Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-02 to 2008-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-01-19
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: white to light yellow powder
- Molecular formula: C16H15NO4
- Molecular weight: 285.3 g/mol


Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, Borchen, Germany
- Age at start of acclimatisation: 8 - 10 weeks
- Weight at study initiation: males: mean value 38.9 g (SD ± 1.2 g); females; mean value 31.2 g (SD ± 2.6 g)
- Assigned to test groups randomly: yes
- Housing: single; Makrolon Type I, with wire mesh top (EHRET GmbH, Emmendingen, Germany); granulated soft wood bedding (Harlan Laboratories GmbH, Borchen, Germany)
- Diet (ad libitum): pelleted standard diet (Harlan Laboratories GmbH, Borchen, Germany)
- Water (ad libitum): tap water (Gemeindewerke, Rossdorf, Germany)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Relative humidity: 30 - 70% (the aim was 50-60%)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: the test item was formulated in 30% DMSO/70% PEG 400
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals. furthermore, the vehicle does not react with the test substance
Details on exposure:
All animals received a single standard volume of 10 mL/kg bw orally.
Duration of treatment / exposure:
1 day
Frequency of treatment:
once
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
6 males and 6 females (except for the highest dose level: 12 males and 12 females)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw (dissolved in deionised water)(volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:
At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.
Ten animals (5 males, 5 females) per test group were evaluated. The remaining 6th animal of each sex in the respective test group is usually evaluated in case an animal dies in its test group spontaneously.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of all dose groups (excepted the positive control) were examined for acute toxic symptoms at intervals of around 1 hour, 2 - 4 hours 6 hours, 24 hours and 48 hours after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively (48 hours only for high dose group).

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum. The cell suspension was centrifuged at for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, Darmstadt, Germany)/Giemsa (Merck, Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.

ACCEPTANCE CRITERIA:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data.
- the positive controls are in the range of our historical control data.
- at least 5 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (Krauth, 1971)*) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

*Reference
- Krauth, J. (1971): Locally most powerful tied rank test in a Wilcoxon situation, Annals of Mathematical Statistics, 42, 1949 - 1956.
Statistics:
Please refer to "Evaluation criteria" above

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
please refer tp "Additional information on results" below
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT FOR TOXICITY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals:
a) ruffled fur:
1- 6 hours after administration: all animals
24 and 30 hours: 2 animals
b) excitement:
1 - 4 hours: one animal

On the basis of these data 2000 mg/kg bw were estimated to be suitable.

TOXIC SYMPTOMS IN THE MAIN EXPERIMENT
2000 mg/kg bw: all animals had ruffled fur 1 to 6 hours after administration. 12 animals had ruffled fur 24 hours after administration.
1000 mg/kg bw: 5 animals had ruffled fur 1 to 4 hours after administration. 6 animals had ruffled fur 6 hours after administration.
Neither the animals treated with the low dose nor the vehicle control treated animals expressed any toxic reactions.

RESULTS OF DEFINITIVE STUDY
The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not mutagenic.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as mutagenic.