the presence of absence of the S9-mix.
- The test substance was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on the bacterial strain type and Experiment number.
- No test substance precipitate was observed on any plates either in the presence of absence of the S9-mix.
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either in the presence or absence of metabolic activation.

Acceptance criteria met: yes

Main experiment

- The master strains were checked prior to use for characteristics, viability and spontaneous reversion rate, which were all found to be satisfactory.

- The culture density of each bacterial strain was also considered to be acceptable.

- The amino acid supplemented top agar and S9 -mix were shown to be sterile.

- The culture density for each bacterial strain was checked and considered to be acceptable.

- The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, the presence and absence of metabolic activation and the experimental methodology.

- The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology.

- A test substance precipitate (oily in appearance) was seen under an inverted microscope on the 5000 µg/plate, but it did not prevent the scoring of the revertant colonies.

Acceptance criteria - the reverse mutation assay may be considered valid if the following criteria are met:

- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.

- All tester strain cultures should show a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.

- All tester strain cultures should be in the range of 0.9-9 x10^9 bacterial per mL.

- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control cehmicals used in the study should induce marked increases in the frequency of revertant colonise, both in the presence and absence of metabolic activation.

- There should be a minimum of four non-toxic test substance dose levels.

- There should be no evidence of excessive contamination.

Conclusions:
Under the conditions of the study and based on the results, the test substance Vetimoss was considered to be non-mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).
Executive summary:

This study was conducted to evaluate the ability of the test substance, 1, 4 -Dimethoxy-2 -tert-butylbenzene (Vetimoss) to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptopjhan locus in the genome of five strains of bacteria. The study was performed in accordance with OECD 471, EU Method B.13/14, the major Japanese Regulatory Authorities including METI, MHLW and MAFF, and the USA EPA (TSCA) OPPTS harmonised guidelines.

The bacterial strains employed in this study were Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. The bacterial strains wree treated with the test substance using the Ames pre-incubation method at up to seven dose levels, tested in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). A preliminary toxicity assay was initially performed in order to select appropriate dose levels for the main experiment. The test ubstance was found to be toxic to S. typhimurium TA100 from 50 -500 µg/plate (depending on bacterial tester strain and the absence or presence of metabolic activation) and non-toxic to E.coli WP2uvrA. The results of the preliminary assay determined the test substance dose range in Experiment 1, which ranged from 0.5 -5000 µg/plate. Experiment 2 was repeated on a separate day using fresh cultures of bacterial strains and test substance formulations. Additional dose levels and an expanded dose range were selected, where applicable, in order to achieve both four non-toxic dose levels and the toxic limit of the test substance.

The test substance caused a visible reduction in the growth of the bacterial background lawns of all Salmonella strains initially from the 15 µg/plate in the absence of the S9 -mix and 150 µg/plate in the presence of the S9 -mix. No toxicity was seen to E.coli WP2uvrA strain at any test substance concentration tested in the presence or absence of metabolic activation. The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains with any dose of the test material, either in the presence or absence of the S9 -mix or in either exposure method employed.

There was no test substance precipitate observed on the plates at any of the doses tested in the absence or presence of the S9 -mix. The vehicle (dimethyl sulphoxide) control plates gave revertant colony counts within the normal range and all positive chemicals induced marked increases in the frequency of revertant colonies, both in the presence and absence of metabolic activation. The acceptance criteria were met and therefore the sensitivity of the assay and effiicacy of the S9 -mix were validated.

Under the conditions of the study and based on the results, it can be concluded that the test substance, 1, 4-Dimethoxy-2-tert-butylbenzene, was considered to be non-mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September - 16 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance I.D.: 1,4-Dimethoxy-2-tert-butylbenzene (Vetimoss)
- Description: clear colourless liquid
- Source and lot No.of test material: 1000537818
- Expiration date of the lot/batch: July 2014
- Purity test date: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Vehicle: DMSO was chosen as the vehicle to deliver the test material to the test system.
- Treatment of test material prior to testing: Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under light.
Species / strain / cell type:
mammalian cell line, other: HPBL
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 -induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.194-1940 µg/mL (10 mM)
Initial Micronucleus assay: Non-activated 4-hr treatment (5-100 µg/mL); Non-activated 24-hr treatment (1-60 µg/mL); S9-activated 4-hr treatment (5-100 µg/mL).
Repeat Micronucleus assay: Non-activated 4-hr treatment (10-70 µg/mL); Non-activated 24-hr treatment (2-60 µg/mL); S9-activated 4-hr treatment (10-70 µg/mL).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was conducted using water and DMSO. The vehicle of choice was selected in order of preference that permitted preparation of the highest soluble or workable concentration, up to 50 mg/mL in aqueous solvents and up to 500 mg/mL in organic solvents. DMSO was selected.
Untreated negative controls:
yes
Remarks:
Cytochalasin B (cytoB)
Negative solvent / vehicle controls:
yes
Remarks:
The DMSO vehicle was used as the vehicle control for each treatment group.
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Viblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: standard conditions for 44-48 hours (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air).
- Exposure duration: with and without S9 activation: 20-hours ± 30 minutes, absence of S9-activation: 24-hours ± 30 minutes.

PRELIMINARY TOXICITY ASSAY
- HPBL were exposed to the vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures in both the absence and presence of the S0 activation system for 4 hours or continuously for 24 hours in the absence of S9 activation.
- The precipitation in the treatment medium was determined by unaided eye at the beginning and end of treatment.
- Measurements made: solvent osmolality, the highest dose level, lowest precipitating dose level, highest soluble dose level in treatment medium was measured.
- Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control.

MICRONUCLEUS ASSAY
- Eight or nine dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance.
- The precipitation in the treatment medium was determined by unaided eye at the beginning and end of treatment.

TREATMENT OF TARGET CELLS
- pH was measured at the highest test substance concentration prior to dosing.
- Cultures were re-fed with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL culture medium + 1 mL S9 cofactor pool) for the S9-activated exposure.
- 50 µL test substance dosing solution or vehicle was then added.
- Positive control cultures were resuspended in either 5 mL complete medium for the non-activaed studies or 5 mL S9 reaction mixture ( 4mL serum free medium + 1 mL S9 cofactor pool), to which 50 µL positive control in solvent was added.
- Following the 4-hour treatment, cells were centrifuged and the treatment medium was aspirated. Cells were washed with calcium and magnesium-free phosphate buffered saline (CMF-PBS), then re-fed with complete medium containing cytoB at 6.0 µg/mL and returned to the incubator.
- For the 24-hour treatment group in the non-activated study, 6.0 µg/mL cytoB was added at the beginning of treatment.

CELL COLLECTION
- Cells exposed to cytoB for 24 hours (±30 minutes): Cells were collected 1.5-2 normal cell cyles to ensure identification and selective analysis of micronucleus frequency in cells that have complete one mitosis, signfied by binucleated cells.
- The cytoB exposure time for the 4-hour treatment in the non-activated and S9-activated studies was 20 hours (± 30 minutes).
- Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol:glacial acetic acid, 25:1 v/v), capped and may be stored overnight or longer at 2-8°C.
- The suspension of fixed cells was applied to glass microscope slides and air-dried and stained with acridine orange.

MICRONUCLEUS SCORING
- The slides from at least 3 test substance treatment groups were coded using random numbers and scored for the presence of micronuclei based on cytotoxicity.
- A minimum of 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
- Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
- the micronuclei should have the same staining characteristics as the main nucleus
- the diameter of the micronuclei must be approximately 1/3 or less the diameter of a main nucleus
- the micronuclei must be non-refractile, located in the cytoplasm, no overlapping with nucleus and not connected by cytoplasmic bridges
- the micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be clearly distinguishable from the nuclear boundary.
Rationale for test conditions:
The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance.
Evaluation criteria:
- The frequency of cells with micronucleus induction in the vehicle control must be within the historical control range.
- The percentage of cells with micronucleus induction must be statistically increased (p<0.05, Fisher's exact test) in the postiive control condition relative to the vehicle control.
- The test substance was considered positive if it induced a statistically significant and dose-dependent increase in the frequency of MN-BN cells (p>0.05). If only one criterion was met (statistically significant or dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
Statistics:
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test, which compared pairwaise, the percentage of micronucleated cells of each treatment group with that of the vehicle control.
Key result
Species / strain:
lymphocytes: HPBL
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Substantial cytotoxicity was observed at some doses
Remarks:
Initial assay: ≥ 60 µg/mL in both 4-hr groups and ≥ 35 µg/mL in the non-activated 24-hr group. Repeat assay: ≥ 65 µg/mL in the non-activated 4-hr group,70 µg/mL in the S9-activated 4-h group, 60 µg/mL in the non-activated 24-hr group.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Repeat micronucleus assay
- The dose levels selected for analysis of micronucleus were 35, 50 and 65 µg/mL.
- At the highest test concentration, 65 µg/mL, cytotoxicity was 58% relative to the vehicle control.
- The percentage of cells with micronuclei in the test substance-treated group was not significantly increased relative to the vehicle control at any dose level (p>0.05, Fisher's Exact test).
- The percentage of micronucleated cells in the MMS (positive control) group (3.9%) was statistically signfiicant (≤ 0.01, Fisher's Eact test).

Controls

- The stability of the vehicle and positive controls and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Solubility test: The test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, which was the maximum concentration tested for solubility. It was soluble in DMSO at all concentrations tested in the preliminary assay.

Preliminary toxicity assay

- Visible precipitate was observed in treatment medium at dose levels ≥ 194 µg/mL, while dose levels ≤ 58.2 µg/mL were soluble in treatment medium at the beginning of the treatment period.

- At the end of the treatment period, visible precipitate was observed in the 4 -hour exposure groups at ≥ 582 µg/mL and dose levels ≤ 582 µg/mL were soluble in treatment medium at the end of the treatment period. Hemolysis was observed at dose levels ≥ 194 µg/mL in all treatment conditions.

- pH of the highest test substance concentration in treatment medium was 7.0.

- The osmolality of the test substance dose levels in the treatment medium is acceptable since it did not exceed the osmolality of the vehicle by more than 20%.

- Treatment medium at the highest concentration tested (1940 µg/mL) = 384 mmol/kg

- Treatment medium at the lowest precipitating concentration (194 µg/mL) = 406 mmol/kg

- Treatment medium of the highest soluble concentration (58.2 µg/mL) = 414 mmol/kg

- Vehicle (DMSO) in the treatment medium = 404 mmol/kg

>Cytotoxicity

- Substantial cytotoxicity [50 -60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 194 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and dose levels

≥ 58.2 µg/mL in the non-activated 24 -hour exposure group.

Initial micronucleus assay

- Substantial cytotoxicity seens at dose levels ≥ 60 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and at dose levels ≥ 35 µg/mL in the non-activated 24 -hour exposure group.

- A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

- The induction of micronuclei in the vehicle control was outside the historical solvent control range, therefore the assay was repeated.

Conclusions:
Under the conditions of the assay, the test substance Vetimoss was determined to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The purpose of this study was to evaluate the potential of the test substance, 1,4 -Dimethoxy-2 -tert-butylbenzene (Vetimoss) and/or its metabolites to induce micronuclei in human blood peripheral lymphocytes (HPBL) in the presence and absence of an exogenous metabolic activation system. The study was performed in accordance with OECD 487 guideline.

The micronucleus assay was employed to order to evaluate the aneugenic and clastogenic potential of the test substance. Dimethyl sulphoxide was used as a vehicle for the test substance, based on the test substance and comptability with the target cells. The test substance was found to be soluble in DMSO at a concentration of approximately 500 mg/mL, which was the maximum concentration tested for solubility. In both the preliminary toxicity and main micronucleus assays, HBPL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9 -activated test system. All cells were harvested 24 -hours after the start of treatment and a minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

A preliminary toxicity assay was performed to establish the dose range for testing in the micronucleus test. The doses tested ranged from 0.194 -1940 µg/mL (10 mM). There was substantial cytotoxicity [50 -60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 194 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and dose levels ≥ 58.2 µg/mL in the non-activated 24 -hr exposure group. Based on the preliminary results, the dose range selected for the micronucleus assay was 5 -100 µg/mL for the non-activated and S9 -activated 4 -hour exposure gorups and 1 -60 µg/mL for the non-activated 24 -hour exposure group.

In the initial micronucleus assay, substantial cytotoxicity was seen at doses ≥ 60 µg/mL in the non-activated and S9 -activated 4 -hour expsosure groups and at doses ≥ 35 µg/mL in the non-activated 24 -hour expsoure group. The highest dose analysed under each treatment condition caused a 50 -60% reduction in CBPI, which met the dose limit, as recommended by the assay testing guidelines. The induction of micronuclei in the vehicle controls were found to be putside of the historical solvent control range, and therefore a repeat micronucleus assay was performed with a dose range of 10 -70 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and 2.5 -60 µg/mL in the non-activated 24 -hour exposure group. Substantial cytotoxicity was also observed in the repeat micronucleus assay at dose levels ≥ 65 µg/mL in the non-activated 4 -hour exposure group, 70 µg/mL in the S9 -activated 4 -hour exposure groups and at 60 µg/mL in the non-activated 24 -hour exposure group. Similarly to the initial assay, a 50 -60% reduction in CBP1 was observed at the highest dose analysed under each treatment condition, which met the dose limit, as recommended by the assay testing guidelines.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p>0.05, Fisher's Exact test). The positive and negative control results were comparable to historical data, inducating that all criteria for a valid assay were met. Under the conditions of the assay, it can be concluded that the test substance, 1,4-Dimethoxy-2-tert-butylbenzene, was determined to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-08-07 to 2019-02-28
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:
29 July 2016
Deviations:
yes
Remarks:
Deviations had no impact on the study on the results or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Limited (United Kingdom); Batch No. A180118C
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤ 70 % relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Dimethyl sulfoxide was used as the solvent/vehicle because it was found to be compatible with the survival of the cells and the S9 activity. A 200 mg/mL concentration of the test material in DMSO was suitable for this study.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, almost colorless liquid
Target gene:
tk+/- (thymidine kinase) locus in L5178Y cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, USA); LOT No. 60797977

- Suitability of cells: While many mammalian cell gene mutation systems are available, however the mouse lymphoma assay (MLA), employing the tk+/- (thymidine kinase) locus in L5178Y cells, has the advantage of detecting both gene mutations and chromosome aberrations. The principle of this assay is based on placing cells under selective pressure so that only mutant cells are able to survive. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-) for the gene that produces the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products but if a toxic base analogue (5-trifluorothymidine) is present in the medium, the enzyme will incorporate the analogue into the cells. Thus, the cells will not survive unless the enzyme is rendered inactive, by mutation. Resistance to 5-trifluorothymidine (TFT) results in a lack of thymidine kinase (TK) activity so the mutants (tk-/-) are unable to incorporate the toxic analogue and therefore survive in its presence.

Two types of TFT-resistant mutant colonies occur and these are designated as large colonies (normal-growing) and small (slow-growing) colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this assay, gene mutations within the tk gene (11 to 13 kilobases) and chromosomal events involving the gene may be detected. The TK system has a high spontaneous mutant frequency and high numbers of cells can be treated and sampled, therefore it is statistically robust.

- Methods for maintenance in cell culture if applicable: Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than three times before used in the main study).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 3 types of RPMI 1640 medium (Table 1); humidified atmosphere containing approximately 5% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver post-mitochondrial fraction (S9 fraction).
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. Three-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments.
The highest concentration tested in the preliminary test was 2000 µg/mL (the recommended maximum concentration).

The highest selected concentration for the main tests was based on the observed cytotoxicity. A total of 8 concentrations with and without metabolic activation were selected for Assay 1. Concentrations for Assay 2 were different. The details of the same are presented in Tables 2 and 3.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Based on the available information (trial formulation of the test item performed in an in vivo study at the Test Facility [17/183-001P]) Distilled water at 200 mg/mL concentration was not a suitable vehicle for this study. However, based on the result of the preliminary solubility test, the 200 mg/mL concentration using Dimethyl sulfoxide (DMSO) was suitable for this study. Thus, DMSO was selected for vehicle (solvent) of this study. This vehicle (solvent) was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Remarks:
Negative (vehicle) control cultures were treated with the selected vehicle alone in the same way as the test item treated cultures.
Negative solvent / vehicle controls:
yes
Remarks:
, untreated control sample was also used in each assay to demonstrate that the selected vehicle (solvent) had no mutagenic effects.
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
For 3-hour treatments, 10^7 cells
For 24-hour treatment, 6 x10^6 cells

DURATION
- Exposure duration: 3 hours (-S9 and +S9) or 24 hours (-S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Plating for survival:
Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for Viability
At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1 x 10^4 cells/mL with RPMI-20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL.

Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 5-trifluorothymidine (TFT) resistance
At the end of the expression period, the cell concentration was adjusted to 1 x 10^4 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2 x 10^3 cells per well.

Microplates were incubated at 37ºC ± 0.5°C containing approximately 5% (v/v) CO2 in air for 12 days and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.

NUMBER OF CELLS EVALUATED: 1x10^4 cells/mL (Viability and 5-trifluorothymidine (TFT) resistance)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: Relative Total Growth (RTG) was calculated as the percentage total growth of the treated cultures compared to the corresponding negative (vehicle/solvent) control value.
Evaluation criteria:
The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:

1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).

2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).

3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.

The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.

Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological significance. Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).
Statistics:
Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.

The negative (vehicle/solvent) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Only in the presence of metabolic activiation (+S9)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In Assays 1 and 2, there were no large changes in pH after treatment.
- Effects of osmolality: In Assays 1 and 2, there were no large changes in osmolality after treatment.
In both Assays, no insolubility was observed in the final treatment medium at the end of the treatment with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Experiment:
Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment using DMSO as vehicle was 2000 µg/mL (the recommended maximum concentration). Tabulated results of the preliminary experiment are given in Appendix 4.

Insolubility and excessive cytotoxicity was detected in the preliminary experiment. Precipitate/ minimal amount of precipitate was detected in the 2000-3.906 µg/mL concentration range with metabolic activation, in the 2000-500 µg/mL concentration range in case of short treatment without metabolic activation and in the 2000-62.5 µg/mL concentration range in case of long treatment without metabolic activation.

Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-125 µg/mL concentration range using the short treatment with and without metabolic activation and in the 2000-62.5 µg/mL concentration range using the long treatment without metabolic activation.

Concentrations up to the cytotoxic range were selected for the main experiments according to the instructions of the relevant OECD No. 490 guideline. At least eight concentrations were selected for the main experiments in each assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Presented in Table 5
- Negative (solvent/vehicle) historical control data: Presented in Table 5.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Relative Total Growth (RTG)

Preliminary Experiment:
Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-125 µg/mL concentration range using the short treatment with and without metabolic activation and in the 2000-62.5 µg/mL concentration range using the long treatment without metabolic activation.

Mutation Experiment: Assay 1:
Presence of S9-mix (3-hour treatment):
In the presence of S9-mix (3-hour treatment), no cells survived the expression period in the samples of 70 µg/mL concentration. Different degree of cytotoxicity was observed at 60-10 µg/mL concentration (relative total growth of the highest evaluated concentration of 60 µg/mL was 19%). An evaluation was made using data of seven concentrations (concentration range of 60-5 µg/mL).

Absence of S9-mix (3-hour treatment):
In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 70, 65 and 60 µg/mL concentration. However, despite the relatively close concentrations tested, no significant cytotoxicity was observed at lower concentrations (although the longer exposure in Assay 2 without S9 did have concentrations with low survival). An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL).

Mutation Experiment: Assay 2:
Presence of S9-mix (3-hour treatment):
In the presence of S9-mix (3-hour treatment) excessive cytotoxicity was observed at the highest tested concentration (70 µg/mL, relative total growth value of 2%). Lower degree of cytotoxicity (range of the relative total growth values were 29-63%) was observed at lower concentrations. An evaluation was made using data of eight concentrations (concentration range of 60-2.5 µg/mL).

Absence of S9-mix (24-hour treatment):
In Assay 2, following a 24-hour treatment without metabolic activation, excessive or marked cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 55 and 50 µg/mL concentration and excessive cytotoxicity was observed at 45 µg/mL. An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL).

Table 4. Results of Mutation Test (Assays 1 and 2)

Assay

Concentration (RTG)

and MF values

Increase in MF compared to vehicle control (GEF)

Concentration related increase was indicated by the linear trend analysis

Reproducible

between cultures

Conclusion

Assay 1
3-hour +S9

60 µg/mL (19%): 390.3*

262.1 (>GEF)

Yes

YesN

 

Positive

 

50 µg/mL (49%): 364.0*

235.8 (>GEF)

40 µg/mL (30%): 336.6*

208.4 (>GEF)

30 µg/mL (19%): 250.5*

122.3 (<GEF)

20 µg/mL (13%): 312.0*

183.8 (>GEF)

Vehicle (100%): 128.2

 

Assay 1
3-hour -S9

40 µg/mL (71%): 123.8

7.9 (<GEF)

No

Yes

Negative

30 µg/mL (112%): 100.5

15.4 (<GEF)

20 µg/mL (89%): 116.2

0.3 (<GEF)

10 µg/mL (103%): 112.4

-3.5 (<GEF)

5 µg/mL (95%): 132.9

17.0 (<GEF)

Vehicle (100%): 115.9

 

 

Assay 2
3-hour +S9

60 µg/mL (38%): 247.1*

157.4 (>GEF)

Yes

Yes

 

Positive

50 µg/mL (42%): 272.9*

183.2 (>GEF)

40 µg/mL (48%): 241.0*

151.3 (>GEF)

30 µg/mL (31%): 314.1*

224.4 (>GEF)

20 µg/mL (29%): 266.4*

176.7 (>GEF)

Vehicle (100%): 89.7

 

Assay 2
24-hour -S9

40 µg/mL (15%): 82.7

11.9 (<GEF)

No

Yes

Negative

30 µg/mL (52%): 73.2

2.4 (<GEF)

20 µg/mL (76%): 85.4

14.6 (<GEF)

10 µg/mL (87%): 87.8

17.0 (<GEF)

5 µg/mL (71%): 86.1

15.3 (<GEF)

Vehicle (100%): 70.8

 

Notes: Results of the five highest evaluated concentrations are shown in the table. Statistical significance labelled by *.

MF: Mutation Frequency (refers to 106 viable cells), GEF: Global Evaluation Factor (=126 per 106 viable cells)

 

N: At 60 µg/mL concentration: incoherence was seen between the replicates, but there wasno significant difference between the individual mutation frequency values (mean MF value was 390.3, individual values of 376.5 and 349.0 was detected for replicates A and B, respectively).Some incoherence was also seen between replicates at 10 µg/mL, however the mutation frequency value of this concentration was below the GEF.

Table 5. Historical Control Data

Mutation Frequency of the Negative Controls (2006-2016)

 

Culture medium

Distilled water

Treatments

3h,S9+

3h,S9-

24h,S9-

3h,S9+

3h,S9-

24h,S9-

Average

94.3

103.6

106.4

90.4

96.6

96.3

SD

26.9

35.3

27.4

22.7

19.0

24.6

Min.

39.3

52.6

41.7

33.4

55.1

43.2

Max.

198.5

235.6

179.1

121.8

125.0

141.1

n

84

43

44

26

13

13

Dimethyl sulfoxide (DMSO)

Treatments

3h,S9+

3h,S9-

24h,S9-

 

 

 

Average

97.3

97.3

98.9

 

 

 

SD

33.7

38.5

26.8

 

 

 

Min.

44.2

33.7

47.1

 

 

 

Max.

269.9

261.6

159.4

 

 

 

n

101

57

50

 

 

 

Mutation Frequency of the Positive Controls (2006-2016)

 

Cyclophosphamide

4-Nitroquinoline-N-oxide

Treatments

3h,S9+

 

 

 

3h,S9-

24h,S9-

Average

1178.7

 

 

 

722.2

831.9

SD

524.7

 

 

 

330.0

337.2

Min.

196.1

 

 

 

223.5

245.0

Max.

2642.5

 

 

 

1687.3

1577.6

n

106

 

 

 

58

52

h = hour

SD = Standard Deviation

S9+ = experiment with metabolic activation

S9- = experiment without metabolic activation

n = number of cases

Mutation Assay Results

 

Assay 1 (Presence of S9-mix (3-hour treatment)):

In Assay 1 with metabolic activation there was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL with the exception of 30 µg/mL, at this concentration the mutation frequency values were slightly below the GEF. Concentration related increase was indicated by the linear trend analysis. Although, at the highest evaluated concentration in Assay 1 (60 µg/mL) incoherence was seen between replicates, but there was no significant difference between the individual mutation frequency values (mean MF value was 390.3, individual values of 376.5 and 349.0 was detected for replicates A and B, respectively) and both values were above the GEF at appropriate degree of cytotoxicity. Incoherence was also seen between replicates at 10 µg/mL, however the mutation frequency value of this concentration was below the GEF. Reproducibility was observed at further evaluated concentrations. This experiment was considered to be positive.

 

Assay 1 (Absence of S9-mix (3-hour treatment)):

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 70, 65 and 60 µg/mL concentration. However, despite the relatively close concentrations tested, no significant cytotoxicity was observed at lower concentrations (although the longer exposure in Assay 2 without S9 did have concentrations with low survival). An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

Assay 2 (Presence of S9-mix (3-hour treatment)):

In Assay 2 with metabolic activation there was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL. Concentration related increase was indicated by the linear trend analysis. Although lower degree of cytotoxicity was observed at several concentrations in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be positive.

 

Assay 2 (Absence of S9-mix (24-hour treatment)):

No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

Conclusions:
A reproducible mutagenic effect of the test material (2-tert-butyl-1,4-dimethoxybenzene) was observed in the presence of a metabolic activation system, and no mutagenic effect was observed in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, the test material was considered to be genotoxic.
Executive summary:

A Key OECD Guideline 490 in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material (2-tert-butyl-1,4-dimethoxybenzene) 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). DMSO was used as vehicle in the study and the following test material concentrations were examined in the mutation assays:

 

Assay 1 (3-hour treatment with metabolic activation): 70, 60, 50, 40, 30, 20, 10 and 5 µg/mL;

Assay 1 (3-hour treatment without metabolic activation): 70, 65, 60, 40, 30, 20, 10 and 5 µg/mL;

Assay 2 (3-hour treatment with metabolic activation): 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 µg/mL;

Assay 2, (24-hour treatment without metabolic activation): 55, 50, 45, 40, 30, 20, 10 and 5 µg/mL.

 

In both Assays, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in the absence and presence of metabolic activation.

 

In Assay 1 and 2, following a 3-hour treatment with metabolic activation, marked or excessive cytotoxicity was seen in several concentrations. In Assay 1 with metabolic activation, no cells survived the expression period in the samples of 70 µg/mL concentration. An evaluation was made using data of seven concentrations (concentration range of 60-5 µg/mL) in Assay 1 and eight concentrations (concentration range of 60-2.5 µg/mL) in Assay 2.

 

In Assay 1, with metabolic activation, there was a statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL with the exception of 30 µg/mL, at this concentration the mutation frequency values were slightly below the GEF. Concentration related increase was indicated by the linear trend analysis. Although, at the highest evaluated concentration in Assay 1 (60 µg/mL) incoherence was seen between replicates, but there was no significant difference between the individual mutation frequency values and both values were above the GEF at appropriate degree of cytotoxicity. Some incoherence was also seen between replicates at 10 µg/mL, however the mutation frequency value of this concentration was below the GEF. Reproducibility was observed at further evaluated concentrations. This experiment was considered to be positive.

 

In Assay 2, with metabolic activation there was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL. Concentration related increase was indicated by the linear trend analysis. Although lower degree of cytotoxicity was observed in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be positive.

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 70, 65 and 60 µg/mL concentration. However, no significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

In Assay 2, following a 24-hour treatment without metabolic activation, excessive or marked cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 55 and 50 µg/mL concentration. An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, a reproducible mutagenic effect of the test material (2-tert-butyl-1,4-dimethoxybenzene) was observed in the presence of a metabolic activation system, and no mutagenic effect was observed in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, the test material was considered to be genotoxic.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A Key OECD Guideline 471 study (Harlan Laboratories Ltd., 2013; K = 1) was conducted to evaluate the ability of the test substance (1, 4-Dimethoxy-2 -tert-butylbenzene (Vetimoss)) to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria. The bacterial strains employed in this study were Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA. The bacterial strains were treated with the test substance using the Ames pre-incubation method at up to seven dose levels, tested in triplicate with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). A preliminary toxicity assay was initially performed in order to select appropriate dose levels for the main experiment. The test substance was found to be toxic to S. typhimurium TA100 from 50 -500 µg/plate (depending on bacterial tester strain and the absence or presence of metabolic activation) and non-toxic to E.coli WP2uvrA. The results of the preliminary assay determined the test substance dose range in Experiment 1, which ranged from 0.5 -5000 µg/plate. Experiment 2 was repeated on a separate day using fresh cultures of bacterial strains and test substance formulations. Additional dose levels and an expanded dose range were selected, where applicable, in order to achieve both four non-toxic dose levels and the toxic limit of the test substance.

 

The test substance caused a visible reduction in the growth of the bacterial background lawns of all Salmonella strains initially from the 15 µg/plate in the absence of the S9 -mix and 150 µg/plate in the presence of the S9 -mix. No toxicity was seen to E.coli WP2uvrA strain at any test substance concentration tested in the presence or absence of metabolic activation. The test substance was tested up to the toxic limit or maximum recommended dose level of 5000 µg/plate depending on the bacterial strain type, the presence or absence of the S9 -mix and the experimental methodology. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains with any dose of the test material, either in the presence or absence of the S9 -mix or in either exposure method employed.

 

There was no test substance precipitate observed on the plates at any of the doses tested in the absence or presence of the S9 -mix. The vehicle (dimethyl sulphoxide) control plates gave revertant colony counts within the normal range and all positive chemicals induced marked increases in the frequency of revertant colonies, both in the presence and absence of metabolic activation. The acceptance criteria were met and therefore the sensitivity of the assay and efficacy of the S9 -mix were validated.

 

Under the conditions of the study and based on the results, it can be concluded that the test substance, 1, 4-Dimethoxy-2-tert-butylbenzene, was considered to be non-mutagenic to Salmonella strains TA98, TA100, TA1535 and TA1537 and E.coli strain WP2uvrA in the presence and absence of metabolic activation (S9-mix).

 

A Key OECD Guideline 487 study (BioReliance Corporation, 2014; K = 1) was conducted to evaluate the potential of the test substance (1,4 -Dimethoxy-2 -tert-butylbenzene (Vetimoss)) and/or its metabolites to induce micronuclei in human blood peripheral lymphocytes (HPBL) in the presence and absence of an exogenous metabolic activation system.

 

The micronucleus assay was employed to order to evaluate the aneugenic and clastogenic potential of the test substance. Dimethyl sulphoxide was used as a vehicle for the test substance, based on the test substance and compatibility with the target cells. The test substance was found to be soluble in DMSO at a concentration of approximately 500 mg/mL, which was the maximum concentration tested for solubility. In both the preliminary toxicity and main micronucleus assays, HBPL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9 -activated test system. All cells were harvested 24 -hours after the start of treatment and a minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

 

A preliminary toxicity assay was performed to establish the dose range for testing in the micronucleus test. The doses tested ranged from 0.194 -1940 µg/mL (10 mM). There was substantial cytotoxicity [50 -60% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 194 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and dose levels ≥ 58.2 µg/mL in the non-activated 24 -hr exposure group. Based on the preliminary results, the dose range selected for the micronucleus assay was 5 -100 µg/mL for the non-activated and S9 -activated 4 -hour exposure groups and 1 -60 µg/mL for the non-activated 24 -hour exposure group.

 

In the initial micronucleus assay, substantial cytotoxicity was seen at doses ≥ 60 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and at doses ≥ 35 µg/mL in the non-activated 24 -hour exposure group. The highest dose analysed under each treatment condition caused a 50 -60% reduction in CBPI, which met the dose limit, as recommended by the assay testing guidelines. The induction of micronuclei in the vehicle controls were found to be outside of the historical solvent control range, and therefore a repeat micronucleus assay was performed with a dose range of 10 -70 µg/mL in the non-activated and S9 -activated 4 -hour exposure groups and 2.5 -60 µg/mL in the non-activated 24 -hour exposure group. Substantial cytotoxicity was also observed in the repeat micronucleus assay at dose levels ≥ 65 µg/mL in the non-activated 4 -hour exposure group, 70 µg/mL in the S9 -activated 4 -hour exposure groups and at 60 µg/mL in the non-activated 24 -hour exposure group. Similarly to the initial assay, a 50 -60% reduction in CBP1 was observed at the highest dose analysed under each treatment condition, which met the dose limit, as recommended by the assay testing guidelines.

 

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p>0.05, Fisher's Exact test). The positive and negative control results were comparable to historical data, indicating that all criteria for a valid assay were met.

 

Under the conditions of the assay, the test substance (1,4-Dimethoxy-2-tert-butylbenzene) was determined to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

 

A Key OECD Guideline 490in vitromammalian cell assay (Citoxlab Hungary Ltd, 2019; K = 1) was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test material (2-tert-butyl-1,4-dimethoxybenzene) 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). DMSO was used as vehicle in the study and the following test material concentrations were examined in the mutation assays:

 

Assay 1 (3-hour treatment with metabolic activation): 70, 60, 50, 40, 30, 20, 10 and 5 µg/mL;

Assay 1 (3-hour treatment without metabolic activation): 70, 65, 60, 40, 30, 20, 10 and 5 µg/mL;

Assay 2 (3-hour treatment with metabolic activation): 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 µg/mL;

Assay 2, (24-hour treatment without metabolic activation): 55, 50, 45, 40, 30, 20, 10 and 5 µg/mL.

 

In both Assays, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in the absence and presence of metabolic activation.

 

In Assay 1 and 2, following a 3-hour treatment with metabolic activation, marked or excessive cytotoxicity was seen in several concentrations. In Assay 1 with metabolic activation, no cells survived the expression period in the samples of 70 µg/mL concentration. An evaluation was made using data of seven concentrations (concentration range of 60-5 µg/mL) in Assay 1 and eight concentrations (concentration range of 60-2.5 µg/mL) in Assay 2.

 

In Assay 1, with metabolic activation, there was a statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL with the exception of 30 µg/mL, at this concentration the mutation frequency values were slightly below the GEF. Concentration related increase was indicated by the linear trend analysis. Although, at the highest evaluated concentration in Assay 1 (60 µg/mL) incoherence was seen between replicates, but there was no significant difference between the individual mutation frequency values and both values were above the GEF at appropriate degree of cytotoxicity. Some incoherence was also seen between replicates at 10 µg/mL, however the mutation frequency value of this concentration was below the GEF. Reproducibility was observed at further evaluated concentrations. This experiment was considered to be positive.

 

In Assay 2, with metabolic activation there was statistically significant increase in the mutation frequency value of the six highest evaluated concentrations (60-10 µg/mL), the difference between the calculated values and the control exceed the Global Evaluation Factor, GEF (thus showing biological relevance) in the concentration range of 60-20 µg/mL. Concentration related increase was indicated by the linear trend analysis. Although lower degree of cytotoxicity was observed in Assay 2 compared to Assay 1, however reproducibility was observed at the evaluated concentrations and the effect was reproducible between assays. Therefore, this experiment was considered to be positive.

 

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at the higher concentration range. No cells survived the expression period in the samples of 70, 65 and 60 µg/mL concentration. However, no significant cytotoxicity was observed at lower concentrations. An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

In Assay 2, following a 24-hour treatment without metabolic activation, excessive or marked cytotoxicity of the test item was observed at higher concentrations. No cells survived the expression period in the samples of 55 and 50 µg/mL concentration. An evaluation was made using data of five concentrations (concentration range of 40-5 µg/mL). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No concentration related increase was indicated by the linear trend analysis. This experiment was considered to be negative.

 

The experiments were performed using appropriate untreated, negative (vehicle/solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle/solvent) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, a reproducible mutagenic effect of the test material (2-tert-butyl-1,4-dimethoxybenzene) was observed in the presence of a metabolic activation system, and no mutagenic effect was observed in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Therefore, the test material was considered to be genotoxic.

 

Note: The final report of the above OECD 490 study is currently awaited from the CRO. A spontaneous update of this dossier will be undertaken by the registrant upon receipt of all pending study reports from the CRO.

A supporting study (Gentronix Limited, 2014; K = 2) was conducted to assess the genotoxicity and cytotoxicity of the test substance (1,4-Dimethoxy-2-tert-butylbenzene (Vetimoss)) using the BlueScreen HC genotoxicity screening assays, following a protocol with and without metabolic activation. Vetimoss was combined with 100% DMSO to give a 1250 mM stock solution. The highest concentration of Vetimoss tested was at 971 µg/mL.

 

The BlueScreen HC assay was performed in a 96 -well microtitre plate without metabolic activation (-S9). A dilution series of the test substance was generated in the microtitre plate. A standard genotoxic compound (4 -nitroquinoline 1 -oxide) was employed as a positive control and was added as an intra-plate quality control check to the microtitre plate at concentrations of 0.5 µg/mL and 0.125 µg/mL. The test system used was a genetically modified strain of cultured human lymphoblastoid TK6 cells (GLuc-T01) with a patented reported system, which explots the proper regulation of the GADD45a gene. This target gene mediates the adaptive response to genotoxic stress. The BlueScreen HC S9 assay was performed in the same cultured TK6 cell strain in a 96 -well microtitre plate as the first assay, but instead in the presence of metabolic activation (+S9). The test material was incubated with the test cells in the presence of 1% (v/v) Aroclor-1254 induced rat liver S9 fraction mix in Exposure Medium at 37°C (5% CO2, 95% humidity) for 3 hours. Following this incubation period, the cells were washed in a phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C (5% CO2, 95% humidity). Cyclophosphate was employed as positive control, tested at concentrations of 25 µg/mL and 5µg/mL. A coelenterazine substrate was added to the microplate wells in both assays just prior to measurement, which reacts with GLuc resulting in luminescence. Cell exposure to a genotoxic compound increases GLuC expression that can be quantified by luminescence detection. Cytotoxicity was measured by lysis of cells and the addition of a fluorescent DNA binding stain, followed by the assessment of the resulting fluorescence. Fluorescence is proportional to cell proliferation, which is lowered by toxic analytes, and luminescence intensity is proportional to the activity of the cell's DNA repair system, which is increased by genotoxic analytes. The quantity of luminescence is normalised to the fluorescence signal to correct for the variation in cell yield caused by cytotoxicity.

 

Based on the results of the study and the evaluation criteria the test substance Vetimoss was negative for genotoxicity at the concentrations tested without metabolic activation, and positive for genotoxicity with metabolic activation. The test material 1,4-Dimethoxy-2-tert-butylbenzene (Vetimoss) was negative for cytotoxicity in the absence of S9 mix and positive in the presence of the S9 mix after a 3-hour exposure.

Justification for classification or non-classification

Based on the results observed in the mouse lymphoma assay, 2-tert-butyl-1,4-dimethoxybenzene meets the criteria to be classified as a Category 2 mutagen (H341: Suspected of causing genetic defects) under EU Regulation (EC) No 1272/2008 (CLP).