Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 02 December 2016
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
30 may 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian erythrocyte micronucleus test

Test material

1
Chemical structure
Reference substance name:
2-heptylcyclopentanone
EC Number:
205-273-1
EC Name:
2-heptylcyclopentanone
Cas Number:
137-03-1
Molecular formula:
C12H22O
IUPAC Name:
2-heptylcyclopentan-1-one
Test material form:
liquid

Test animals

Species:
mouse
Strain:
other: Hsd: ICR (CD-1®)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, B.V., Horst, The Netherlands
- Age at study initiation: approximately six to ten weeks
- Weight at study initiation: 23 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: The animals were housed in groups of five in solid-floor polypropylene cages with wood-flake bedding.
- Diet: Free access to food (Envigo Teklad 2014C Global Certified Rodent Diet supplied by Envigo Laboratories UK Ltd., Oxon, UK)
- Water: Free access to mains drinking water
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approximately fifteen
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: arachis oil
- Justification for choice of solvent/vehicle: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil. 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.

VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Dosed once.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
150 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
600 mg/kg bw (total dose)
Remarks:
24 and 48 hours exposure time
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide:
- Route of administration: orally at 50 mg/kg bw
- Doses / concentrations: Dissolved in sterile distilled water at a concentration of 5 mg/mL

Examinations

Tissues and cell types examined:
The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the observed mortality and clinical signs in the toxicity assay, dose levels were selected for the micronucleus assay.

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
A positive mutagenic response is demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
The data was analysed following a transformation using Student's t-test (two tailed).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Groups of mice were dosed orally as follows: 1000 mg/kg bw (3 males and 1 female), 800 mg/kg bw (2 male) and 600 mg/kg bw (4 males and 2 females).
The clinical signs of hunched posture, lethargy, ataxia, ptosis, decreased respiration and labored respiration was observed at 1000 mg/kg bw. One of the animals dosed at 1000 mg/kg bw exceeded the severity band therefore was killed in extremis. The dose level was decreased to 800 mg/kg bw and the clinical signs of hunched posture, ptosis, ataxia and tiptoe gait were observed. The clinical signs were considered to be too severe and therefore the dose level was reduced to 600 mg/kg bw. The following clinical signs were observed, hunched posture, and ptosis. This was selected as the considered maximum tolerated dose level and the main test was initiated using this dose level. Acceptable bone marrow toxicity was observed at 600 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Mortality Data and Clinical Observations:
There were no premature deaths in any of the dose groups. Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis. The clinical signs were considered to be indicative of systemic absorption.
- Evaluation of Bone Marrow Slides:
The test item Fleuramone did not show any indication of toxicity to bone marrow. The PCE/NCE ratio had no marked reductions when compared to the vehicle control. No statistically significant, decreases in the PCE/NCE ratio were observed in any of the exposure groups when compared to the vehicle control.
There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.


Applicant's summary and conclusion

Conclusions:
A micronucleus study with the substance was performed equivalent to OECD 474 guideline and GLP principles, in male mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.
Executive summary:

In an in vivo micronucleus study, male mice were exposed to 150, 300 and 600 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles. Based on the results of the range finding test in which clinical signs of hunched posture, lethargy, ataxia, ptosis, decreased respiration and labored respiration were observed at 1000 mg/kg. One of the animals dosed at 1000mg/kg exceeded the severity band therefore was killed in extremis. The dose level was decreased to 800mg/kg and the clinical signs of hunched posture, ptosis, ataxia and tiptoe gait were observed. The clinical signs were considered to be too severe and therefore the dose level was reduced to 600 mg/kg. The following clinical signs were observed, hunched posture, and ptosis. This was selected as the considered maximum tolerated dose level and the main test was initiated using this dose level. At this dose levels and below there were no premature deaths in any of the dose groups. Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.