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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
1000 bone marrow cells (type not specified). No distinction between immature and mature erythrocytes.
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(2,4-ditert-butylphenyl) phosphite
EC Number:
EC Name:
Tris(2,4-ditert-butylphenyl) phosphite
Cas Number:
Molecular formula:
tris(2,4-ditert-butylphenyl) phosphite
Details on test material:
- Substance type: organic- Physical state: solid

Test animals

hamster, Chinese
Details on test animals or test system and environmental conditions:
- Weight at study initiation: 18 - 26 g (females) and 19 - 27 g (males)
- Diet: ad libitum, standard diet: NAFAG No.924
- Water: ad libitum, tap water

- Temperature (°C): 21 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Concentration of test material in vehicle: 500, 1000, and 2000 mg/kg in 20 mL/kg PEG 400
- Amount of vehicle (if gavage or dermal): 20 mL/kg
Duration of treatment / exposure:
48 h
Frequency of treatment:
once daily
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, 128 mg/kg in 20 mL/kg PEG 400
- Route of administration: gavage
- Doses / concentrations: 128 mg/kg


Tissues and cell types examined:
Bone marrow from the shafts of both femurs
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times.

Small drops of the above mentioned mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. At the next day the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40 % for 20 min. After being rinsed in methanol 55 % for 5 - 8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying the slides were cleared in Xylol and mounted in Eukitt.

The slides of three female and three male animals each of the negative control group and the treatment groups were examinedIn the positive control group the slides of four female and two male animals were analysed. 1000 bone marrow cells each were scored per animal and the following anomalies were registered:a) Single Jolly bodies, b) fragments of nuclei in erythrocytes,c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.
The significance of difference was assessed by χ2- test

Results and discussion

Test results
not examined
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:

Any other information on results incl. tables

In the control group one female and one male animal died after the second application. In the positive control group one male animal died after the second application.

In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 7.9, whereas the negative control yielded a percentage of 0.1. The difference was highly significant (p<0.05).

percent of cells with anomalies of nuclei
number of animal sex of animal single jolly bodies frasgments of nuclei in erythrocytes micronuclei in erythroblasts micronuclei in leucopoietic cells polyploid cells total
(PEG 400)
1 f 0.1 0.1
2 f 0.1 0.1
3 f 0.1 0.1 0.2
4 m 0
5 m 0.1 0.1
6 m 0.1 0.1
(128 mg/kg)
1 f 5.2 0.2 0.6 0.2 0.3 6.5
2 f 4.4 0.9 0.7 0.3 6.3
3 f 3 1.2 0.7 4.9
4 m 4.1 1.9 0.1 0.1 6.2
5 m 10.6 1.5 1.5 0.8 0.3 14.7
6 m 6.8 0.7 1.1 0.1 0.1 8.8
test article (500 mg/kg) 1 f 0.2 0.2
2 f 0.3 0.3
3 f 0
4 m 0.1 0.1
5 m 0
6 m 0.2 0.2
test article (1000 mg/kg) 1 f 0.2 0.1 0.3
2 f 0.1 0.1
3 f 0.2 0.2
4 m 0.1 0.1
5 m 0.1 0.1
6 m 0.1 0.1
test article (2000 mg/kg) 1 f 0.1 0.1
2 f 0
3 f 0.3 0.3
4 m 0.1 0.1
5 m 0.1 0.1
6 m 0.1 1

Applicant's summary and conclusion

The MNT assay in chinese hamster was found to be negative.
Under the conditions of this experiment, no evidence of mutagenic effects was obtained with the test article.