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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 / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2006
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:
according to guideline
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
according to guideline
other: EEC Directive 2000/32 L 136 Annex 4C, B 12
Version / remarks:
GLP compliance:
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
Colour: Light yellow

Test animals

Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann
- Age at start of treatment: minimum 7 weeks
- Weight at study initiation: male: 30.7 - 38.2g; female: 23.2 - 32.3g
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex per cage (Macrolon Type III; Hereto)
- Diet: Altromin 1324 maintenance diet for rats and mice, TPF
- Water: tap water, ad libitum
- Acclimation period: adequate acclimatisation period

- Temperature (°C): 19-25°C
- rel. Humidity (%): 55±10%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
Cotton Seed Oil
Details on exposure:
The dosing formulations were prepared 1 hour before treatment (positive control formulation was prepared on the same day). All animals received a single volume of 10 mL/ kg bw.
Duration of treatment / exposure:
Single dose
Post exposure period:
Sampling of the peripheral blood was done at 44 (all groups, including solvent control and positive control) and 68 hours (solvent control and high dose group only) after treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
400 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:
5 (main study)
6 males and 3 females (pre-experiment)
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg/kg bw cyclophosphamide dissolved in physiological saline. Intraperitoneal injection at 10 mL/kg bw


Tissues and cell types examined:
Blood from tail vein.
Details of tissue and slide preparation:
Blood was immediately fixed in ultracold methanol. At least 16 hours after fixation blood cells were washed in Hank's balanced salt solution, centrifuged at 600 g for 5 minutes. Blood cell populations were discriminated against using CD71 and CD61 and DNA content of micronuclei was determine dby the use of a DNA specific strain (propidium iodid).
Evaluation criteria:
Evaluation was performed using FACScan BD flow cytometer. At least 10,000 immature erythrocytes/animals were scored for the incidence of micronucleated immature erythrocytes.
To assess cytotoxicity, the ratio between immature and mature erythrocytes was determined and expressed as relative PCE (proportion of polychromatic (immature) erythrocytes among total erythrocytes.

Criteria for determining a positive result:
- dose related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups

A test item is considered negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
The nonparametric Mann-Whitney Test was used.

The data generated were considered acceptable, if:
- at the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20% of the mean weight of each sex,
- the background frequency of micronucleated cells is in the normal range as reported in the literature or within the laboratory's historical range,
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.

Results and discussion

Test results
Reduction rel. PCE
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
The results are included in tabular form in the attached document.

The results of the positive control group and the solvent control group were within the range of the corresponding historical control data.

The relative PCE values observed in the male and female group were reduced compared to the corresponding negative control. Although the reduction was not statistically significant, the outcome was considered to be a good indication of systemic exposure of the mice.

The mean values of micronuclei observed after treatment were within the range of the negative control, except for the high dose group samples 68 hours after treatment. The values for males and females were slightly increased compared to the negative control (0.26% (0.25%) in the male (female) controls vs. 0.35% (0.30%) for the high dose males (females). As the increase was minimal and not statistically significant this was not considered to be toxicologically significant.

Historical control data ranges are attached below.

Any other information on results incl. tables

Results pre-experiment to find MTD:

Two males died within 72 hours after application of the test item. They showed toxic symptoms as reduction of spontaneous activity, rough fur, constricted opistosoma, palpebral closure and loss of weight. 

All other animals survived.

The females and three males did not show signs of toxicity. 

One male mouse showed toxic symptoms including reduction of spontaneous activity, rough fur, constricted opisthosoma, palpebral closure and loss of weight. 


Applicant's summary and conclusion

Based on the results of an in vivo Micronucleus study performed according to OECD/EC guideline and GLP principles, the test substance is considered to be non-cytogenic.