Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.04.2010 - 17.05.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Methylbuten-3-ol-1
- Analytical purity: 99%
- Lot/batch No.: 31-0342

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Since no gender specific differences concerning signs of toxicity in the pre-experiment were observed, the main study was performed using males only.
Sex:
male
Details on test animals and environmental conditions:
Age at start of experiment: 8-9 weeks
Mean inital body weight: 39.3 g (SD +/- 2.3 g)

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type 111111, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding (Rettenmaier & Sohne GmbH + Co. KG, 73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum (Hartan Laboratories B.V. Postbus 6174,5960 AD Horst,The Netherlands)
Water: tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
Environment: temperature 22 +/- 2 GC
relative humidity 45 - 65 %
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
Corn oil
Details on exposure:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test item, the vehicle or the positive control substance once orally.
Frequency of treatment:
Once
Post exposure period:
24 h
Additional sample for the highest dose level at 48 h after treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
7 males per group (5 for contol groups)
Control animals:
yes, concurrent vehicle
Positive control(s):
5 males, 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 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.
At least one slide was made from each bone marrow sample.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
Statistical significance at the five per ceAt level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, Isoprenol is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of Isoprenol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mLlkg b.w.. 24 hand 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the vehicle and positive control groups with 5 males each) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Isoprenol

did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide as positive control showed a substantial increase of induced micronucleus frequency.