Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1998-07-31 to 1999-03-04
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted: 1997
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Molecular formula:
Not applicable as multi-const. substance. Please refer to IUCLID section 1.2 for details on constituents.
2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol; 2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate; 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethan-1-ol; bis{2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl} carbonate; methyl 2-[(2R)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate; methyl 2-[(2S)-2-(propan-2-yl)-1,3-oxazolidin-3-yl]ethyl carbonate
Test material form:
liquid: viscous

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 4 - 8 weeks old
- Weight at study initiation: 25 - 32 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: yes
- Housing: appropriate caging
- Diet:ad libitum ,Special Diets Services Ltd, RM1.[E].SQC.
- Water: ad libitum, tab water
- Acclimation period: 7 days

- Temperature (°C): 19 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes: 15 air changes per hour
- Photoperiod : light for 12 hours, from 6:00 to 18:00

Administration / exposure

Route of administration:
oral: gavage
Corn oil
Details on exposure:
Dosing preparations were made by suspending Incozol LV (with homogenisation) in corn oil to give the top concentrations specified below and dilutions were made using corn oil. The test article preparations in the main study were protected from light, maintained as an even suspension (by multiple inversion) and used within 2.5 hours of initial formulation.
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily o two consecutive days
Post exposure period:
Micronucleus assay: 24 or 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) was freshly dissolved in saline at 2 mg/mL to serve as the positive control.


Tissues and cell types examined:
Bone marrow erythrocytes (polychromatic erythrocytes, PCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Observed toxicity in Dose Range Finding assay
An initial range finding study was performed using groups of three male and three female mice. Animals were dosed once daily for two consecutive days with the test article. Observations were made over a two day period following the first administration. Clinical signs of toxicity and body weight over this period were recorded and a maximum acceptable dose determined based on these data. This was used as the highest dose level in the main study.

SAMPLING TIME: Bone marrow was sampled 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: Bone marrow was removed from the femurs of the animals in a dose group. Following centrifugation to pellet the tissue, the supematant was removed by aspiration and portions of the pellet were spread on slides, air dried and stained in May-Grünwald solution followed by Giemsa.

Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue/purple enucleate cells, and normochromatic erythrocytes (NCE), seen as yellow/grey stained enucleate cells, were detennined until a total of at least 1000 cells (PCE plus NCE) had been analysed. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.
Evaluation criteria:
The test article was to be considered as positive in this assay if:
1) a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, and
2) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.

Results and discussion

Test results
Key result
no effects
No toxicity was seen at the maximum level of 2000 mg/kg in male or female mice.
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Additional information on results:
There was no sign of ill effect to the animals treated.
- Dose range: 500, 1000 and 2000 mg/kg bw/day
- Clinical signs of toxicity in test animals: No clinical signs of toxicity observed.
- Evidence of cytotoxicity in tissue analyzed: no

Groups of male mice treated with Incozol LV exhibited PCE/NCE ratios which were similar to vehicle controls. Group mean frequencies of micronucleated PCE were also similar to those seen in the vehicle control group and were not significantly different by 2 % analysis, except at 1000 mg/kg bw/day, where a small but significant increase was observed (p<0.05). Additionally, the statistical test for linear trend indicated a small, but significant dose-response relationship (p<0.05) ( Appendix 5). Insofar as the frequency of micronucleated PCE both at 1000 mg/kg bw/day and the other doses analysed fell within the historical negative control range , these effects were considered to be of no biological relevance.

Applicant's summary and conclusion

It is concluded that Incozol LV did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw/day.
Executive summary:

 A study was conducted according to OECD TG 474 and Directve 92/69/EEC method B.12 to assess the induction of micronuclei in the bone marrow of treated mice of Incozol LV. Incozol LV was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study in which Incozol LV, made up in corn oil, was administered to mice orally by gavage. The test article was administered once daily on two consecutive days to groups of three male and three female mice at 500, 1000 and 2000 mg/kg bw/day. Observations were made over a 2 day period following the first administration and signs of toxicity and body weight recorded. For the micronucleus test, Incozol LV was made up as described and administered at 500, 1000 and 2000 mg/kg bw/day. Since no difference in toxicity was observed between the two sexes, male mice in groups of eight, killed 24 hours after the second administration were treated in the main study. The highest dose used (2000 mg/kg bw/day) was considered an acceptable top dose for the rodent micronucleus assay. The negative (vehicle) control in the study was corn oil also administered orally by gavage once daily on two consecutive days. A groups of eight male mice treated with this was killed and sampled 24 hours after the second administration. Cyclophosphamide (CPA), the positive control, was dissolved in saline and administered orally as a single dose at 40 mg/kg to a group of eight male mice which was killed after 24 hours. All positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in the positive control group was significantly greater than in concurrent controls (2x2 contingency x2 test).

Seven mice from each group were analysed. Negative (vehicle) control mice exhibited normal group mean ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control (normal) ranges. Male mice treated with Incozol LV at all doses exhibited group mean ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to the values for the vehicle control group and also fell within normal ranges. It is concluded that Incozol LV did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 2000 mg/kg bw/day.