1,4-Dioxaspiro[4.5]decane, 8-[4-(4-methylphenyl)-3-cyclohexen-1-yl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 07, 2003 - October 22, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Communities L136, 8. June 2000

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:

603-101-3

EC Number:

603-101-3

Cas Number:

125962-78-9

Molecular formula:

C21 H28 O2

IUPAC Name:

603-101-3

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 6

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

uvrA pkM101

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- Source of S9: Liver S9 from Aroclor 1254-pretreated rats

- Method of preparation of S9 mix: Male Wistar, HSdCpb:Wu rats aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of 0.15 M KCI per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about + 4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C

- Volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 10% (1st test series) or 30% (2nd test series S9) in 3.11 - 3.1316 mL final culture medium

- Quality controls of S9: Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 2.81, 8.89, 28.1, 88.9, 281, 889 and 2810 µg/plate (with and without S9).
2nd series: 8.89, 28.1, 88.9, 281 and 889 μg/plate (with and without S9).
In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.

Vehicle / solvent:

Tetrahydrofuran (test item), H2O (daunomycin), DMSO (N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-aminoanthracene, benzo[a]pyrene), ethanol (9-amonoacridine)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

For TA 100 (5 µg/plate), TA 1535 (10 µg/plate) and E.coli WP2 uvrA (5 µg/plate), without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

For TA 102, (10 µg/plate), with S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For TA 98 (2 µg/plate), 100 (2 µg/plate), 1535 (2 µg/plate), 1537 (5 µg/plate) and E.coli WP2 uvrA (10 µg/plate), with S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

For TA 102, without S9, 200 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For TA 1537, without S9, 50 µg/plate

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin

Remarks:

For TA 98, without S9, 4 µg/plate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Plate incorporation method

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Scoring of revertant colonies using an Artek MiniCount colony counter or manually

Evaluation criteria:

See "Any other information on materials and methods incl. tables".

Statistics:

n.a.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 281 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation: See "Any other information on materials and methods incl. tables".

Ames test:
- Signs of toxicity: No signs of toxicity were observed.
- Individual plate counts: See "attached background material".
- Mean number of revertant colonies per plate and standard deviation: See "attached background material"

HISTORICAL CONTROL DATA
See "Any other information on materials and methods incl. tables".

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Executive summary:

The purpose of this assay according to OECD guideline 471 was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man. The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test material was dissolved in tetrahydrofuran and tested at concentrations ranging from 2.81 to 2810 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations of >= 281 μg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.