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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
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:
solid: granular
Specific details on test material used for the study:
- Lot/batch No.: 004140
- Purity: 99.79%

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: no information
- Weight at study initiation: ranged from 30.2 to 32.4 for males and 25.2 to 26.6 for females
- Assigned to test groups randomly: allocated to treatment groups as they came to hand from delivery boxes
- Fasting period before study: no information
- Housing: labeled cages containing purified sawdust bedding
- Diet: Pelleted laboratory animal diet (Altromin), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
corn oil
Details on exposure:
Four groups, each comprising 5 males and 5 females, received a single intraperitoneal injection. This route of administration was chosen to maximize the chance of the test
article reaching the target tissue.
Two groups were dosed with 300 mg/kg body weight, one group was dosed with 150 mg/kg and one group was dosed with 75 mg/kg body weight.
Appropriate positive and negative control groups were included.
The dosing volume was 10 mg/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.
Duration of treatment / exposure:
24 and 48 h
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dissolved in physiological saline.
- Route of administration: single intraperitoneal injection
- Doses / concentrations: 50 mg/kg bw


Tissues and cell types examined:
Micronucleated polychromatic erythrocytes and normochromatic erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus test was based on a dose range finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Corn oil sampled at 24 hours
Tribromophenol (300 mg/kg) sampled at 24 and 48 hours
Tribromophenol (150 mg/kg) sampled at 24 hours
Tribromophenol (75 mg/kg) sampled at 24 hours
Cyclophosphamide (50 mg/kg) sampled at 48 hours

Animals were sacrificed by cervical disolcation 24 or 48 hours after dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum. Cell suspension was collected and centrifuged at 1000 rpm for 5 minutes. Supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned and marked. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45 degrees over the slide with the drop of bone marrow suspension. Preparations were air-dried, fixed for 5 minutes in 100% methanol and air-dried overnight. Two slides were prepared per animal. Slides were stained using the Wright-stain-procedure, and then dipped in xylene before mounting with a coverslip.

All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide . At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e., where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromated erythrocytes. The ratio of polychormatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronucle were only counted in polychromatic erythrocytes.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) the positive control induced a statistically significant incresae in frequence of micronucleated polychromatic erythrocytes (Wilcoxon Rank Sum Test, 2-sided, p < 0.05).
b) the incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.
Wilcoxon Rank Sum Test on the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (treatment/control comparison)

Results and discussion

Test results
Key result
After dosing, all animals of the dose level of 300 mg/kg body weight were lethargic, showed ataxia and tremors. Within 17 hours all animals had recovered from the treatment.
Vehicle controls validity:
Positive controls validity:
Additional information on results:
The animals of the groups treated with 150 and 75 mg/kg body weight and the animals of the negative and positive control groups showed no abnormalities.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.
No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with TBP.
The groups that were treated with TBP showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with CP showed a decrease in the ratio of polychromatic erythrocytes compared to the vehicle controls.

Applicant's summary and conclusion

It is concluded that TBP is not mutagenic in the micronucleus test under the experimental conditions described in this report.