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:
15 July 2009 to 06 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Thiazol Blau

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: CRL: (WI) BR rats
Source: TOXI COOP Ltd., Cserkesz u. 90. 1103 Budapest, Hungary
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of strain: Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies
Number of animals: 35 male and 35 female rats
5 rats/sex/group, 4 Main groups (Groups 1 to 4); 5 rats/sex/group, 2 Recovery groups (Groups 1 and 4)
5 rats/sex/group, 1 Positive control group (Group 5)
Age of animals: Young adult rats, 7-8 weeks old at onset of treatment
Body weight: The weight variation did not exceed +/- 20 percent of the mean weight/sex at onset of treatment with the test item; 243-293 g males, 161-217 g females
Acclimation period: 14 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Cage type: Type III. polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG, D-73494 Rosenberg Holzmühle 1 Germany, and Laboratory Animal Bedding produced by Brandenburg Holzfaserstoffe Gmbh& Co.KG, Arkeburger Str. 31, 49424 Goldenstedt, Germany.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.6 – 24.1°C
Relative humidity: 44-70%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed (up to 5 animals/sex/cage), to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

The temperature and relative humidity were recorded twice daily; no deviations from the intended range occurred during the study.


Food and water supply

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The test item was formulated in PEG400 at concentrations of 15.6, 62.5 and 250 mg/mL in the Central Dispensary of LAB Research Ltd. Formulations were prepared at the appropriate frequency for use within 72 hours as documented in the raw data, while stored refrigerated, according to stability assessment results

Name: Polyethylene glycol 400 (PEG400), Ph. Eur
Lot No.: 1421464
Manufacturer: Fluka / Sigma-Aldrich
Expiry Date: September 2010
Storage: Room temperature
Details on exposure:
Dosing procedure
The dosing solutions were administered daily for at least 28 days by oral gavage, using a bulb tipped gastric gavage needle attached to a syringe. The first day of treatment was regarded as Day 0. The oral route of administration was selected because it is the most likely route for possible human exposure. A constant volume of 4 mL/kg body weight was administered to all test-item treated and the vehicle/control rats.

The positive control rats (Group 5) for the Mammalian Erythrocyte Micronucleus Test were administered Cyclophosphamide by oral gavage, at a dose level of 15 mg/kg bw on Day 27, at 10 mL/kg, 1.5 mg/mL, approximately 24 hours prior to scheduled necropsy on Day 28.

The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.




Duration of treatment / exposure:
28 days

Frequency of treatment:
Once daily, 7 days per week.
Post exposure period:
Positive control animals were euthanised approximately 24 hours after administration of cyclophosphamide.

Doses / concentrations
Remarks:
Doses / Concentrations:
62.5, 250 and 1000 mg/kg bw/day
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
other: Positive control: cyclophosphamide
Positive control(s):
Positive Control Micronucleus Test (MNT) animals
Group 5 animals were treated with 15 mg/kg bw/day Cyclophosphamide by oral gavage at 10 mL/kg, 1.5 mg/mL on Day 27, approximately 24 h prior to scheduled necropsy on Day 28.

Name: Cyclophosphamide
Lot No.: 068K1131
Manufacturer: Fluka / Sigma-Aldrich
Expiry Date: August 2011
Storage: In refrigerator (2-8 °C)

Examinations

Tissues and cell types examined:
Bone marrow slides were prepared from all animals, including the vehicle control and the positive control groups. The bone marrow was obtained from the right femur of the rats immediately after euthanasia and flushed with foetal bovine serum (5 mL).
Details of tissue and slide preparation:
Cells were concentrated by a gentle centrifugation. Smears of the cell pellet were made on standard microscope slides and the slides were then air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for at least 5 minutes and allowed to air-dry.

One set of Giemsa-stained slides was given unique code numbers for blinded evaluation (the code labels covered all unique identification markings on the slides to ensure that they were scored without bias). All slides were blinded; only those of the Control (Gr. 1), Positive Control (Gr. 5) and High dose (Gr. 4) Main animals were sent for evaluation.

2000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated (MN) cells. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei was recorded in both types of erythrocytes.

Criteria for Identification of Micronucleated Erythrocytes

A micronucleus is defined in following way:

- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

The Micronucleus Test is considered acceptable/valid in the conditions of this study, as it met the following criteria:

-the frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls fell within the range of historical laboratory control data.
-the positive control item produced biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
-each treated and control group included at least 5 analysable animals.

Evaluation criteria:
Criteria for a positive response: The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related.

Criteria for a negative response: The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response: It may be necessary to perform further investigations or to score additional cells if equivocal results are obtained which do not meet the criteria for a positive or negative response. In this study there were no equivocal results, therefore no additional scoring was required.
Statistics:
Data were collected by completing a pre-prepared sheet by hand. The data were tabulated using appropriate forms for reporting. The frequencies of micronucleated polychromatic erythrocytes in animals in the test groups were compared to the values found in the corresponding negative control group. Statistical analysis was performed using Kruskal Wallis Non Parametric ANOVA test (level of significance 5%). Statistical analysis of the positive control data was not necessary as all values were higher than any of the corresponding negative control values.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
discoloration of urine
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Exposure of the animals to the test item was proved by blue discoloration of the urine during the treatment perion at 250 and 1000 mg/kg bw.

No induction of micronuclei in bone marrow erythrocytes was observed following administration of Thiazol Blau to Wistar rats daily by oral gavage for 28 days at up to and including 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Comparison of the vehicle control data and the high dose of the test agent (1000 mg/kg) using the Kruskal-Wallis test gave a value of H = 0.147. This is non-significant, giving a negative response.

The positive and negative control results were also compared, and gave a value of H = 10.089 (p<0.01>0.001). This confirms a positive response in the group treated with cyclophosphamide.

The positive and negative control data were considered to give adequate data to confirm the validity of the study. The evaluation showed a clear negative result for the test item at 1000 mg/kg bw/day, thus, no further slide examination was considered required.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Thiazol Blau to Wistar rats daily by oral gavage for at least 28 days at up to and including 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
Executive summary:

The objective of this study was to assess the potential genotoxic effect of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals.

This study was conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of Thiazol Blau to Wistar rats daily by oral gavage for at least 28 days at up to and including 1000 mg/kg bw/day, thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.