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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Link to relevant study records
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
GLP compliance:
Type of assay:
micronucleus assay
Details on test animals or test system and environmental conditions:
- Source: Charles River Ltd, Margate, UK
- Age at study initiation: 35-42 weeks
- Weight range at study initiation: males 25-31 g; females 20-28 g
- Housing: in groups of no more than 3 animals of the same sex in appropriate caging
- Diet and water: ad libitum
- Acclimation period: 6 days

- Temperature (°C): 19-21°C
- Humidity (%): 51-59
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
1% (m/v) methylcellulose
Details on exposure:
The test substance was injected intraperitoneally.
Duration of treatment / exposure:
Animals were treated once.
Frequency of treatment:
Single intraperitoneal administration of the test substance or the positive or negative control substance.
Post exposure period:
The animals were sacrificed for bone marrow preparation 24 hours after test substance administration. Additional groups of animals receiving 150 mg/kg were sacrificed 48 hours after test substance administration.
Doses / Concentrations:
125, 250 and 375 mg/kg bw
other: main study, trial 1
Doses / Concentrations:
25, 75 and 150 mg/kg bw
other: main study, repeat
No. of animals per sex per dose:
5 for sacrifice time 24 hours; only for 150 mg/kg bw: 5 for sacrifice time 48 hours
Control animals:
other: yes, for negative control concurrent vehicle, sacrifice time 24 and 48 hours... (see attached file)
Positive control(s):
cyclophosphamide, dissolved in physiological saline
- Route of administration: intraperitoneally
- Doses / concentrations: single dose of 40 mg/kg bw, sacrifice time 24 hours after substance administration.
Tissues and cell types examined:
Bone marrow smears examined.
Details of tissue and slide preparation:
Data from the range-finding study and the main study, trial 1 were combined, and a maximum acceptable dose determined. This was used as the highest level for the repeat of the main study.

Both femurs from each animal were used for slide preparation. Staining of the slides was achieved according a modification of the method of Gollapudi and Kamra, Mutation Res 64, 1979, 45-46.

METHOD OF ANALYSIS: Initially the relative proportions of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were determined 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. The ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequency of micronucleated PCE/1000 were also determined.

Evaluation criteria:
The test article was to be considered as positive in this assay if a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time, and the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
The group mean frequencies of micronucleated PCE in vehicle control animals were compared with historical negative control ranges to determine whether or not the assay was acceptable. For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity Chi²-test. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine Chi². Probability values of p
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
Groups of 3 male and female mice received a single i.p. administration of the test substance at doses covering the range of 357-2000 mg/kg bw. Within the 4-days observation period following administration all animals exhibited clinical signs. Mortalities were observed at all doses except 357 mg/kg.

Excessive numbers of mortalities at 250 and 375 mg/kg in this trial resulted in an insufficient number of animals available for sampling.

No significant increases in frequencies of micronucleated cells were seen in the test groups. At the maximum dose levels, systemic toxicity and findings suggestive of effects on bone marrow (a trend towards reduced PCE/NCE ratios) were seen.
Executive summary:

A mouse bone marrow micronucleus test (according to EU Method B.12) was conducted with 5 male and 5 female mice receiving each a single intraperitoneal injection of a test substance dose in vehicle (1% methylcellulose). For the first main testing doses of 125, 250 and 375 mg/kg bw were employed, but excessive numbers of mortalities at the higher dose groups resulted in an insufficient number of animals available for sampling. Therefore the testing was repeated with doses of 25, 75 and 150 mg/kg bw. Animals were sacrificed for preparation of bone marrow smears 24 and 48 hours after test substance administration, the latter only for control and high dose group animals.

In the dose group 150 mg/kg bw clinical signs and mortalities could be observed, and also findings suggestive of effects on bone marrow (a trend towards reduced PCE/NCE ratios). No statistically significant increases in frequencies of micronucleated cells were seen in the test-groups, therefore it was concluded that the test substance was not genotoxic in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro toxicity testing revealed negative results for gene mutation assays (Ames, OECD TG 471; HPRT, OECD TG 476), but a positive result for an in vitro chromosomal aberrations test with and without metabolic activation (OECD TG 473). A micronucleus in vivo test (EU Method B.12) with single intraperitoneal injections of the test substance up to doses of 150 mg/kg yielded a negative result and does not confirm a clastogenic potential. As clinical signs and mortalities were observed in the in vivo experiment an adequate systemic availability could be assumed. Therefore, the substance is not expected to be genotoxic in vivo.

Justification for selection of genetic toxicity endpoint
Highest tier study available for the endpoint

Justification for classification or non-classification

Not classified for genetic toxicity according to Regulation (EC) No 790/2009 (Amendment to Regulation (EC) No 1272/2008) and based on the criteria set out in Annex I to Regulation (EC) No 1272/2008 or in Annex VI to Council Directive 67/548/EEC (June 1967).