Reddish Blue

Genetic toxicity: in vivo

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 for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

Name: Reddish Blue

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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; 289-349 g males, 194-247 g females
Acclimation period: 14 days


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: 20.8 – 24.9°C
Relative humidity: 32%-69%
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 quality control results are retained in the archives at LAB Research Ltd. and are reported in Appendix 9.
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.

Animal identification

Each parental animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at LAB Research Ltd. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.
The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

Randomization

All parental (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The test item was formulated in the vehicle (1:1 PEG 400 and Sterile Distilled Water mixture) as it is not water soluble. The control groupe received the vehicle only. The dose volume administered was 8 mL/kg.

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. TheThe test item was formulated in the vehicle (1:1 PEG 400 and Sterile Distilled Water mixture) at 7.81, 31.25 and 125 mg/mL in the Central Dispensary of CiToxLAB Hungary 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.

The dose volume administered was 8 mL/kg.
Ten Wistar rats (5 male and 5 female), Group 5, served as the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT). They were treated with 30 mg/kg bw/day Cyclophosphamide by oral gavage (dose volume, 10 mL/kg, concentration, 3 mg/mL) on Day 27, 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.

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:

yes

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 grey discoloration of the urine during the treatment perion at 250 and 1000 mg/kg bw.

Haematological changes considered related to treatment and suggesting a regenerative anaemia were observed in the 1000 mg/kg bw/day animals at the end of the treatment period although the findings were not present following the 14 day recovery period.

Macroscopic changes including small testes and/or epididymides and spleen enlargement were observed at 1000 mg/kg bw/day and correlated with the organ weight and microscopic changes. The microscopic changes in the testes consisted in bilateral diffuse mild tubular atrophy/degeneration in the testes and bilateral mild to moderate reduction of the sperm content accompanied with ductal cell debris and apoptotic bodies in the epididymides. These changes were also noted in animals following the recovery period, however with a slightly reduced severity in recovery animals, indicating a possible tendency to recover after cessation of the treatment. In the spleen, a slight increase in the severity of extramedullary haematopoiesis was noted at 1000 and 250 mg/kg bw/day and was considered correlated with the spleen response to the slight regenerative anaemia in these animals. Recovery animals showed no such effects.

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

Any other information on results incl. tables

Statistical analysis of the frequencies of micronuclei in the high dose and control males gave a value of H = 0.082 (not significant). The frequencies in the high dose females were lower than the control females, so no statistical analysis was appropriate. The evaluation thus showed a clear negative result for the test item at 1000 mg/kg bw/day in both sexes, thus, no further slide examination was considered required. 

 

The positive and negative control results were also compared, and while the females gave a marginally significant response, with a value of H = 4.158 (p<0.05), the males gave a non-significant response (H = 0.676,not significant). However, the PCE:NCE ratios showed high levels of toxicity, particularly in the males, in which situation the PCE population observed at the time of sacrifice is considered to be potentially non-representative. 

 

While the animals treated with the positive control compound had not therefore shown an adequate response, the positive control data from a concurrently run study under identical conditions for the same sponsor (CiToxLAB study code 09/067-100P) were used for comparison purposes with Sponsor’s approval (amendment 3 to the study plan) and gave a value ofH = 5.963 (p<0.05) when compared with the negative control animals in this study, giving a significant positive response to cyclophosphamide at 30 mg/kg bw/day considered suitable for the study purposes. 

 

In view of the potential effects on the haematopoiesis, which appeared to be an adaptive response to haematology changes, it should be noted that no effect was observed on the erythrocytes used to evaluate the micronucleus test in the animals treated with the test item. Comparison of the PCE/NCE ratios in the negative control animals and those treated with 1000 mg/kg bw/day, and the negative and positive controls using a Student’s t-test showed no effect at 1000 mg/kg in either sex, but a reduction in the proportion of PCEs in the positive control animals, as follows: 

 

COMPARISON OF PCE/NCE RATIOS USING STUDENT’S T-TEST

 

Group 1	Group 2	Probability	Significance
Negative control males	High dose males 1000 mg/kg	0.256	Not significant
Negative control males	Positive control males	<<0.001	***
Negative control females	High dose females 1000 mg/kg	0.416	Not significant
Negative control females	Positive control females	0.003	**

 

The above statistical analysis and the observation that there was no increase in the numbers of micronucleated PCEs in either sex confirm that no additional analysis of lower doses was 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.