(3E)-dec-3-en-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-04-16 to 2009-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21th July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated 30th May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-dec-3-en-2-one

Cas Number:

18402-84-1

Molecular formula:

C10H18O

IUPAC Name:

(3E)-dec-3-en-2-one

Test material form:

liquid

Specific details on test material used for the study:

- Name: AMW-1018
- Purity: 98% w/w
- Batch No: 3D2-2009/01
- Physical state: liquid
- Colour: pale yellow
- Storage conditions: refrigerator 5 +/-3 °C; protected from light keep under nitrogen
- Expiry date: December 2010

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: Mouse Lymphoma L5178Y cells which are heterozygous at the Thymidine Kinase (TK) locus
- Source: BSL stock cultures

MEDIA USED
- RPMI 1640 supplemented with: 9.0 µg/mL hypoxanthine, 15.0 µg/mL thymidine, 22.5 µg/mL glycine and 0.1 µg/mL methotrexate.
The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days.
- Complete Culture Medium:
RPMI 1640 medium supplemented with 15% horse serum, 100U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2mM L-glutamine, 25 mM HEPES, 250 µg/mL amphotericin B
- Treatment Medium:
RPMI 1640 medium supplemented with 3% horse serum (short-term exposure), 7.5% horse serum (long-term exposure), 100U/100 µg/mL penicillin/streptomycin, 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES and 250 µg/mL amphotericin B
- Selective Medium: RPMI 1640 complete culture medium supplemented with TFT (5 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Pre-test for toxicity:
Short term:
with S9 mix
0, 0.005, 0.01, 0.05, 0.10, 0.20, 0.40 mM
without S9 mix
0, 0.005, 0.01, 0.05, 0.10, 0.20 mM
Long-term (24 hours):
without S9 mix: 0.001, 0.005, 0.01, 0.05, 0.1 and 0.2 mM

Main test:
Experiment I:
with S9 mix
0.05, 0.15, 0.22, 0.25, 0.28, 0.31, 0.34, 0.37 mM
without S9 mix
0.005, 0.01, 0.02, 0.05, 0.10, 0.12, 0.14, 0.16 mM

Experiment II:
with S9 mix
0.10, 0.15, 0.19, 0.23, 0.27, 0.31, 0.35, 0.38 mM
without S9 mix
0.0001, 0.0007, 0.004, 0.007, 0.014, 0.028, 0.034, 0.04 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, final concentration 200 µg/mL and 500 µg/mL

Controls 2

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, final concentration 10 µg/mL

Controls 3

Untreated negative controls:

yes

Remarks:

Treatment medium plus S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation, final concentrations 2.5 - 3.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1x10^7 cells suspended in 11 mL RPMI medium with 3% horse serum

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (Experiment I, Experiment II with S9); 24 hours (Experiment II, without S9)
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 11-14 days

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutation frequencies were calculated from the data obtained from cultures used for the coloning efficiency and those used for selection in the following number:
- Mutation frequency= ((-ln [NC/TC (selective medium)])/ (-ln [NC/TC (non selective medium)]) x 800

Rationale for test conditions:

n.a.

Evaluation criteria:

There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency
- biological relevant response (at least 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups
- combined with a positive effect in the mutant frequency, an increased occurence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (1.5 times the ratio of clatogenic controls MMS and/or B[a]P is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guidelines, the biological relevance is the criterion for the interpretation of results. A statistical evaluation of the results is not regarded as necessary. A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

Statistics:

Not required

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment I

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment I, the relative total growth (RTG) was 12.22% and 13.71% for the highest concentrations (0.37 and 0.16 mM) evaluated with and without metabolic activation respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

Experiment II

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II, the relative total growth (RTG) was 9.20% and 13.16% for the highest concentrations (0.38 and 0.04 mM) evaluated with and without metabolic activation respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment II

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II, the relative total growth (RTG) was 9.20% and 13.16% for the highest concentrations (0.38 and 0.04 mM) evaluated with and without metabolic activation respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity: In experiment I, the relative total growth (RTG) was 12.22% and 13.71% for the highest concentrations (0.37 and 0.16 mM) evaluated with and without metabolic activation respectively. In experiment II, the relative total growth (RTG) was 9.20% and 13.16% for the highest concentrations (0.38 and 0.04 mM) evaluated with and without metabolic activation respectively.

Mutagenicity: In experiment I with metabolic activation, all mutant values found were within the historical control data of the test facility BSL BIOSERVICE, no dose-relationship was observed and the mutation frequencies found in the treated groups did not show a biologically relevant increase compared to solvent controls. Without metabolic activation, all mutant values found were within or slightly above the historical control data of the test facility. Even though a slight dose-response relationship was observed, the higher mutant value at 0.16 mM was considered as biologically not relevant due to the lack of mutagenicity. However, this should be verified in an independent repetition experiment.
In experiment II with metabolic activation, some of the mutant values found were within the historical control data of the test facility. Some of the mutant values (at doses of 0.27, 0.31 and 0.38 mM) clearly exceeded the range of historical control data. Two dose groups (0.31 and 0.38 mM) the threshold value of 2 for the mutation factor was slightly exceeded and a slight dose response relationship could be observed. However, since in experiment I no mutagenicity was evaluated up to an RTG of 12.22% these results are considered to be equivocal. In experiment II without metabolic activation all mutant values found up to the dose of 0.0014 mM were within the historical control data of the test facility. At doses from 0.028 mM the data exceeded the historical control range. In addition, in these dose groups the threshold value of 2 for the mutation factor was exceeded and a dose-response relationship could be observed

Relationship of large to small colonies: Colony sizing was performed for the highest concentrations of the test item and for the controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies is an indication for potential clastogenic effects and/or chromosomal aberrations.
In experiment I with metabolic activation in the highest dose groups tested and increased number of small colonies was noted. However, no clear corresponding mutagenicity was found in these dose groups so no clear conclusion could be drawn. To clarify the findings an independent repetition was performed. In experiment I without metabolic activation, all dose groups were considered to be not clastogenic.
In experiment II with metabolic activation, the increases in the number of small colonies noted at doses of 0.31 mM and 0.38 mM showed clastogenicity since in this experiment for these dose groups the threshold value for mutagenicity was exceeded. Without metabolic activation, an increase in small colonies noted at a dose of 0.034 mM suggests clastogenicity since corresponding mutagenicity was found in this dose group.
Ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS) and benzo-a-pyrene (BaP) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and BaP significantly increased the number of small colonies, thus validating the test system.

Any other information on results incl. tables

Table 1: Results experiment I with metabolic activation

Dose (mM)	RSG (%)	RCE (%)	RTG (%)	Mutants/10^6 cells	MF	No. Large Colonies		No. Small Colonies
NC1	94.32	103.5	97.19	121.78	-	84	22
NC2	91.36	106.10	96.93	147.76	-	111	25
S1	100.00	100.00	100.00	162.23	-	98	90
S2	100.00	100.00	100.00	168.06	-	93	31
0.05	97.41	103.05	10.38	142.69	0.86	-	-
0.15	90.97	98.17	89.31	191.81	1.16
0.22	78.42	107.93	84.64	146.18	0.89
0.25	70.58	106.71	75.32	163.45	0.99
0.28	51.20	103.66	53.07	192.50	1.17
0.31	36.61	101.83	37.28	225.79	1.37	95	73
0.34	21.19	100.61	21.32	244.20	1.48	100	73
0.37	13.09	93.29	12.22	315.01	1.91	101	78
BaP 3.5 µg/mL	42.03	103.66	43.57	432.60	2.62	133	132

 Table 2: Results experiment I without metabolic activation

Dose (mM)	RSG (%)	RCE (%)	RTG (%)	Mutants/10^6 cells	MF	No. Large Colonies	No. Small Colonies
NC1	115.95	105.04	121.79	96.88	-	74	28
NC2	115.95	105.04	118.26	88.96	-	78	19
S1	100	100	100	127.92	-	108	13
S2	100	100	100	158.58	-	105	15
0.005	93.55	90.80	84.94	178.64	1.25	-	-
0.01	95.89	99.70	95.60	124.27	0.87
0.02	88.87	104.45	92.83	97.12	0.68
0.05	71.75	93.77	67.28	141.04	0.98
0.10	38.14	96.14	36.67	200.76	1.40
0.12	27.05	97.92	26.49	193.38	1.35	91	54
0.14	18.60	93.18	17.33	234.81	1.64	100	51
0.16	13.96	94.36	13.17	265.61	1.85	90	80
EMS 500 µg/mL	77.55	100.89	78.24	738.31	5.15	dng	dng
MMS 10 µg/mL	86.60	100.30	86.86	388.56	2.71	103	144

 

Table 3: Results experiment II with metabolic activation

Dose (mM)	RSG (%)	RCE (%)	RTG (%)	Mutants/10^6 cells	MF	No. Large Colonies	No. Small Colonies
NC1	11.68	95.91	107.11	168.48	-	107	21
NC2	103.73	100.58	104.33	151.80	-	115	19
S1	100	100	100	138.41	-	111	32
S2	100	100	100	190.20	-	116	23
0.1	97.06	100	97.06	188.45	1.15	-	-
0.15	98.39	94.74	93.22	222.77	1.36
0.19	85.13	95.32	81.15	181.65	1.11
0.23	74.59	99.42	74.15	219.37	1.34
0.27	53.95	95.91	51.74	307.97	1.87
0.31	42.47	85.96	36.51	365.24	2.22	125	61
0.35	21.44	97.66	20.93	246.30	1.50	110	69
0.38	10.42	88.30	9.20	353.79	2.15	135	55
BaP 3.5 µg/mL	42.03	93.74	33.30	1322.40	8.05	183	105

 

Table 4: Results experiment II without metabolic activation

Dose (mM)	RSG (%)	RCE (%)	RTG (%)	Mutants/10^6 cells	MF	No. Large Colonies	No. Small Colonies
NC1	141.19	95.43	134.73	179.57	-	119	22
NC2	120.38	92.57	111.44	176.43	-	115	14
S1	100	100	100	122.40	-	96	23
S2	100	100	100	117.46	-	92	23
0.0001	100.59	98.86	99.44	162.56	1.36	-	-
0.0007	97.97	99.43	97.41	115.30	0.96
0.004	98.53	95.43	94.03	163.74	1.37
0.007	80.10	95.43	76.44	153.03	1.28
0.014	61.51	95.43	58.70	177.95	1.48
0.028	33.36	89.71	29.93	340.70	2.84	141	57
0.034	20.43	85.71	17.51	435.14	3.63	104	112
0.04	15.05	87.43	13.16	495.78	4.13	132	109
EMS 200 µg/mL	82.51	80.57	66.48	1172.73	9.78	-	-
MMS 10 µg/mL	54.50	52.57	28.65	1986.63	16.57	123	185

 

NC Negative Control; S Solvent control; RSG Relative Suspension Growth; RCE Relative Cloning Efficiency; RTG Relative Total Growth; MF (mutation frequency); dng data not given; BaP Benz(a)pyrene; EMS = ethyl methane sulfonate; MMS = methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 3-decen-2-one is considered to be mutagenic in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence of metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay using the Thymidine Kinase Gene conducted according to OECD Guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to (3E)-dec-3-en-2-one (98% purity) for 4 hours at concentrations of 0.05, 0.15, 0.22, 0.25, 0.27, 0.31, 0.34, 0,37 mM with S9 metabolic activation and at concentrations 0.005, 0.01, 0.02, 0.05,0.10,0.12, 0.14 and 0.16 mM without S9 metabolic activation in experiment I. In experiment II (4-hour exposure, with S9) the following concentrations were tested: 0.10, 0.15, 0.19, 0.23, 0.27, 0.31, 0.35 and 0,38 mM. Experiment II without S9 metabolic activation was performed as a 24-hour long-term exposure assay with the following concentrations tested: 0.0001, 0.0007, 0.004, 0.007, 0.014, 0.028, 0.034 and 0.4 mM. The selection of the concentrations used in the main experiment was based on data from the pre-experiment.

No precipitation of the test item was noted in the experiments. Growth inhibition was observed in experiments I and II with and without metabolic activation. In experiment I, the relative total growth (RTG) was 12.22% and 13.71% for the highest concentrations (0.37 and 0.16 mM) evaluated with and without metabolic activation respectively. In experiment II, the relative total growth (RTG) was 9.20% and 13.16% for the highest concentrations (0.38 and 0.04 mM) evaluated with and without metabolic activation respectively.

In experiment I (both with and without metabolic activation) no biologically relevant increase in mutants was found after treatment with the test item. A second experiment was performed to confirm these results. In experiment II with metabolic activation, the effect of the test item on mutation frequency was considered equivocal. In experiment II without metabolic activation, a biologically relevant increase in mutants was found after treatment with the test item. Additionally, a dose-response relationship was observed, and colony sizing showed clastogenic effects induced by the test item under the experimental conditions (without metabolic activation). The positive controls showed distinct and biologically relevant effects in mutation frequency, thus validating the test system.

In conclusion, in the described test under the experimental conditions reported 3-decen-2-one is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y without metabolic activation.