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:
2008-07-08 to 2008-08-07
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-01-19
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SymHelios 1031
- Physical state: solid, powder, light yellow
- Storage condition of test material: At ambient temperature, dark, dry, in original container.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, 33178 Borchen, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males mean value 38.3 g (SD ± 1.7 g) and females mean value 30.3 g (SD ± 2.0 g)
- Assigned to test groups randomly: yes, under following basis: The animals were distributed into the test groups at random and identified by cage
number.
- Housing: single; The animals were kept conventionally. Animals were kept in Makrolon Type I cages, with wire mesh top and a granulated soft wood bedding.
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.
No further details are given.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO + PEG 400
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: In the main experiment SymHelios 1031 was formulated in 30% DMSO + 70% PEG 400. The volume administered was 10 mL/kg b.w..

DIET PREPARATION
not applicable
Duration of treatment / exposure:
once
Frequency of treatment:
single application
Post exposure period:
24 and 48 hours after treatment, respectively
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg b.w.
Basis:
nominal conc.
24 hours treatment
Remarks:
Doses / Concentrations:
1000 mg/kg b.w.
Basis:
nominal conc.
24 hours treatment
Remarks:
Doses / Concentrations:
2000 mg/kg b.w.
Basis:
nominal conc.
24 and 48 hours treatment
No. of animals per sex per dose:
Six males and six females were assigned to each dose group. The animals were identified by their cage number.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CAS 6055-19-2); dissolved in deionised water
- Justification for choice of positive control(s): according to guideline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: prliminary test; The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

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 using a syringe. The cell suspension was centrifuged 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/Giemsa. Cover slips were mounted with EUKITT. 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. 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. The analysis was performed with coded slides.

OTHER: 10 animals (5 males, 5 females) per dose group were evaluated as described. The remaining 6th animal of each sex in the respective test group is usually evaluated in case an animal dies in its dose group spontaneously.
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. 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.
Statistics:
Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
The mean values of micronuclei observed after treatment with SymHelios 1031 were near to the value of the vehicle control group.
Toxicity:
no effects
Remarks:
After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that SymHelios 1031 did not exert any cytotoxic effects in the bone marrow.
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Four experiments were conducted each in a total of 4 animals (2 males and 2 females).
- Dose range: The animals received orally a single dose of 100, 500, 1000 or 2000 mg/kg b.w. SymHelios 1031 formulated in 30% DMSO + 70% PEG 400. The volume administered was 10 mL/kg b.w..
- Clinical signs of toxicity in test animals: Male animals that received up to 1000 mg/kg b.w. test item showed ruffled fur 1 hour to 6 hours after treatment. Male test animals receving 2000mg/kg b.w. test item showed a reduction of spontaneous activity (2-30 hours after treatment) and ruffeled fur (up to 48 hours after treatment). No toxic reactions were observed in female test animals. All test animals' urine was coloured orange after treatment with 1000 and 2000 mg/kg b.w. (6-24 hours after treatment). On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 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 SymHelios 1031 were near to the value of the vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): see under "any other information on results incl. tables".
- Appropriateness of dose levels and route: The urine of the treated animals was discoloured and the spontaneous activity of the animals was reduced, thus indicating the systemic distribution of the test item and its bioavailability.

Any other information on results incl. tables

A summary of the test results is attached as background material.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
This study was performed to investigate the potential of SymHelios 1031 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, SymHelios 1031 is considered to be non-mutagenic in this micronucleus assay.