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:
2012/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 1997
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
adopted August 1998
GLP compliance:
yes (incl. certificate)
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): Laromer LR8869
- Physical state: clear colorless liquid
- Analytical purity: The test substance is a complex mixture of isomers and homologous components (see analytical report, study code 11L00263).
- Purity test date: 2012-01-11
- Lot/batch No.: 110009P040
- Expiration date of the lot/batch: 11/2012
- Stability under test conditions: confirmed indirectly by dose formulation analytics
- Storage condition of test material: room temperature, below 25°C, protected from light

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 35.6g (SD +/- 1.5g)
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: relative non-toxicity
- Amount administered: 10ml/kg b.w.
- Lot/batch no. (if required): MKBF8603V
Duration of treatment / exposure:
24h and 48h (only highest dose tested)
Frequency of treatment:
once
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
7 (5 for vehicle and positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral, gavage in sterile water
- Doses / concentrations: 40mg/kg b.w.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the maximum recommended dose of 2000mg/kg b.w. was tolerated without causing toxic reactions

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 at 1500 rpm (390 x g) 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-Gruenwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 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. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate 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.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

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.

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:
nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
only clinical signs evaluated
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): not altered
- Statistical evaluation: no significant increase detected

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance is considered to be non-mutagenic in this micronucleus assay.