2H-Pyran-2-one, tetrahydro-5-pentyl-

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:

From 2 to 23 November 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2006

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 2004-12-16 / Signed on 2005-03-11.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(5R)-5-pentyloxan-2-one

Molecular formula:

C10 H18 O2

IUPAC Name:

(5R)-5-pentyloxan-2-one

Constituent 2

Reference substance name:

(5S)-5-pentyloxan-2-one

Molecular formula:

C10 H18 O2

IUPAC Name:

(5S)-5-pentyloxan-2-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: room temperature.

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

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% v/v S9 fraction; S9 fraction was prepared from liver homogenates of Sprague Dawley rat induced with Aroclor 1254

Test concentrations with justification for top dose:

Mutagenicity test:
Main test: 20, 60, 200, 600 and 2000 μg/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: 20, 60, 200, 600 and 2000 μg/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: Ethanol
- Justification for choice of solvent/vehicle: For the first bacteriostatic activity control, the highest dose studied is 5000 μg/plate. A solution at 200 mg/mL is prepared with ethanol.
For the two other bacteriostatic activity controls and mutagenic assays, the highest dose studied is 2000 μg/plate. A solution at 80 mg/mL is prepared with ethanol.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: cis-Platinum (II) Diammine Dichloride, 1 µg/plate for Escherichia coli cis WP2 (uvr A-) (pKM101). Beta-Propiolactone, 50 µg/plate for TA 1535.

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine & Dimethyl-benzanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Strains of Salmonella typhimurium and Escherichia coli are obtained from "Unité de Programmation Moléculaire et Toxicologie Génétique" (CNRS UA 144) (Institut Pasteur).

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: Pre-incubation assay where the test substance was preincubated with the test strain, and 500 μL of S9-mix fraction for 1 hour at 37° C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.
- Exposure duration: Plates were incubated at 37 °C over a 48 hour period

NUMBER OF REPLICATIONS: 3 plates/dose for all groups

DETERMINATION OF CYTOTOXICITY
- Method: reduced number of spontaneous reversions indicating the bacteriostatic activity.

- OTHER:
- Checking strains: The genotype of bacterial strains was checked for Histidine and tryptophan requirements; Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains; Ampicillin resistance for the strains which have the pKM 101 plasmide; Δuvr B mutation i.e. U.V.B sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A sensitivity for Escherichia coli; Spontaneous revertant rate
- After a 48 hour incubation period at 37° C, revertant colonies per plate are counted (n = 3).
- Sterility tests: The highest concentration and dilutions of the test substance (100 μL) were tested for sterilit

Evaluation criteria:

The result of the test is considered as negative if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101) strains with and/or without metabolic activation.
The validity criteria are as follows :
- bacteriostatic activity of the highest concentration shall be equal to or less than 75 %, - the spontaneous reversion rate of the absolute negative control shall comply with the laboratory’s historical control data.
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with laboratory’s historical control data.
The result of the test is considered positive if a concentration – related increase is obtained in one, or several of the 5 strains, with and/or without metabolic activation; a mutagenic effect is taken into account for a given concentration of the test substance if the number of revertant colonies is at least two fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

bacteria, 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:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: None
- Precipitation: None
- Other confounding effects: None


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 of the test facility.

OTHERS:
- 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 condition, the test material is not mutagenic in the presence and absence of 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 item diluted in ethanol at the following concentrations both in the presence and absence of metabolic activation system (10% v/v S9).

 

Main test: 20, 60, 200, 600 and 2000 μg/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: 20, 60, 200, 600 and 2000 μg/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 showed valid ratios (R) above 2.5.

 

Cytotoxic effect was observed at 5000 µg/plate. 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 condition, the test material is not 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.