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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-tert-butyl-1,4-dimethoxybenzene
EC Number:
244-216-5
EC Name:
2-tert-butyl-1,4-dimethoxybenzene
Cas Number:
21112-37-8
Molecular formula:
C12H18O2
IUPAC Name:
2-tert-butyl-1,4-dimethoxybenzene
Test material form:
liquid
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)

Method

Target gene:
Histidine or tryptophan locus
Species / strain
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.
Controls
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

Results and discussion

Test results
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).