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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2020
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Supplier: Sigma-Aldrich Chemie GmbH
Batch: BCCB1352
Purity: 100.0%
Molecular weight: 98.14 g/mol
Physical state / Appearance: Colourless liquid
Retest Date: 30 June 2023
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor

Test animals

Details on species / strain selection:
The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.
Details on test animals or test system and environmental conditions:
Strain: Mouse (NMRI)
Source: Charles River Laboratories Research Models and Services Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of Animals in the pre-test: 2 males and 2 females
Number of Animals in the main study: 43 males (no gender differences in pre study)
Initial Age at Start of Experiment: 6 – 10 weeks (main study)
Acclimation: minimum 5 days
Body Weight at Start of Treatment: mean value 33.2 g (SD ± 1.4 g); range 30.1 – 35.4 g
Body Weight at End of Treatment: mean value 33.8 g (SD ± 1.6 g); range 30.8 – 37.7 g
According to the supplier’s assurance, the animals were in healthy condition. The animals were under acclimatisation in the animal house of ICCR-Roßdorf GmbH for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.
Housing: Single
Cage Type: Macrolon Type II / III, with wire mesh top
Bedding: Granulated soft wood bedding
Feed: Pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Environment: Temperature: 22 ± 2°C
Relative humidity: 45 – 65%, except for deviations (with the aim of 50 – 60%)
Artificial light: 6.00 a.m. – 6.00 p.m.
Ventilation: at least eight air changes per hour
Environmental enrichment: nesting material, wooden chewing blocks

Administration / exposure

Route of administration:
oral: gavage
olive oil
Details on exposure:
The animals received the test substance, the negative or the positive control substance once by oral gavage, using a stainless steel feeding needle with rounded tip (1.2 Gauge) and disposable syringe at a dose volume of 10 mL/kg b.w.
Duration of treatment / exposure:
24h (negative control, low, mid and high dose, positive control)/48h (negative control and high dose)
Frequency of treatment:
Post exposure period:
Sampling of the bone marrow was done 24 h and 48 h after treatment, respectively.
Doses / concentrationsopen allclose all
Dose / conc.:
312.5 mg/kg bw (total dose)
7 male animals
Dose / conc.:
625 mg/kg bw (total dose)
7 male animals
Dose / conc.:
1 250 mg/kg bw (total dose)
14 male animals (2 x 7 males per test group)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: Cyclophosphamide (CPA)
Supplier: Acros Organics
Batch: A0406976
Expiry Date: 14 August 2020
Dissolved in: sterile water
Batch No.: 19NCB170
Expiry Date: February 2022
Dosing: 40 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 mL/kg b.w.


Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a disposable syringe. The cell suspension was centrifuged at 1500 rpm (3900 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 4000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined from the same slide by counting until 500 PCEs had been determined among total erythrocytes and expressed as polychromatic erythrocytes per total erythrocytes counted. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells.
Evaluation criteria:
A test substance is classified as positive in the assay if
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
A test item that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying the above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure.
nonparametric Mann-Whitney test, linear regression analysis

Results and discussion

Test results
hunched posture, decreased activity
Vehicle controls validity:
Positive controls validity:

Any other information on results incl. tables

An additional study has been carried out to demonstrate proof of systemic exposure in the mouse after oral (gavage) administration of the test item. Therefore, three groups of 3 male mice of the Crl:CD-1 strain were dosed with 312.5, 625 or 1250 mg/kg cyclohexanone in olive oil on one occasion, by gavage, at a dose volume of 10 mL/kg body weight.

All animals were observed for any visible signs of reaction to treatment and body weights were recorded on Days 1 and 2. Blood samples were taken at 1, 4 and 24 hours after dosing for bioanalysis.

There were no clinical signs observed following administration of cyclohexanone at 312.5 mg/kg/day (Group 1) or 625 mg/kg/day (Group 2). Clinical signs observed following administration at 1250 mg/kg/day (Group 3) included excessive salivation, piloerection, partially closed eyes and coldness to the touch. There was minor body weight loss in all 3 groups.

Exposure to cyclohexanone was confirmed in all 3 groups by detectable concentrations of the test item in circulating plasma at 1 hour and 4 hours after dosing using a validated bioanalysis method. Cyclohexanone was below the limit of quantification 24 hours after dosing.

In summary, proof of exposure was demonstrated by detectable plasma concentrations of cyclohexanone in male Crl:CD-1 mice after a single administration of cyclohexanone at 312.5, 625 and 1250 mg/kg.

Applicant's summary and conclusion