Methyl 3-oxo-2-pentylcyclopentaneacetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 July 2000 to 21 December 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: UKEMS Sub-Committee on Guidelines for Mutagenicity testing: Report Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

24851-98-1

IUPAC Name:

24851-98-1

Constituent 2

Reference substance name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

EC Number:

246-495-9

EC Name:

Methyl 3-oxo-2-pentylcyclopentaneacetate

Cas Number:

24851-98-7

Molecular formula:

C13H22O3

IUPAC Name:

methyl 3-oxo-2-pentylcyclopentaneacetate

Details on test material:

- Substance type: pure active substance
- Sponsor's identification: ST 41 C 00
- Physical state: clear colourless liquid
- Analytical purity: 98.1%
- isomer composition: ca. 10-12 % cis- and 86-88 % trans isomers
- Batch No.: F02214-01D
- Date received: 22 June 2000
- Storage condition of test material: room temperature, in the dark under nitrogen

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK
- Age at study initiation: approximately nine weeks
- Weight at study initiation: 208 g to 250 g.
A few animals, two in experiment 1 and five in experiment 2, were marginally below the lower weight limit of 220 g; however, this was considered not to affect the purpose or integrity of the study.

- Housing: solid-floor polypropylene cages with woodflake bedding in groups of up to four
- Diet: ad libitum (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


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

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: arachis oil
- Supplier's lot number: T44 (Safepharm lot number: V-1940)
- Description: straw coloured slightly viscous liquid
- Storage conditions: room temperature
- Concentration of test material in vehicle: 33.3 & 100 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg

Details on exposure:

Intraperitoneal. A range-finding toxicity study was performed to determine a suitable dose level/route of administration for the main UDS study.
In animals dosed with the test material via the oral route there were no premature deaths and no clinical signs were observed. In animals dosed with the test material via the intraperitoneal route there were premature deaths at 2000 mg/kg and clinical signs were seen at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration and splayed gait. Based on the above data the intraperitoneal route was selected for use in the main study as adequate evidence of systemic absorption had been demonstrated. The maximum tolerated dose (MID) of the test material 1000 mg/kg, was selected for use in the main study, with 333.3 mg/kg as the lower dose level.

Duration of treatment / exposure:

Single dosing.
The study was performed in two parts, in Experiment 1 the livers were perfused approximately 16 hours after dosing and, in Experiment 2, perfusion was performed approximately 2 hours after dosing.

Frequency of treatment:

Dosing of the test substance occured once in two groups of rats for Experiment 1 and 2.

Post exposure period:

Clinical signs were observed within the 16 and 2 hours exposure time for Experiment 1 and 2 respectively

Doses / concentrations

Remarks:

Doses / Concentrations:
1000 and 333.3 mg/kg
Basis:
nominal conc.
Experiment 1 and 2

No. of animals per sex per dose:

See Animal Assignement in "Any information on materials and methods incl.tables"

Control animals:

yes, concurrent vehicle

Positive control(s):

- 2-acetylaminofluorene (Experiment 1) and N,N'-dimethyl hydrazine dihydrochloride (NDHC) (Experiment 2)
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg (2-AA) and 20 mg/kg (NDHC)

Examinations

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Dose selection was established according to range-finding toxicity results. In animals dosed with the test material via the oral route there were no premature deaths and no clinical signs were observed.
In animals dosed with the test material via the intraperitoneal route there were premature deaths at 2000 mg/kg and clinical signs were seen at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration and splayed gait.
Based on the above data the intraperitoneal route was selected for use in the main study as adequate evidence of systemic absorption had been demonstrated. The test material 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 tolerated dose (MID) of the test material 1000 mg/kg, was selected for use in the main study, with 333.3 mg/kg as the lower dose level.

DETAILS OF SLIDE PREPARATION & METHOD OF ANALYSIS:
Isolation of hepatocytes:
The isolation of the hepatocytes was carried out using a two stage in situ method, and was performed immediately after the termination of the animal. The post-aortic vena cava was ligatured, and then cannulae were introduced into the hepatic portal vein and superior vena cava. This allowed a flow of liquid through the liver and out through the heart. Livers were first perfused with a buffered medium containing chelating agents to remove metal ions, this was followed by a digest medium containing collagenase and calcium causing the liver to diassociate into a single cell population.
The liver was removed from the body and the capsule opened and the liver cells were suspended in transport medium. The suspended cells were then passed through nylon gauze to remove large particles and debris. The cells were centrifuged and washed three times using a buffered medium and finally suspended in attachment medium at 3 x 10e5 viable cells/ml. They were then seeded onto 22 mm coverslips (Nunc Thermonox) in 5 mm six-well plates at 3 ml/well (six coverslips per animal). The plates were incubated at 37 C in 5% CO2 : 95% air in a humidified incubator for 1.5 to 2 hours to allow cell attachment.

Radiolabelling of cells:
After cell attachment, the medium was aspirated using aseptic technique. The hepatocytes were washed with serum-free medium which was replaced with 2 ml of serum-free medium containing 10Ci/ml (370 kBq/ml) of [metyl-3H] thymidine. The cultures were then incubated for a further 3 hours at 37 °C in 5% CO2 / 95 % air.
Cultures were washed three times with serum-free medium containing 0.25 mM unlabelled thymidine solution. This was a 'cold chase' procedure to remove excess radiolabel from the cultures. The cells were incubated overnight with 2 ml of "cold chase" medium.
Autoradiography

The medium was aspirated and the cells washed with phosphate buffer solution. The cells were fixed in freshly prepared 1:3 acetic acid: methanols, three changes of fixative were used. Finally cells were washed with distilled water and the coverslips allowed to air dry and then mounted onto the ends of glass slides, cell side up, and left to dry overnight in a dust free environment. The coverslips were coated with Ilford K2 autoradiographic emulsion and incubated at 4 C for 7 to 14 days in a sealed light proof container.

The emulsion is sensitive to the emission of radioactive particles, causing a deposit of silver, visualized as black grains when development is complete.
Following the exposure period the slides were processed using photograhic developer and fixative, and then the cells were stained using haematoxylin/eosin. When the cells were dry they were coverslipped using a mounting medium. The slides were then assessed for obvious signs of toxicity, reduced number of cells and poor labeling.

Three good quality slides from each animal were selected and coded using a code supplied by the "Grain" computer programme.

Evaluation criteria:

The coded slides were scored using an automated image analysis system linked to a computer program (Grain) which followed the UKEMS guidelines for statistical analysis. The method used to score the slides was an area counting method which complies with the UKEMS guidelines. Ideally a minimum of three slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total of 150 cells per animal. However, it is acceptable where slide quality is poor to score a minimum of 100 cells.
The number of silver grains within the nucleus were first observed and recorded as the Nuclear Count (N). Three cytoplasmic areas (each equal to the nuclear area) were also scored to give the Mean Cytoplasmic Grain Count (C). A net Nuclear Grain Count (N-C) was then calculated.
Cells in 'S'-phase were not scored for grain counts, these were easily recognisable due to the dense 'graining' appearance of the nucleus. Mean values (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, although variations in experimental conditions can give values outside of this range. 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:

The coded slides were scored using an automated image analysis system linked to a computer programme (Grain) which followed the UKEMS guidelines for statistical analysis (see Evaluation criteria)

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs observed (hunched posture, lethargy)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg oral route, 1000 and 2000 mg/kg intraperitoneal route
- Clinical signs of toxicity in test animals:
oral route: no premature deaths and no clinical signs;
intraperitoneal route: premature deaths at 2000 mg/kg and clinical signs were seen at and above 1000 mg/kg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration and splayed gait.
- Rationale for exposure: intraperitoneal route was selected for use in the main study as adequate evidence of systemic absorption had been demonstrated. The test material did not show 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 tolerated dose (MTD) of the test material 1000 mg/kg was selected for use in the main study, with 333.3 mg/kg as the lower dose level.

DEFINITIVE STUDY
There were no premature deaths seen in any of the dose groups, clinical signs were observed in both experiment: hunched posture and lethargy.
See "Remarks on results including tables and figures" for Group mean grain count values and percentage of cells in repair. To see complete data see "background attached material"

Any other information on results incl. tables

Experiment 1

The test material did not induce any marked increases in the incidence of cells in repair at either dose level. The vehicle control gave acceptable values for net nuclear grain count. The positive control 2-Acetylaminofluorene (2AAF) induced a marked increase in the incidence of cells in repair, demonstrating that the test method was operating satisfactorily. As can be seen from the data (see attached background material for more details), five out of six animals from the vehicle control group were successfully processed to give scorable slides, four out of four animals from both of the test material groups were processed to provide scorable slides and three out of four positive control group animals were processed to provide scorable slides. It was considered that the loss of an animal from vehicle and positive control groups did not affect the integrity of the study because the minimum number of animals specified in the test guideline was achieved in the vehicle control dose group.

Table 1. Group mean net grain count values and percentage cells in repair after 16 hours harvest time (experiment 1)

Dose level (mg/kg)	Net grain count (NG)		Net grain count of cells in repair		Percentage of cells in repair (NG ≥ 5)
	Mean	SD	Mean	SD	Mean	SD
0 (vehicle)	-0.2	0.4	6.3	0.6	3.6	1.7
333.3	0.8	1.0	7.5	4.1	5.3	3.6
1000	0.2	0.4	6.8	1.5	3.0	2.1
50 (2-AAF)	7.2	1.9	9.2	1.1	68.2	14.0

Experiment 2

The test material did not induce any marked increases in the incidence of cells in repair at either dose level. The vehicle control gave acceptable values for net nuclear grain count, and the positive control N,N'-dimethylhydrazine dihydrochloride (NDHC) induced a marked increase in the percentage of cells in repair demonstrating that the test method was operating satisfactorily. As can be seen from the data (see "background attached material" for more details) all six vehicle control animals, all four of the 333.3 mg/kg and 1000 mg/kg test material animals and four positive control (20 mg/kg NDHC) animals provided slides suitable for scoring.

Table 2. Group mean net grain count values and percentage cells in repair after 2 hours harvest time (experiment 2)

Dose level (mg/kg)	Net grain count (NG)		Net grain count of cells in repair		Percentage of cells in repair (NG ≥ 5)
	Mean	SD	Mean	SD	Mean	SD
0 (vehicle)	0.3	0.4	7.4	0.8	7.4	3.7
333.3	-0.4	0.4	6.6	0.7	7.2	2.3
1000	0.2	0.9	6.5	0.3	7.3	3.3
20 (NDHC)	7.6	2.8	10.4	2.2	63.2	13.9

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material 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 2 and 16 hours. Therefore the test material was considered to be nongenotoxic under the conditions of this study.

Executive summary:

Introduction. A study was performed to assess the potential of the test material to induce DNA repair in isolated rat hepatocytes following administration to rats. The method used has been designed to comply with the OECD Guidelines for Testing of Chemicals N° 486 and follows the recommendations of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993).

Methods. A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male rats.

The UDS assay was conducted using the intraperitoneal route of administration. The test material was administered at the maximum tolerated dose (MTD) of 1000 mg/kg with 333.3 mg/kg as the lower dose level. The study was performed in two parts, in Experiment 1 the livers were perfused approximately 16 hours after dosing and, in Experiment 2, perfusion was performed approximately 2 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. The method used for scoring the hepatocytes was an area counting method which complies with the UKEMS guidelines.

Further groups of rats were given a single intraperitoneal dose of arachis oil, or dosed orally with 2-acetylaminofluorene (2-AAF) at 16 hours orN,N'-dimethylhydrazinedihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively.

Results & Conclusions. There was no marked increase in the incidence of unscheduled DNA synthesis in animals dosed with the test material at either time point. The positive controls both produced marked increases in the incidence of cells in repair.

The test material was considered to be non-genotoxic under the conditions of the test.