4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

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:

24-31 March 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation. However, the isomers ratio is not reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Directive 2000/32/EC.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2007-07-18 / Signed on 2007-07-26

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (2R,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2S,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Molecular formula:

C11H22O2

IUPAC Name:

Reaction mass of (2R,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2S,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Constituent 2

Reference substance name:

Reaction mass of (2S,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2R,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Molecular formula:

C11H22O2

IUPAC Name:

Reaction mass of (2S,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2R,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Test material form:

liquid

Details on test material:

- Physical state: Colourless to pale yellow, limpid/translucent liquid

Specific details on test material used for the study:

- Test item was considered at 100% for the study.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% v/v S9 fraction; rat liver microsome fraction (S9)

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.
Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% ethanol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TESTER STRAINS: Strains of salmonella typhimurium and E. Coli were obtained from Moltox. Stock plates were stored at about -80 °C and master plates at about 4 °C.
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 37 °C for 72 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Inhibition of growth by the test item suggests a cytotoxic activity. A cytotoxic effect at high concentrations only would require lower concentrations of the test item in the main test. Cytotoxic activity at lower concentrations could rule out the bacterial reverse mutation test for the evaluation of mutagenicity.

OTHER:
- After an incubation of about 72 hours at about 37ºC, the number of colonies per plate was counted. Data are presented as the number of colonies present per plate (mean ± standard deviation) per plate. The R ratio is calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
- Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. For this purpose, the highest concentration of test item and a probe of the S9 mix were added respectively to top agar preheated at about 45 °C and poured over plates. The plates were then incubated for about 72 hours at about 37 °C. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix.

Rationale for test conditions:

Tested up to limit concentration

Evaluation criteria:

Several criteria were used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or reading frame shifts in the genome of either Salmonella Typhimurium and/or Escherichia Coli.
Negative results from the test indicate that under the test conditions, the test item is not mutagenic and non-promutagenic in the tested species.

An independent confirmation test was performed with the test item. If the first assay is positive, the confirmation test is performed in the same manner. If the first assay is negative, the confirmation test is performed according to the preincubation procedure.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY TEST
- No cytotoxic effect was observed.

MUTAGENICITY TEST
- No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation.
− No dose response was observed in any of the tested bacterial strains.

HISTORICAL CONTROL DATA
- Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. See "Attached background material" section for further details.

OTHER
Sterility test: Sterility test showed no contamination during the study.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2(pKM101) were exposed to the test substance diluted in ethanol (1%) at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

Main test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the direct plate incorporation method.

Confirmation test: 0.05, 0.15, 0.5, 1.5 and 5 μL/plate in TA 98, TA 100, TA 1535, TA 1537 and WP2(pKM101) with and without S9 under the pre-incubation method.

 

Negative and positive control groups were also included in mutagenicity tests.

 

Positive and negative controls showed absolute numbers of revertant colonies comparable to historical data. All positive controls performed showed valid ratios (R) above 2.5.

 

No cytotoxic effect was observed. No concentration of the test item showed a biological significant increase (R ≥ 2.5) of the number of revertant either with or without S9 metabolic activation. No dose response was observed in any of the tested bacterial strains.

 

Under the test conditions, the test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.