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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

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: 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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.