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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of (3E)-dec-3-en-2-one was investigated in two in vitro genotoxicity tests (OECD 471 and OECD 476). There was no increase of mutant colonies compared to the negative control observed in all strains in the bacterial reverse gene mutation test conducted according to OECD test guideline 471. In the in vitro mammalian cell gene mutation assay conducted according to OECD test guideline 476, the substance was considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y without metabolic activation. Based on these contradictory results, additional in vivo tests were conducted to further assess the genotoxic potential of the test substance.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21th July 1997

Deviations:

no

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

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

Target gene:

Thymidine kinase (TK)

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

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

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-04-27 to 2009-07-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 30th May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name: AMV-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

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
MOLTOX, INC., NC 28607, USA (TA98, TA 100, TA1535, TA 1537 and TA102).

MEDIA USED
- Type and identity of media:
- Nutrient media: 8 g Nutrient Broth, 5 g NaCl, 125 µL ampicillin (for TA98, 100 and 102)
- Vogel-Bonner Medium E: 2% glucose, 10 g/L MgSO4 x 7H2O, 100 g/L citric acid, 175 g/L NaNH4HPO4 x 4H2O, 500 g/L K2HPO4
-Overlay Agar: 7 g/L Agar Agar, 6 g/L NaCl, 10.5 mg/L L-histidine x HCl x H2O, 12.2 mg/L Biotin

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see box "Any other information on materials and methods incl. tables"; Results: see box "Any other information on results incl tables", Table 2). Two independent main experiments were performed with the following concentrations: 0.0316, 0.100, 0.316, 1.0, 2.5, and 5.0 μL/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: purified water (A. dest.)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A. dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 (10 µg/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A. dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

TA98 (10 µg/plate), TA1537 (40 µg/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A. dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA102 ( 1µL/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

A. dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

All strains (2.5 µg/plate), except TA102 (10 µg/plate), with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), pre-incubation (Experiment II)

EXPERIMENTAL PERFORMANCE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension, 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 µL of the test solution was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

DURATION
- Pre-incubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "B" in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs.
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA 98,TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Evaluation criteria:

Criteria of Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (see box “Any other information on material and methods incl. tables”, Table 1)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Remarks:

Experiment 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment 2 toxic effects of the test item were noted in all tester strains at a dose of 5.0 μL/plate (without metabolic activation) and in tester strain TA102 at a dose of 5.0 µL/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. No precipitation of the test item was observed in any tester strain used in experiments I and II (with and without metabolic activation). No cytotoxic effects of the test item were noted in any of the five tester strains used up to the highest concentration evaluated (with and without metabolic activation) in experiment I. In experiment II toxic effects of the test item were noted in all tester strains at a dose of 5.0 μL/plate (without metabolic activation) and in TA102 at a dose of 5.0 µL/plate (with metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 1535 at a dose of 0.316 μL/plate (without metabolic activation) was regarded as not biologically relevant due to the lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 3-decen-2one at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Table 2: Pre-experiment results

Substance	Dose (µL/plate)	TA98				TA100
		Mutation Factor*				Mutation Factor*
		without S9		with S9		without S9		with S9
Solvent Control (A. dest.)		1.0		1.0		1.0		1.0
4-NOPD	10.0 µg	19.2		-		-		-
NaN3	10.0	-		-		11.7		-
2-AA	2.50 µg	-		105.8		-		19.0
Test Item	0.00316	1.2		0.9		1.0		0.8
	0.0100	1.3		1.1		1.1		0.9
	0.316	1.2		1.2		1.1		0.8
	0.100	1.2		1.3		1.1		0.9
	0.316	1.2		1.2		1.2		0.8
	1.0	1.2		1.0		1.1		0.8
	2.5	0.8		1.3		1.1		0.8
	5.0	1.0		1.3		0.9		0.9

* Mutation factor = mean revertants (test item) / mean revertants (solvent control)

Conclusions:

Under the experimental conditions reported, the test item did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA98, TA100, TA102, TA1535 and TA1537 of Salmonella typhimurium were exposed to 3-decen-2-one (98% purity) in A. dest. at concentrations of 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Genetic toxicity in vivo

Description of key information

The mutagenic potential of (3E)-dec-3-en-2-one was investigated in three in vivo genotoxicity tests (OECD 474, OECD 486 and OECD 489). The test substance was tested negative in all three in vivo tests, namely in the in vivo Micronucleus Test (according to OECD 474), the Unscheduled DNA Synthesis Assay (according to OECD 486) and in the in vivo Comet Assay (according to OECD 489). In a weight of evidence approach, based on the results from all in vitro and in vivo genotoxicity tests, it can be concluded, that the substance is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-08-21 to 2016-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

- Name: AMV-1018
- Purity: 99.5% w/w
- Batch No: HA-2013/02
- Physical state: liquid
- Colour: pale yellow
- Storage conditions: under nitrogen, room temperature
- Expiry date: September 2016

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: Preliminary toxicity test: Day 1 Males and females ca. 47-54 days old; Comet test: Day 1 Males ca. 48-55 days old
- Weight at study initiation: Preliminary toxicity test: Males weighed between 178 g to 185 g, Females weighed between 134 g to 143 g; Comet test: Males weighed between 184 g to 204 g
- Assigned to test groups randomly: yes
- Housing: Each group was kept with the sexes separated in cages
- Diet (e.g. ad libitum): pelleted Harlan Teklad 2014C diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period:Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12 /12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test substance were prepared in purified water obtained in house prepared on 27 Apr 15 and 27 Jul 15, expiry 27 Apr 2020 and 27 Jul 2020 respectively. An accurately measured amount of purified water was added to an accurately measured quantity of test material and magnetically stirred for 5 minutes. Ethyl Methanesulphonate (EMS) obtained from Sigma, batch number BCBM2272V was used as the positive control compound. A solution was prepared using purified water at a concentration of 20 mg/mL just prior to administration. All animals were dosed orally by gavage using a dose volume of 10 mL/kg.

Duration of treatment / exposure:

The test substance was administered on two occasions approximately 24 hours apart

Frequency of treatment:

Two times, 24 hours apart

Post exposure period:

3 hours after the second dose, the animals were killed. In addition, animals in the positive control group were killed 3 hours after a single dose.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

6 males for the test item dosage and negative control groups; three males for the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulphonate
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/mL equivalent to 200 mg/kg/day

Tissues and cell types examined:

Liver and duodenum

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary toxicity test was performed to identify the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia up to a standard limit of 2000 mg/kg/day. From the results obtained in the preliminary toxicity study dose levels of 500, 1000 and 2000 mg/kg/day were used for the comet test.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Following dosing, the animals were examined regularly during the working day for a period of 24 hours after the first dose and for 3 hours after the second dose any mortalities or clinical signs of reaction during the experiment were recorded. Animals from the vehicle control and test substance groups were killed 3 hours after administration of the second dose. In addition, animals in the positive control group were killed 3 hours after a single dose. Animals were killed by exposure to rising levels of carbon dioxide. Sections of the liver and duodenum were placed into ice cold mincing solution all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method. Comet slides were prepared from all cell suspensions. Sections of the liver and duodenum were stored in 10% buffered formalin and stored within Genetic Toxicology. On finalisation of the study report, these tissues (if not required) will be disposed of. These tissues and slides will be retained and will only be examined in the event that the Comet result is positive.

DETAILS OF SLIDE PREPARATION:
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer. For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75μL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored refrigerated overnight prior to electrophoresis.

METHOD OF ANALYSIS:
Electrophoresis:
The slides were rinsed in electrophoresis buffer and randomly placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each treatment were spread across the platform to avoid any positional effects. The buffer reservoir of the unit was topped up with electrophoresis buffer until the surfaces of the slides were covered. The slides were left for 20 minutes to allow the DNA to unwind. After alkali unwinding, the slides were electrophoresed at 18V with a starting current of approx. 300 mA (between 0.7 to 1.0 V/cm) for 30 minutes. The temperature of the electrophoresis solution during unwinding and electrophoresis was maintained at a low temperature, usually 2 – 10 °C. When electrophoresis was complete, the slides were washed 3 times, for 5 minutes with neutralisation buffer and stored, refrigerated, in lightproof boxes with moistened tissues (to prevent agar dehydration).

OTHER: Microscopic examination
Coded slides were examined by staining with SYBR GOLD® and visualised under a fluorescence microscope. The comet images from the microscope were projected onto a computer display screen via a CCD camera and measured using Perceptive Instruments COMET IV(TM) image analysis system. Initially the slides were examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. ‘hedgehog’ or ‘ghost’ cells). Data presented in Table 1 in box "Any other information on materials and methods incl. tables". These cells were excluded from the analysis, along with any cells that had unusual staining artefacts. Fifty cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by: % TAIL INTENSITY, defined as the percentage of DNA in the tail compared to the DNA contained in the whole comet.

Evaluation criteria:

- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if: a) at least one of the test doses exhibits a statistically significant increase compared with the concurrent negative control, b) the increase is dose-related when evaluated with an appropriate trend test, and c) any of the results are outside the distribution of the historical negative control data. When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, a) none of the test concentrations exhibits a statistically significant increase comapred with the concurrent negative control, b) there is no concentration-related increase when evaluated with an appropriate trend test and c) all results are inside the distribution of the historical negative control data. The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this system.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

Bartlett’s test for variance homogeneity, Williams’ test & Dunnett's test were used for statistical analysis of the data.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TEST:
- Mortalities & clinical signs:
To determine suitable dose levels for use in the comet test, one group consisting of two male and two female animals were administered (3E)-dec-3-en-2-one at 2000 mg/kg bw/day on two consecutive days approximately 24 hours apart. No mortalities were observed throughout the duration of the preliminary test. Clinical signs observed in male animals included decreased activity, flattened posture, partially closed eyelids, unsteady gait and splayed hind limbs. Clinical signs observed in female animals included decreased activity, piloerection and flattened posture. (3E)-dec-3-en-2-one administered at 2000 mg/kg bw/day was therefore considered to be the maximum tolerated dose in both male and female animals. On the basis of these results, 2000 mg/kg bw/day (the standard limit dose for the comet test) was considered to be the maximum dose in both male and female animals. No substantial differences in toxicity were observed between the sexes, therefore, in line with current guidelines, the comet test was performed using male animals only.
Body weight was unaffected throughout the preliminary toxicity testing. Dose levels of 500, 1000 and 2000 mg/kg bw/day were selected for the main test.

COMET TEST:
- Mortalities & clinical signs:
No mortalities were observed throughout the duration of the comet test. Animals were treated with (3E)-dec-3-en-2-one at dose levels of 500, 1000 and 2000 mg/kg bw/day. No clinical signs of toxicity were observed for the vehicle control, and positive control or animals administered (3E)-dec-3-en-2-one at 500 mg/kg bw/day over the duration of the test. At 1000 mg/kg bw/day clinical signs of toxicity observed included salivation, flattened posture, decreased activity, unsteady gait and piloerection. At 2000 mg/kg bw/day clinical signs of toxicity included chin rubbing and piloerection. Some incidences of body weight loss were observed throughout the comet test.

- Tail intensity:
No statistically significant increases in the median % TI were observed in either the liver or duodenum from male Wistar Han rats administered (3E)-dec-3-en-2-one at any dose level, compared to vehicle control values. The positive control compound, ethyl methanesulphonate, produced statistically significant increases in the median % TI when compared to vehicle control values (p< 0.001, t-test).

- “Hedgehog” and “ghost” cell data:
No “hedgehog or “ghost” cells were observed in either duodenum or liver of male Wistar Han rats.

Table 2. Male animal data – Liver

Treatment (mg/kg bw/day)		Number of cells scored		Group mean tail intensity%* (SD)		Group mean of median rail intensity %*(SD)
Vehicle (-)		900		3.55 (0.58)		1.05 (0.81)
(3E)-dec-3-en-2-one (500)		900		3.12 (0.49)		1.21 (0.32)
(3E)-dec-3-en-2-one (1000)		900		3.18 (0.36)		1.37 (0.58)
(3E)-dec-3-en-2-one (2000)		900		3.16 (0.54)		1.76 (0.74)
EMS (200)		450		50.17 (8.70)		49.88 (9.49)***L

Vehicle: Purified water

EMS: Ethyl methanesulphonate (positive control), dosed once only, approx. 3 hours prior to termination

SD: Standard deviation

*Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Statistical analysis performed on median tail intensity values only. p values for comparisons with control using Williams’ test, unless indicated otherwise (+t-test)

***p< 0.001 (significant), otherwise p > 0.05 (not significant)

L Analysis performed upon logarithmically transformed data

 

Table 3: Male animal data -Duodenum

Treatment (mg/kg/day)	Number of cells scored	Group mean tail intensity%* (SD)	Group mean of median rail intensity %*(SD)
Vehicle (-)	900	4.43 (0.55)	1.00 (0.36)
(3E)-dec-3-en-2-one (500)	900	4.10 (0.54)	1.00 (0.55)
(3E)-dec-3-en-2-one (1000)	900	3.60 (0.18)	0.80 (0.22)
(3E)-dec-3-en-2-one (2000)	900	3.75 (0.52)	1.19 (0.95)
EMS (200)	450	61.63 (6.92)	64.03 (8.23)***L

 

EMS: Ethyl methanesulphonate (positive control), dosed once only, approx. 3 hours prior to termination

 

SD: Standard deviation

 

* Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Statistical analysis performed on median tail intensity values only. p values for comparisons with control using Williams’ test, unless indicated otherwise (+t-test)

 

*** p < 0.001 (significant), otherwise p > 0.05 (not significant)

Conclusions:

It is concluded, that (3E)-dec-3-en-2-one has not shown any evidence of causing an increase in DNA strand breaks or cytotoxicity in either the duodenum or liver of male Wistar Han rats when administered orally in this in vivo test procedure.

Executive summary:

In an in vivo Comet assay conducted in accordance with OECD Guideline 489, male Wistar Han rats (6 animals/dose group) were exposed orally to 3-decen-2-one (99.5% purity) by gavage on two occasions, the second dose being administered approximately 24 hours after the first dose and 3 hours before tissue sampling. A preliminary toxicity test demonstrated that a dose of 2000 mg/kg bw/day was tolerated. No substantial differences in toxicity between sexes were observed in the preliminary toxicity test, therefore, in line with current guidelines, the main test was performed using male animals only. Based on the results, dose levels of 500, 1000 and 2000 mg/kg bw/day were selected for the main study. The vehicle control group received purified water and the positive control group ethyl methanesulphonate at 200 mg/kg bw. Cell suspensions from the liver and duodenum tissues were obtained from male animals in the vehicle control group and in each of the test substance groups 3 hours after administration of the second dose. Cell suspensions from male animals in the positive control group were obtained approximately 3 hours after a single dose.

Following electrophoresis, three slides per animal per tissue were analysed for comets by assessing 150 morphologically normal cells. DNA strand breaks were assessed by comparing the mean and median % tail intensities from (3E)-dec-3-en-2-one treated animals compared to vehicle control values.

No statistically significant increases in the median % tail intensity were observed in either the duodenum or liver of male Wistar Han rats administered (3E)-dec-3-en-2-one at any dose level, compared to vehicle control values. The positive control compound produced statistically significant increases in the median % tail intensity in the duodenum and liver (p< 0.001) when compared to vehicle control values, showing the validity of the experiment.

No “hedgehog” or “ghost” cells were observed in either the duodenum or liver of male Wistar Han rats administered (3E)-dec-3-en-2-one at any dose level, compared to vehicle control values.

It is concluded that (3E)-dec-3-en-2-one did not demonstrate any evidence of causing an increase in DNA strand breaks or cytotoxicity in either the duodenum or liver of male Wistar Han rats when administered orally in this in vivo Comet test.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-05-28 to 2011-07-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

other: RCCHan:WIST

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 176-242 g
- Assigned to test groups randomly: yes
- Housing: Group housed in solid-floor polypropylene cages with woodflake bedding
- Diet (e.g. ad libitum): Harlan Teklad 2014 Rodent Pelleted Diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): At least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water
- Lot/batch no. (if required): 300730602, supplied by Laboratoire Aguettant Ltd

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in sterile distilled water. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.

Duration of treatment / exposure:

Experiment I: 16 hours
Experiment II: 4 hours

Frequency of treatment:

Single treatment

Post exposure period:

N.a.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (distilled water)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

In experiment I: 2-acerylaminofluoerene (2-AAF) (vehicle: arachis oil)
In experiment II: N-(2-Fluorenyl)acetamide (NDHC) (vehicle: distilled water)
- Route of administration: orally
- Doses / concentrations: 2 AAF: 50 mg/kg bw; NDHC: 40 mg/kg bw

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

- Isolation of hepatocytes: Hepatocyte isolation was performed immediately after the termination of the animal. The post-aortic vena cava was ligatured and cannulae were introduced into the hepatic portal vein and superior vena cava. Livers were perfused with a buffered medium containing chelating agents to remove metal ions followed by a digest medium containing collagen. The liver was then removed from the body and the liver cells suspended in attachment medium and passed through a nylon gauze to remove large particles. Cells were then centrifuged and washed three times in a buffered medium and suspended in attachment medium at 1 x 10^5 viable cells/mL. Cells were then seeded onto cover slips in 35 mm six well plates at 3 mL/well (six cover slips per animal). Plates were incubated at 37 °C in 5% CO2, 95% air in a humidified incubator for 1.5-2 hours to allow cell attachment.

- Radiolabelling of cells: After cell attachment, the medium was aspirated using aseptic techniques. Hepatocytes were washed with serum-free medium which was replaced with 2 mL serum-free medium containing 10 µCI/mL of [methyl-3H] thymidine. The cultures were incubated for a further 3 hours at 37 °C in 5% CO2, 95% air before being washed three times with a serum-free medium containing 0.25 mM unlabelled thymidine solution and incubated overnight in 2 mL of the same medium to remove excess radiolabel from the cultures.

- Autoradiography: The medium was aspirated from the cultures and the cells washed with phosphate buffer solution. The cells were fixed in freshly prepared 1:3 acetic acid:methanol. Cells were washed with distilled water, the cover slips allowed to air dry and then mounted on the ends of glass slides, cell side up and left to dry overnight. The cover slips were coated in an autoradiographic emulsion and incubated at approximately 4 °C for 7-14 days in a sealed lightproof container. Following the exposure period slides were processed using photographic developer and fixative and then the cells were stained using haematoxylin/eosin and once dry a cover slip was applied using a mounting medium. The slides were assessed for obvious signs of toxicity, reduced number of cells and poor labelling. Two good quality slides from each animal were selected and coded.

- Scoring of slides and interpretation of data: The coded slides were scored using an automated image analysis system linked to a computer programme, which utilised an area counting method. Where possible, two slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total of 100 cells per animal. The number of silver grains within the nucleus were first observed and recorded as the Nuclear Grain Count (N). Three cytoplasmic areas were scored to give the Mean Cytoplasmic Grain Count (C). A Net Nuclear Grain Count (N-C) was calculated. Cells in S-phase were not scored. Mean values of N, C, N-C and percentage cells in repair (%R) were calculated. Values of N-C are typically in the range of -6 to -2 for vehicle controls and in positive controls at least 20% of all cells assessed should be in repair, having a N-C value of +5 or greater.

Evaluation criteria:

The UKEMS guidelines suggest that in positive responses at least 20% of all cells assessed should be in repair, ie undergoing unscheduled DNA synthesis, having a (N-C) value of +5 or greater

Statistics:

Not required

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
There were no clinical signs observed in animals dosed with the test item at 2000 mg/kg bw in the initial sighting work, therefore a repeat of this dose level was used to confirm the suitability for experiments 1 and 2. The test item showed no marked difference in its toxicity to male or female rats. it was therefore considered to be acceptable to use males only for the main study. The maximum recommended dose (MRD) of the test item, 2000 mg/kg bw was selected for use in the main test, with 1000 mg/kg bw as the lower dose level.

MAIN STUDY:
There were no premature deaths in any of the dose groups. No clinical signs were observed with the test item at either of the dose levels. For a number of animals in Experiment 1, the cell viability was substantially less than 50%, this was considered to be due to high collagenase potency, and though the cells were processed and scored this experiment was repeated. For the repeated Experiment 1 and Experiment 2 cell viability was considered acceptable.
The test item did not induce any marked increases in the incidence of cells in repair at either dose level. In Experiment 1 and Experiment 2 the net nuclear gain counts (N-C) were outside the typical range of -2 to -6 in the vehicle and test item dose groups, however these values were considered to be due to experimental variation and therefore acceptable. The positive controls induced a marked increase in the percentage of cells in repair thus demonstrating the viability of the test.

Table 1: Group Mean Net Nuclear Grain Count Values and % Cells in Repair

Dose level (mg/kg)		Net Nuclear Grain Count (N-C)		Net Nuclear Grain Count of Cells in Repair		MFPercentage of Cells in Repair (N-C ≥5)
Experiment 1
		Mean	SD	Mean	SD	Mean	SD
0	Vehicle	-0.3	0.2	6.6	1.1	1.5	1.3
1000	3-decen-2-one	-0.4	0.6	5.5	0.8	2.5	2.4
2000	3-decen-2-one	-0.5	0.3	9.3	5.8	0.8	0.5
50	2AAF	5.6	0.6	10.5	2.7	44.3	11.4
Experiment 1 repeat
0	Vehicle	-2.4	0.5	5.0	-	0.2	0.5
1000	3-decen-2-one	-1.7	0.2	5.7	-	0.2	0.5
2000	3-decen-2-one	-1.6	0.6	5.3	0.0	0.8	1.0
50	2AAF	7.6	2.0	11.4	1.4	60.8	9.6
Experiment 2
0	Vehicle	-0.6	0.2	5.7	0.3	2.0	2.2
1000	3-decen-2-one	-0.7	0.4	6.3	-	0.2	0.5
2000	3-decen-2-one	-0.4	0.1	5.4	0.4	1.3	0.6
50	2AAF	5.3	4.3	10.5	3.7	42.8	24.9

SD = standard deviation

Vehicle = distilled water

2AAF = 2-acetylaminofluorine

NDHC = N,N’-dimethylhydrazine dihydrochloride

Conclusions:

In conclusion, the test item did not induce any marked or toxicologically significant increases in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure for 4 to 16 hours. Therefore, (3E)-dec-3-en-2-one is considered to be non-genotoxic under the conditions of this study.

Executive summary:

In an in vivo liver unscheduled DNA synthesis (UDS) assay conducted in accordance with OECD Guideline 486, male Wistar:Han rats (4/dose groups) were exposed orally once to (3E)-dec-3-en-2-one (98.6% purity) for 4 or 16 hours to 1000 and 2000 mg/kg bw. The study was performed in two parts. In Experiment 1 and Experiment 1 (repeat), liver perfusion started at approximately 16 hours after dosing. In Experiment 2, perfusion started at approximately 4 hours after dosing. Following perfusion, the hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. Scoring utilised an area counting method. Due to some quality issues with data from Experiment 1, at the request of the sponsor a repeat experiment was performed. Data from both experiments are presented. Further groups of rats were given a single oral dose of distilled water or 2-acetylaminofluorene (2-AAF) at 16 hours or N, N’-dimethylhydrazine dihydrochloride (NDHC) at 4 hours to serve as vehicle and positive controls respectively. There were no marked increases in the incidence of unscheduled DNA synthesis in animals dosed with the test item at either time point. The positive controls produced marked increases in net nuclear grain counts and in the incidence of cells in repair, whilst the vehicle control groups gave acceptable values for net nuclear gain counts. Under the conditions of the study (3E)-dec-3-en-2-one was considered to be non-genotoxic.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2009-04-22 to 2009-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 21th July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

dated 30th May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Name: AMV-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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was prepared and diluted in Cottonseed Oil before treatment.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN WINKELMANN
- Age at study initiation: miniumum 7 weeks
- Weight at dosing: Males: 28.6-33.5 g, females: 22.9-28.0
- Assigned to test groups randomly: yes and individually marked for identification by ear and tail drawing
- Housing: Group housed in an IVC Type II L cage with Altromin saw fibre bedding
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice, ad libitum.
- Water (e.g. ad libitum): Tap water (sulphur acidified to approximately pH 2.8), ad libitum
- Acclimation period: yes but not clearly specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): at least 10x per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

intraperitoneal

Vehicle:

Cottonseed oil

Details on exposure:

After a dose range finding study, five animals per sex per dose group were administered a single i.p. dose of the test item formulated in cottonseed oil at a dose rate of 10 mL/kg bw. Peripheral blood was sampled at 44 and 68 hours after dosing. The dose groups were as follows: 1 MTD (50% solution/kg bw), 0.5 MTD (25% solution/kg bw), 0.2 MTD (10% solution/kg bw).

Duration of treatment / exposure:

- 44 hours for all dose groups
- 68 hours for the additional negative control and highest dose group

Frequency of treatment:

One single treatment

Post exposure period:

44 and 68 hours

Dose / conc.:

10 other: % solution/ kg bw

Remarks:

0.2 MTD

Dose / conc.:

25 other: % solution/kg bw

Remarks:

0.5 MTD

Dose / conc.:

50 other: % solution kg/bw

Remarks:

1 MTD

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Cyclophosphamide (CPA)
- Route of adminstration: ip, single
- Dose: 40 mg/kg bw
- Volume administered: 10 mL/kg bw

Tissues and cell types examined:

Erythrocytes derived from the bone marrow of the mouse

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results from a pre-experiment, three dose groups were chosen.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals received the test item once via i.p.. Sampling of the peripheral blood was carried out on animals 44 and 68 hours after treatment.

DETAILS OF SLIDE PREPARATION:
Blood was obtained from tail vein after its incision. Blood cells were immediatly fixed in ultracold methanol. Before analysis (at least 16 h after fixation), fixed blood cells were washed in Hank's balanced salt solution, centrifuged at 600 x g for 5 minutes and the supernatant discarded. Blood cell populations were discriminated using specifc antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei was determined by the use of a DNA specific stain (propidium iodide, PI).

METHOD OF ANALYSIS:
Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer. Anti-CD71 antibodies were labelled with fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity was recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes.)

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups
According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.

Statistics:

Data were subject to the nonparametric Mann-Whitney Test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

All animals treated with the highest dose group (50% solution/kg bw) showed toxic effects. Animals treated with 25% solution/kg bw showed slight toxic effects. No abnormalities were detected in the animals treated with 10% solution/kg bw.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the pre-experiment 100% of the test item solution was evaluated. One male animal received a single dose of 100% solution/kg bw i.p. and showed very toxic symptoms as catalepsis, reduction of spontaneous activity, prone position, ataxia, constricted abdomen, piloerection, closed eyes, loss of weight and nasal discharge, but survived 72 hours after the treatment. Due to these very severe toxic symptoms at 100%, three male and three female animals received 50% solution/kg bw i.p.. All animals showed toxic symptoms. Due to the results obtained in the pre-experiment 50% solution/kg bw was chosen as the maximum tolerable dose (1 MTD) in the main experiment.

RESULTS OF DEFINITIVE STUDY
The relative PCE values measured for negative control animals were within historical controls. For the 0.2 MTD dose group male PCE values were within the control range, females were reduced compared to controls but the reduction was not statistically significant. The 0.5 MTD and 1 MTD dose groups showed reduced and increased values compared to control in males and females respectively, the differences were not statistically significant. The decrease and/or increase of PCE values in treated animals compared to control animals are an indication of a target cell exposure of the test item.
The micronucleated polychromatic erythrocyte values obtained for the negative control were within historical control data. For the 0.2 and 0.5 MTD dose groups the values were within the range of corresponding negative controls. For the 1 MTD dose group values were within negative control values 44 hours after dosing. By 68 hours after dosing male values were reduced compared to control values and females were increased, but the difference was not statistically significant. No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated. CPA used as a positive control demonstrated the validity of the assay.

Table 1: Toxicity observed at 1 MTD dose level (males/females)

Toxicity	Time after dosing (hours)
	4	24	44	68
Reduction of spontaneous activity	5/5	5/5	0/0	0/0
Prone position	5/5	0/0	0/0	0/0
Colonic convulsion	5/5	0/0	0/0	0/0
Ataxia	5/5	0/0	0/0	0/0
Constricted abdomen	5/5	0/0	0/5	0/0
Piloerection	5/5	5/5	5/5	5/5
Half eyelid closure	5/5	0/0	0/0	0/0
Diarrhoea	0/0	5/5	0/0	0/0
Cramps	5/5	0/0	0/0	0/0
Loss of weight	0/0	5/5	0/0	0/0

Conclusions:

Under the experimental conditions reported, (3E)-dec-3-en-2-one did not induce structural and/or numerical chromosomal damage in immature erythrocytes of mice. Therefore, 3-decen-2-one is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

A mammalian micronucleus test in murine peripheral blood cells was conducted in accordance to OECD Guideline 474 to assess the clastogenicity/aneugenicity of (3E)-dec-3-en-2-one (98% purity). Five NMRI mice per sex per dose were treated once intraperitoneally with the test item at doses of 0, 10%, 25%, and 50% solution/kg bw. The vehicle was cottonseed oil. Peripheral blood samples derived from the bone marrow were collected for micronuclei analysis 44 and 68 hours after application of the test item.

The animals treated with 10% solution/kg bw showed no signs of systemic toxicity. In the mid and high dose group signs of systemic toxicity were observed. The signs of toxicity noted were reduction of spontaneous activity, prone position, clonic convulsion, ataxia, constricted abdomen, piloerection, half eyelid closure diarrhoea, cramps and loss of weight.

All mean values of micronuclei observed for the male groups after treatment with the test item were decreased compared to the corresponding negative control. All mean values of micronuclei observed for the female groups after treatment with the test item were within the range of the corresponding negative control or slightly decreased compared to the corresponding negative control. Thus, no biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated. The non-parametric Mann-Whitney Test was performed to verify the results. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated. CPA (40 mg/kg bw) administered i.p. was used as positive control and induced a significant increase in micronucleus frequency. This demonstrates the validity of the assay.

Under the experimental conditions reported, the test item (3E)-dec-3-en-2-one did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, (3E)-dec-3-en-2-one is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genotoxic potential of (3E)-dec-3-en-2-one was assessed in a weight-of-evidence approach based on two in vitro genotoxicity studies (OECD 471 and OECD 476, GLP) and three in vivo genotoxicity studies (OECD 474, OECD 486 and OECD 489, GLP).

The target substance was tested negative with and without metabolic activation in a bacterial reverse gene mutation test conducted according to OECD 471. In an in vitro mammalian cell gene mutation assay conducted according to OECD 476, the substance was considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y without metabolic activation. In contrast, with metabolic activation, the substance did not induce any mutagenic effects. Based on these contradictory results from the two in vitro assays, additional in vivo tests were conducted to further assess the genotoxic potential of the test substance.

The genotoxic potential of (3E)-dec-3-en-2-one was investigated in three in vivo genotoxicity studies (OECD 474, OECD 486 and OECD 489, GLP). The test substance was tested negative in all three in vivo assays, namely in the in vivo Micronucleus Test (according to OECD 474), the Unscheduled DNA Synthesis Assay (according to OECD 486) and in the in vivo Comet Assay (according to OECD 489). Thus, in a weight of evidence approach, based on the results of the in vitro and in vivo genotoxicity studies, it can be concluded, that the substance is non-mutagenic. Therefore, no classification for mutagenicity is warranted in accordance with the CLP Regulation 1272/2008.

Justification for classification or non-classification

Based on the results from several in vitro and in vivo genotoxicity studies conducted in accordance with current OECD testing guidelines, the target substance 3 -decen-2-one is considered negative for mutagenicity. Therefore, no classification for mutagenicity is warranted in accordance with the CLP Regulation 1272/2008.