5-hept-6-enyloxolan-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to the maximum concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April to 26 May 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. The substance is adequately identified. Therefore full validation applies.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

LEMI operating procedure MB08/45, version 1997

Deviations:

yes

Remarks:

2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. No information on the characterisation of S9 with a mutagen that requires metabolic activation by microsomal enzymes has been included in the report

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

Specific details on test material used for the study:

- Date of reception: 19 April 2005

Target gene:

Salmonella typhimurium histidine (his) reversion system and the Escherichia coli tryptophan (trp) reversion system

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:

Type and composition of metabolic activation system:
- source of S9: MOLTOX TM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 -USA)
- method of preparation of S9 mix : not detailed
- concentration of S9 in the final culture medium: S9-fraction 10% v/v was prepared from liver homogenates of Sprague Dawley rat induced with Aroclor 1254

Test concentrations with justification for top dose:

Cytotoxicity test (plate incorporation): 50, 150, 500, 1500 and 5000 μg/plate, with or without metabolic activation in S. typhimurium TA 100.
Mutagenicity test:
- Assay No. 1 (plate incorporation):
50, 150, 500, 1500 and 5000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)
Assay No. 2 (pre-incubation): 50, 150, 500, 1500 and 5000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Test substance preparation:
The highest concentration studied is 5 000 μg/plate. A solution at 500 mg/mL is prepared with acetone (Merck - Ref 14 - Lot K13208414). The test substance is stable in solvent used, under similar assay conditions, according to the sponsor.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: Diammine Dichloride: 1 µg/plate for E.Coli. Beta propiolactone: 50 µg/plate for TA1535

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

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 medium; 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 sterility.
- Data were presented as the number of revertant colonies (mean ± standard deviation) per plate. The following ratio was calculated: R = Number of revertant colonies in the presence of the product / Number of revertant colonies in the absence of the product.

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

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:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

BACTERIOSTATIC ACTIVITY CONTROL (STRAIN TA 100)
A bacteriostatic activity of 60.5 % is observed in the presence of the test substance 5 000 μg/plate (the maximun acceptable level is 75 %). The test substance is tested at the following doses 5 000, 1 500, 500, 150 and 50 μg/plate.

HISTORICAL CONTROL DATA
- See historical control table in "Attached background material" section

MUTAGENICITY RESULTS
There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (5000, 1500, 500, 150, 50 μg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).
Results were confirmed in a second independent experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of the test substance at 5 000 μg/plate we observe for all bacterial strains a significant decrease of the number of spontaneous reversions which confirmed the bacteriostatic activity observed at this dose.

OTHERS:
Sterility test did not show any bacterial growth in presence of all substance doses / S9 mix.
Genotoxicity testing: see tables in "Attached background material" section.

Conclusions:

Under the test condition, test substance is not 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 in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to test substance at the following concentrations. 

 

Assay No. 1 (plate incorporation method): 50, 150, 500, 1500 and 5000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-)

Assay No. 2 (pre-incubation method): 50, 150, 500, 1500 and 5000 μg/plate, with or without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (uvr A-) 

 

Metabolic activation system used in this test was10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

In the presence of the test substance at 5 000 μg/plate we observe for all bacterial strains a significant decrease of the number of spontaneous reversions which confirmed the bacteriostatic activity observed at this dose.

There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental historic values obtained in the laboratory..

There is no evidence of any increase in the number of revertant colonies in the presence of the test substance (5000, 1500, 500, 150, 50 µg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101).

Results were confirmed in a second independent experiment.

 

Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in these bacterial systems.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   LEMI, 2005	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	up to limit concentration	-S9: non mutagenic +S9: non mutagenic

 

Gene mutation Assay:

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance(See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test conditions, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The substance is therefore considered as non-mutagenic according to the Ames test.

Justification for classification or non-classification

 

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the CLP and to the GHS.