2,6,6-trimethoxy-2-vinyltetrahydropyran

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 November 2003 and 12 December 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: US EPA (TSCA) OPPTS harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6,6-trimethoxy-2-vinyltetrahydropyran

EC Number:

230-983-3

EC Name:

2,6,6-trimethoxy-2-vinyltetrahydropyran

Cas Number:

7392-19-0

Molecular formula:

C10H18O

IUPAC Name:

2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran

Test material form:

other: Liquid

Details on test material:

Sponsor’s identification: Limetol (batch # 9000523633)
Description: Colourless liquid
Chemical name: 2-ethenyl-2,6,6-trimethyltetrahydropyrane
Purity: 99 % (sum of 2 peaks)
Batch number: 9000523633
Date received: 10 November 2003
Storage conditions: Room temperature in the dark

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 prepared from the livers of male Sprague-Dawley rats weighing ~250 g which had each received daily oral doses of phenobarbitone / β-napthoflavone prior to S9 preparation.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 1: 10, 33.3, 100, 333, 1000, 2500, 5000 µg/plate.
Experiment 2:
1.5, 5, 15, 50, 150 and 500 µg/plate in the absence of S9 mix.
5, 15, 50, 150, 500 and 1500 µg/plate in the presence of S9 mix.

Vehicle / solvent:

Dimethyl suphoxide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene; 1,8-dihydroxyanthraquinone

Details on test system and experimental conditions:

Preparation of test and control materials
The test material was accurately weighed and appropriate dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was therefore not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumina-silicate) i.e. 2 mm pellets with a normal pore diameter of 4E-04 microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials were as follows:
N-ethyl-N’nitro-N-nitrosoguanidine (ENNG): 3 µg/plate for TA100 and 5 3 µg/plate for TA1535.
9-Aminacridine (9AA): 80 µg/plate for TA1537.
Mitomycin C (MMC): 0.5 µg/plate for TA102.
4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98.
In addition, 2-Aminoanthracene (2AA), Benzo(a)pyrene (BP) and 1,8-Dihydroxyanthraquinone (DAN) which are non-mutagenic in the absence of metabolising enzymes, were used in the S9 series of plates and the following concentrations:
2AA: 1 µg/plate for TA100.
2AA: 2 µg/plate for TA1535 and TA1537.
BP: 5 µg/plate for TA98.
DAN: 10 µg/plate for TA102.

Microsomal enzyme fraction
S9 was prepared in-house on 19 July 2003 from the livers of male Sprague-Dawley rats weighing ~ 250 g. These had each orally received three consecutive daily doses of phenobarbitone / β-naphthoflavone (80 / 100 mg/kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames test. The S9 was stored at -196 °C.

S9-mix and agar
The S9 mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65 M KCl / 0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADPH: 2.0 mL
0.1 M NADH: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 12.5 mL
A 0.5 mL aliquot of S9 mix and 2 mL molten, trace histidine supplemented top agar was overlaid on to a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9 mix. This procedure was repeated in triplicate on the day of each experiment.
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution added to each 100 mL of top agar. Vogel-Bonner minimal agar plates were purchased form ILS Ltd.

Test procedure
Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material using both the plate incorporation and pre-incubation methods. The concentrations tester were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The plate incorporation test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten trace histidine supplemented top agar, 0.1 mL of test material formulation, 0.5 mL of S9 mix or phosphate buffer and overlaying onto sterile agar plates of Vogel-Bonner Minimal agar (30 mL / plate). The pre-incubation modification was performed by mixing 0.1 mL of bacterial culture (TA100) 0.1 mL of test material formulation and 0.5 mL of S9mix of phosphate buffer for 20 minutes at 37 °C prior to the addition of 2 mL of molten trace histidine supplemented top agar. Ten doses of the test material and a vehicle control (dimethyl suphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten trace histidine supplemented top agar was over laid onto a sterile nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation test – Experiment 1
Seven concentrations of the test material (10, 33.3, 100, 333, 1000, 2500 and 5000 µg/plate) were assayed in triplicate against each tester strain using the direct plate incorporation method.
Additional dose levels were included to allow for test material induced toxicity, ensuring that a minimum of four non-toxic doses were achieved.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten trace histidine supplemented top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9 mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9 mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation test – Experiment 2 (pre-incubation method)
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was amended following information obtained from the Preliminary Toxicity Test and was 1.5, 5, 15, 50, 150 and 500 µg/plate for hte strains dosed in the absence of S9 and 5, 15, 50, 150, 500 and 1500 µg/plate for hte strains dosed in the presence of S9. The experimental method followed the pre-incubation modification where 0.1 mL of bacterial culture, 0.1 mL of vehicle or test material formulation and 0.5 mL of S9 mix or phosphate buffer were mixed for 20 minutes at 37 °C prior to the addition of 2 mL of molten trace histidine supplemented top agar and plating onto the surface of Vogel-Bonner Minimal agar plates.
The positive and negative controls were plated out using the plate incorporation method in both experiments.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA100 -S9 at and above 1500 µg/plate in the plate incorporation method. A clear toxic response in the pre-incubation method at 150 and 500 µg/plate with and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mutation test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The S9 mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered acceptable. These data are for concurrent untreated control plates performed on the same day as the mutation test.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level employing the direct plate incorporation method. In Experiment 2 however (pre-incubation method) toxicity was noted to all of the tester strains, initially at 150 and 500 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella at any dose level, either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test

The test material exhibited slight toxicity to TA100 (without S9 only) at and above 1500µg/plate employing the plate incorporation method. A clear toxic response was noted employing the pre-incubation method with weakened lawns initially observed at 150 and 500µg/plate in the absence and presence of S9 respectively. The test material formulation and the S9mix used in this experiment were both shown to be effectively sterile.

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) metabolic activation	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100*	110	100	104	117	107	101	126	96	123	78S	101SP
+	TA100*	91	96	117	103	119	98	91	122	118	93	117P
-	TA100**	90	115	94	108	84	88	76	105S	0V	0T	0TP
+	TA100**	111	102	106	101	104	119	119	92	106S	0V	0VP

*: Plate incorporation method

**: Pre-incubation method

S: Sparse bacterial background lawn

V: Very weak bacterial background lawn

T: Toxic, complete absence of bacterial background lawn

P: Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13 / 14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Methods

Salmonella typhimurium strains TA1535, TA1537, TA 102, TA98 and TA100 were treated with the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the additional f a rat liver homogenate metabolising system (19 % liver S9 in standard cofactors). The dose range for the first experiment (plate incorporation) was determined in a preliminary toxicity assay and was 10 to 45000µg/plate. The experiment was repeated on a separate day using an amended dose range (1.5 to 1500µg/plate), fresh cultures of the bacterial strains and fresh test material formulations. The pre-incubation modification was employed for the second experiment.

Additional dose levels were included in both experiments to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved.

 

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level employing the direct plate incorporation method. In Experiment 2 however (pre-incubation method), toxicity was noted to all of the tester strains, initially at 150 and 500µg/plate in the absence and presence of S9 respectively. The test material was therefore tested up to the maximum recommended dose level of 5000µg/plate when employing the direct plate incorporation method (Experiment 1) and the toxic limit when employing the pre-incubated method (Experiment 2). A light, oily in appearance, precipitate was observed at 5000µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion

The test material was considered to be non-mutagenic under the conditions of the test.