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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study. According to ECHA guidance, a study with a read-across substance can have no reliability of higher than 2. The study itself is valid without restriction.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
(only 1000 erythrocytes/animal were counted)
Type of assay:
micronucleus assay

Test material

Constituent 1
Details on test material:
- Batch #: Vers. 31-33
- Purity: pilot sample
- Storage: room temperature
- Physical state: liquid
- Expiry date: September 1991

Test animals

hamster, Chinese
other: Cricetulus griseus
Details on test animals or test system and environmental conditions:
- Source: Ciba Geigy Tierfarm, Sisseln, Switzerland
- Age at study initiation: females 6- 10 weeks, males 4-9 weeks
- Weight at study initiation: females 22-32 g, males 22-34 g
- Diet: ad libitum; Standard diet: NAFAG No.924
- Water: ad libitum
- Acclimation period: 4-5 days

- Temperature (°C): 20-21
- Humidity (%): 60-64
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle used: Arachis oil
- Source: Siegfried AG, Switzerland
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle:
- Amount of vehicle: 10 ml/kg bw
- Purity: no data
Details on exposure:
Application volume: 10 ml/kg bw
Duration of treatment / exposure:
single dose application
Frequency of treatment:
Post exposure period:
sacrifice was performed 16, 24 and 48 hours after gavage
Doses / concentrations
Doses / Concentrations:
5000 mg/kg bw
actual ingested
No. of animals per sex per dose:
8/sex/dose/sampling time
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: Cyclophosphamide (CPA)
- Source: ASTA-Werke, Germany
- Justification for choice of positive control(s): guideline recommended
- Number of animals: 8/sex/dose/sampling time
- Route of administration: gavage
- Vehicle: Arachis oil
- Application volume: 10 ml/kg bw
- Doses: 64 mg/kg bw
- Time for sacrifice: 24 hours after dosing


Tissues and cell types examined:
Bone marrow; erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay

DETAILS OF SLIDE PREPARATION: Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS: Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment are examined. From the animals of the positive control group which are sacrificed 24 hours after application, the slides of five female and five male animals are scored. 1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.
The significance of difference is assessed by X^2-test

Results and discussion

Test results
no effects
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Dose: 200, 1000, 5000 mg/kg bw
- Number of animals/sex/dose: 2 per sex per dose
- Frequency of application: once
- Rationale for exposure: Determine the highest dosage of the test substance to be applied in the mutagenicity assay
- Harvest times: 16, 24, 48 and 72 hours after gavage

- Induction of micronuclei (for Micronucleus assay): There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg as compared with the negative control animals.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 1.73. In comparison with the vehicle control (0.16) this value is highly significant (p <0.05).
- Percent of polychromatic erythrocytes with micronuclei:
16 h: 0.20 for males (control: 0.20); 0.14 for females (control: 0.10)
24 h: 0.26 for males (control: 0.18); 0.14 for females (control: 0.14)
48 h: 0.30 for males (control: 0.18); 0.04 for females (control: 0.06)
pos. control, 24 h: 2.32 for males (control: 0.18); 1.14 for females (control: 0.14)
- Ratio of PCE/NCE (for Micronucleus assay):
16 h: 1.0 for males; 0.9 for females
24 h: 1.1 for males; 0.9 for females
48 h: 1.1 for males; 1.0 for females
pos. control, 24 h: 1.1 for males; 0.8 for females

Applicant's summary and conclusion