Pentadecan-15-olide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2001-07-23 to 2001-09-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for justification).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP (inspection date: 2000-02-28)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: (E)-oxacyclohexadec-12-en-2-one; (E)-oxacyclohexadec-13-en-2-one; a) (Z)-oxacyclohexadec-(12)-en-2-one and b) (Z)-oxacyclohexadec-(13)-en-2-one

EC Number:

422-320-3

EC Name:

A mixture of: (E)-oxacyclohexadec-12-en-2-one; (E)-oxacyclohexadec-13-en-2-one; a) (Z)-oxacyclohexadec-(12)-en-2-one and b) (Z)-oxacyclohexadec-(13)-en-2-one

IUPAC Name:

422-320-3

Test material form:

other: viscous liquid

Details on test material:

- Name of test material (as cited in study report): ST 12 C 00
- Physical state: colourless viscous liquid
- Storage condition of test material: room temperature under nitrogen, in the dark

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: L5178Y TK+/- 3.7.2c mouse lymphoma cell line obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: yes, stored in liquid nitrogen at -196°C.
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, by culturing in THMG medium for 24 hours. For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

PB/betaNF S9: S9-mix

Test concentrations with justification for top dose:

Preliminary toxicity test using 36.88 to 2360 µg/mL
Exp 1: without S9-mix, six doses levels 2.5 to 40 µg/mL
Exp 1: with S9-mix, six doses levels 5 to 80 µg/mL
Exp 2: without S9-mix, six doses levels 5 to 50 picog/mL
Exp 2: with S9-mix, six doses levels 10 to 70 picog/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

800 µg/mL in Exp 1 and 150 µg/mL in Exp 2

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

at 2.5 µg/mL

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 hours in Experiment 1, 3 hours with metabolic activation cultures and 24 hours without metabolic activation in Experiment 2
- Expression time (cells in growth medium): two days
- Selection time (if incubation with a selection agent): 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: 2, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: the cells were counted and processed to give 1* 10exp6 cells/mL in 10 ml aliquots in R10 medium

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - After the preliminary toxicity test, the cultures were incubated and sub-cultured after 24 hours by counting and diluting to 2 x 10EXP5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate suspension growth values (SG). The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth Value (%RSG).

OTHER
- Other: Determination Plating Efficiency: Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the plating efficiency (P.E.) was calculated using the zero term of the Poisson distribution [P(O)] method.
- Other: Determination of Calculation of Mutation Frequency - M.F. per survivor = [(-In P(O) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium. The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Evaluation criteria:

For a test material to give a 'significant' result then 2 or more of the following criteria should be met:
i) A greater than three-fold increase in the mutant frequency per survivor over the negative control value.
ii) A dose-related increase in the mutant frequency per survivor.
iii) An increase in the absolute number of mutants.
A test material may be reported as equivocal if only one of the above criteria is met. Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Statistics:

Statistics will be found in the attached tables.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no change in pH at maximum dose level
- Effects of osmolality: 10 mM maximum dose level used in preliminary toxicity test
- Precipitation: not seen

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10EXP5 cells/ml, using a 3-hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10EXP5 cells/ml using a continuous 24-hour exposure without S9. The dose range used in the preliminary toxicity test was initially 36.88 to 2360 ug/ml. However, the test material proved to be very toxic and the preliminary toxicity test was repeated using the. dose range 0.625, 1.25, 2.5, 5, 10, 20 and 40 ug/ml in the 3-hour exposure group, both with and without S9, and 1.25, 2.5, 5.0, 10, 20, 30 and 40 ug/ml in the 24-hour exposure without S9. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a coulter counter and then serially diluted to 10 cells/ml.

COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle control mutation frequencies were within historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

(All the tables mentioned in this section will be found as attachment)

Preliminary Toxicity Test The dose range of the test material used in the preliminary toxicity test was initially 36.88 to 2360 µg/mL. However, due to excessive toxicity it was repeated using the dose range of 0.625 to 40 µg/mL. The results of the Day 0 Relative Suspension Growth (RSG) were as follows:

Dose (ug/ml)		% RSG (-S9) 3 -hour Exposure	% RSG (+S9) 3 -hour Exposure	% RSG (-S9) 24-hour exposure
0		100	100	100
(1.25)^a	0.625	101	113	91
(2.5)^a	1.25	118	123	96
(5)^a	2.5	119	108	98
(10)^a	5	114	107	98
(20)^a	10	117	115	90
(30)^a	20	102	112	93
(40)^a	40	3	101	55

^a=24 -h exposure dose levels

It can be seen, in both of the groups in the absence of metabolic activation (S9), that there was a marked reduction in the %RSG value of cells treated with the test material at the maximum dose level when compared to the concurrent vehicle controls indicating a steep toxicity curve. In the presence of metabolic activation, there was no reduction in %RSG. However in the initial preliminary toxicity test (data not shown) there was no survival at dose levels greater than 36.88 µg/mL. No precipitate of test material was observed in any dose level up to 40 µg/mL. In the mutagenicity test the maximum dose was limited by the toxicity of the test material.

Mutagenicity Test

Experiment 1 The results of the Day 0 and Day 2 microtitre plate counts and their analysis are presented in Tables 1 to 6. The test material showed some evidence of toxicity (reduced %RSG values) at the upper two dose levels only. The toxicity dose response curve was observed to be extremely steep. The toxicity observed at 80 µg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, the dose was excluded from the statistical analysis. Toxicity was seen with both positive control substances (Table 2 and Table 5). Neither of the vehicle control mutant frequencies were outside the range of 25 to 150 x 10EXP-6 viable cells that is acceptable for L5178Y cells at Safepharm Laboratories. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 2 and 5). The test material did not induce any significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the presence or absence of metabolic activation (Tables 2 and 5). No precipitate of test material was observed at any dose level. The numbers of small and large colonies and their analysis are presented in Tables 3 and 6.

Experiment 2 The results of the Day 0 and Day 2 microtitre plate counts and their analysis are presented in Tables 7 to 12. The 24-hour continuous exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test material. As was seen in Experiment 1 there was evidence of a steep dose-related reduction in the %Relative Suspension Growth values for cultures dosed with the test material in both the absence and presence of metabolic activation. There was no evidence of a reduction in Day 2 (%V) viability, however, there was some evidence of a reduction in relative total growth (RTG), indicating that some residual toxicity had occurred, in both exposure groups. The toxicity observed in the presence of metabolic activation was within the optimum maximum range of 80 to 90%. In the absence of metabolic activation the maximum toxicity observed was just below the optimum range of 80 to 90%. It was considered that the test material had been adequately tested because of the extremely steep dose-response curve. Both positive controls produced reductions in both the %RSG value and Day 2 (%V) viabilities and RTG values (Tables 8 and 11). Neither of the vehicle control mutant frequencies were outside the acceptable range of 25 to 150 x 10EXP-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 8 and 11). The test material did not induce any statistically significant or dose-related increases in the mutant frequency per viable cell in either the absence or presence of metabolic activation (Table 8 and 11). No precipitate of test material was observed at any dose level tested. The numbers of small and large colonies and their analysis are presented in Tables 9 and 12.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, L5178Y mouse lymphoma cells were exposed to the test material diluted in DMSO at 6 dose levels, in duplicate in the presence and absence of metabolic activation (S9-mix). The entire experiment was repeated to confirm the result of the first experiment. Three-hour exposures were used both with and without activation (S9) in Experiment 1. In Experiment 2 the exposure time without activation was increased to 24 hours. The dose range of test material, plated out for expression of mutant colonies, was selected on the results of a preliminary toxicity test and was 2.5 to 40 µg/mL in the absence of S9 and 5 to 80 µg/mL in the presence of S9 for the first experiment. For the second experiment the dose range was 10 to 70 µg/mL with activation and 5 to 50 µg/mL without activation. The maximum dose level used was limited by the presence of precipitate. A precipitate of the test material was not observed at any dose level used in the main study.

 

The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus.

 The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

 

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

The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for justification).