Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP regulation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
SANDOLAN BLUE E-HRL
IUPAC Name:
SANDOLAN BLUE E-HRL
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf; Switzerland
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 40.0 g (SD ± 3.4 g); females mean value 31.5 g (SD± 1.6 g)
- Housing: Makrolon Type I, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 24 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water is a known inocciousstandard vehicle
- Concentration of test material in vehicle: 50 to 200 mg/ mL
- Amount of vehicle: 10 ml/kg b.w

Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
single
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/mL
Basis:
other: actual injected
Remarks:
Doses / Concentrations:
1000 mg /mL
Basis:
other: actual injected
Remarks:
Doses / Concentrations:
2000 mg/ mL
Basis:
other: actual injected
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): usual standard
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw.

Examinations

Tissues and cell types examined:
bone marrow, polychromatic arythrocytes
Details of tissue and 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 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 resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITI (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 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 normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.03 - 0.15 %; mean = 0.086 ± 0.027 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (1.0 - 2.71 %; mean = 1.653 ± 0.409 PCEs with micronuclei).
- at least 80 % of animals are evaluable

A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 2000 mg/kg: reduction of spontaneous activity, apathy and closure of eyes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that the test item had no cytotoxic properties in the bone marrow. However, the analytics with the bone marrow samples showed that the item was bioavailable. As shown in the analytics report the bone marrow samples of the male mice contained 5.27, 5.12 and 3.75 µg and the samples of the female mice had 3.56, 6.65 and 6.27 µg of the test item one hour post treatment. The test item concentrations were comparatively lower in the bone marrow samples taken 4 hours after the treatment (test item concentrations in the bone marrow samples were 1.40, 1.30 and 0.847 µg/sample for the male mice and 0.525, 1.37 and 2.59

µg/sample in the female mice), indicating the excretion or metabolism of the test item.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered intraperitoneally was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w ..

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that the test item had no cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as positive control which showed a substantial increase of induced micronucleus frequency.

CONCLUSION

In conclusion, it can be stated that during the study described and 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, the test item is considered to be non-mutagenic in this micronucleus assay.

Categories Display