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:
September 09 - December 14, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles. Limited information available to verify the composition of the used test substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1983)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."
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): KY-ET
- Physical state: Light yellow solid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: minimum 11 weeks
- Weight at study initiation: 28 - 32 g
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment
- Housing: Single in Makrolon Type I cages with wire mesh top containing granulated soft wood bedding.
- Diet (e.g. ad libitum): Free access to standard pelleted diet ( ALTROMIN 1324, D-4937 Lage/Lippe)
- Water (e.g. ad libitum): Free access to tap water (Gemeindewerke, D-6101 Rosdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70 (The upper limit was exceeded for short periodes; maximum 90%)
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose, 1% aqueous solution)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in 1% aqueous CMC solution.

Dosing volume administered:

Test substance solution and negative control (vehicle): 20 mL/kg body weight.
Positive control: 10 mL/kg body weight.
Duration of treatment / exposure:
Test substance and negative control: 24, 48 and 72 hours.
Positive control: 24 hours.
Frequency of treatment:
Single,
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Pre test: 2
Main study: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
1% Cyclophosphamide (CPA) in physiological saline
- Route of administration: Orally once
- Doses / concentrations: 30 mg/kg body weight (10 mL/kg body weight)

Examinations

Tissues and cell types examined:
Measuring the increase of the number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in mouse bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selection of an adequate dose for the Micronucleus test was based on a preliminary study. It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals were weighed and the individual volume to administered was adjusted to the animal's body weight. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and than stained with May-Grunwald/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 microscopes with 100x oil immersion objectives. 1000 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 normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg body weight
- Solubility: the test substance was formulated in 1% CMC
- Clinical signs of toxicity in test animals: the treated animals expressed no toxic reactions.

RESULTS OF DEFINITIVE STUDY, see "Any other information on results incl. tables"

- Induction of micronuclei: In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test substance. The mean values of micronuclei observed after treatment with the test substance were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls.

Any other information on results incl. tables

Summary of results

Test group

Dose mg/kg bw

Sampling time (h)

PCEs with micronuclei (%)

Range

PCE/NCE

Vehicle

0

24

0.11

0 - 3

1000/788

Test substance

5000

24

0.09

0 - 4

1000/761

Cyclophosphamide

30

24

2.49

9 - 44

1000/749

Vehicle

0

48

0.10

0 - 3

1000/710

Test substance

5000

48

0.08

0 - 2

1000/706

Vehicle

0

72

0.10

0 - 3

1000/652

Test substance

5000

72

0.04

0 - 2

1000/682

 

Cyclophosphamide showed a distinct increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
An in vivo micronucleus study with KY-ET in the mouse (5 animals per sex/dose, orally administration) was conducted according to OECD 474 and GLP guidelines. It is concluded that KY-ET is not clastogenic in the micronucleus test under the experimental conditions described in this report.
Executive summary:

An in vivo micronucleus study with KY-ET in the mouse (5 animals per sex/dose, orally administration) was conducted according to OECD 474 and GLP guidelines. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test substance. The mean values of micronuclei observed after treatment with the test substance were in the same range as compared to the negative control groups. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that KY-ET has no cytotoxic properties. It is concluded that KY-ET is not clastogenic in the micronucleus test under the experimental conditions described in this report.