Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to OECD 407 and GLP guideline

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 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

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


Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524
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.


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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
The test item was formulated in PEG400 at concentrations of 15.6, 62.5 and 250 mg/mL. 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle under the conditions employed in the study was performed in the Analytical Laboratory of LAB Research Ltd.

Concentration and homogeneity of formulations were evaluated by UV-HPLC method on duplicate samples collected from the top, middle and bottom of test item solutions, and one sample from the control taken and analyzed fresh during the first and last weeks of treatment. Dose formulations were homogenous. The measured (actual) concentrations varied between 100% and 105% of the nominal concentrations. No test item was detected in the control solution samples.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
15.625, 62.5, and 250 mg/mL
Basis:
other: formulation analytics (UV-HPLC ): 91% to 103% of nominal concentrations
No. of animals per sex per dose:
Groups 1 to 4: 5 animals/sex/main group
Groups 1 and 4: 5 animals/sex/recovery group
Group 5: 5 animals/sex positive control
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-selection: Acute oral toxicity testing of Thiazol Blau at a dose level of 2000 mg/kg in the rat (LAB study code: 09/068-001P) did not result in any signs of test item-related toxicity. The median lethal dose level (LD50) was above 2000 mg/kg for oral administration in the female rat. Based on these results and due to experience with similar compounds, dose levels of 62.5, 250, and 1000 mg/kg/day were selected for use in the study.

During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight. SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Main animals underwent necropsy on Day 28 (start of treatment, Day 0), after 28 days of treatment. Recovery animals were treated for 28 consecutive days, followed by a 14-day recovery/observation period after the last dose and were then euthanized and subjected to a necropsy examination.
Positive control:
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.

Examinations

Observations and examinations performed and frequency:
Clinical Observations and Functional Observation Battery
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). General clinical observations were made once a day, after the treatment at approximately the same time, and daily during the recovery period. Detailed clinical observations were made on all animals outside the home cage in a standard arena, once prior to the first exposure and once weekly thereafter. Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on all animals towards the end of treatment period on Day 26. General physical condition and behaviour of animals were tested. A modified Irwin test was performed. A detailed assessment of any potentially treatment-related neurotoxic effects was made on the basis of these measurements.


Oestrus cycle evaluation
Prior to necropsy, vaginal smears were taken from all females, stained with 1% aqueous methylene blue solution and examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Body weight measurement
Individual body weight was recorded with precision of 1 g at randomization (Day 7), on the first day of treatment (Day 0), then at least weekly and prior to necropsy (fasted for scheduled necropsy).


Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 0 (pre-treatment, start on Day -7) then at least weekly. The weekly and mean daily food consumption was calculated.


CLINICAL PATHOLOGY
Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia. After an overnight period of food deprivation of animals, three samples were taken from each animal: one for haematology (1.2 mL blood; tube contained K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for determination of blood clotting times (1.2 mL blood; APTT and PT; tube contained sodium citrate as anticoagulant) and the third one to obtain serum samples (at least 2.4 mL blood as practical, in a tube with no anticoagulant) for clinical chemistry.

Haematology and Blood Clotting Times
The following parameters were evaluated in all animals:
Red Blood Cell (erythrocyte) count, White Blood Cell (leukocyte) count, Haemoglobin concentration, Haematocrit, Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume (%) Distribution Width, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Partial Thromboplastin Time, Prothrombin Time,


Clinical chemistry
The following parameters were evaluated in all animals:
Glucose Blood sugar concentration, T-BIL Total Bilirubin concentration, Urea Urea concentration, Chol. Cholesterol concentration, Creat. Creatinine concentration, Phos. Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ratio, Aspartate Aminotransferase activity, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids
Sacrifice and pathology:
Terminal procedures
Following blood sample collection for clinical pathology evaluation, necropsy and macroscopic examination was performed on every Main Study or Recovery animal euthanized at the end of the treatment or recovery periods, with the exception of positive control Group 5 animals (Mammalian Erythrocyte Micronucleus Test in Bone Marrow Cells), for which bone marrow was collected from the right femur immediately after euthanasia, after which the carcasses were discarded without further examination. Surviving animals were euthanized by exsanguination under pentobarbital anaesthesia.

Macroscopic examination
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically.
Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

On completion of the macroscopic examination and organ weight measurements according to OECD 407 were evaluated.

Organ weight measurement
The following organs were weighed and recorded: with precision of 0.01 g, liver, kidneys, testes, epididymides, uterus, including cervix, prostate, seminal vesicle with coagulating glands as a whole, thymus, spleen, brain and heart; with precision of 0.001 g, ovaries, adrenals.
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and are reported.

Histopathology
Full histopathology was performed in Group 1 (Control) and Group 4 (High Dose), main and recovery animals and on all organs and tissues with macroscopic findings. Representative tissue samples were embedded into paraffin and slides were prepared, then stained with haematoxylin-eosin/phloxine and examined with light microscope. As no microscopic findings recorded in High Dose animals were considered potentially treatment-related, no further histology examination was performed on other tissues or organs from the lower dose levels.
Statistics:
Data were collected using the software PROVANTIS v.7 or recorded on the appropriate forms from the relevant SOPs of LAB Research Ltd., then tabulated using the software PROVANTIS v.7, Microsoft Office Word and/or Excel, as appropriate.

Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations. The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The frequency of clinical observations and necropsy and histopathology findings was calculated as applicable.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
There were no changes that could be ascribed to Thiazol Blau administration noted in the body weight and body weight gain, animal food consumption, or clinical pathology parameters (haematology, coagulation and clinical chemistry) evaluated at the completion of the 28-day treatment period (all dose levels) or at the end of a 14 day recovery period (control, and 1000 mg/kg bw/day animals).

There were no test item related changes in the female oestrus cycle examined prior to necropsy. No macroscopic or microscopic changes or changes in the organ weights related to Thiazol Blau administration were recorded in either main or recovery animals examined. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects in parameters investigated

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Thiazol Blau administered daily by oral gavage for 28 days in Wistar rats did not lead to test item related mortality or clinical adverse effects, including animal behaviour, general physical condition, reactions to different type of stimuli, grip strength or motor activity, at dose levels of 62.5, 250, or 1000 mg/kg bw/day during either the treatment or 14-day recovery/observation periods.

There were no changes that could be ascribed to Thiazol Blau administration noted in the body weight and body weight gain, animal food consumption, or clinical pathology parameters (haematology, coagulation and clinical chemistry) evaluated at the completion of the 28-day treatment period (all dose levels) or at the end of a 14 day recovery period (control, and 1000 mg/kg bw/day animals).

There were no test item related changes in the female oestrus cycle examined prior to necropsy. No macroscopic or microscopic changes or changes in the organ weights related to Thiazol Blau administration were recorded in either main or recovery animals examined. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for Thiazol Blau is considered to be 1000 mg/kg bw/day.
Executive summary:

The objective of the study was to assess the toxicity of Thiazol Blau when administered daily for 28 days by oral gavage to CRL:(WI)BR rats. The reversibility of any treatment-related changes was evaluated following a 14-day recovery period. In addition, the test item was evaluated for genotoxic effects by examiningthe induction ofmicronuclei in bone marrow erythrocytes of treated and control animals.

Thiazol Blau, the vehicle or cyclophosphamide were administered to seventy Wistar rats (20 male and 20 female Main animals, 10 male and 10 female Recovery animals, and 5 male and 5 female positive control animals)according to the following experimental design:

 

Group No.

Group Designation
Dose Level
(mg/kg/day)

Conc. (mg/mL)

Dose volume

(mL/kg)

Number of Animals

Main

Recovery

Male

Female

Male

Female

1

Control

0

0

4

5

5

5

5

2

Low Dose

62.5

15.6

5

5

-

-

3

Mid Dose

250

62.5

5

5

-

-

4

High Dose

1000

250

5

5

5

5

Mammalian Erythrocyte Micronucleus Test Positive Control Group Animals*

Group

Cyclophosphamide

Male

Female

No

Designation

(mg/kg/day)

(mg/mL)

(mL/kg)

5

Positive Control

15

1.5

10

5

5

*administered approximately 24 hours prior to scheduled necropsy

 

Main animals underwent necropsy on Day 28 (start of treatment, Day 0), after 28 days of treatment. Recovery animals were treated for 28 consecutive days and were euthanized and subjected to necropsy after a 14-day recovery period. Control animals received the vehicle item solution only (PEG400), at the same volume as the high dose animals.No correction for purity ofThiazol Blauwas applied.

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 15 mg/kg bw/day Cyclophosphamide by oral gavage (dose volume, 10 mL/kg, concentration, 1.5 mg/mL) on Day 27, approximately 24 hours prior to scheduled necropsy on Day 28.

Parameters measured during the study includedsigns of morbidity and mortality twice daily,observation of clinical signs, performed daily (general, cage side observations, after the dose administration), or weekly (detailed observations), a modified Irwin test conducted on Day 26, weekly determination of body weight and food consumption. Prior to necropsy, the oestrus cycle of all females was evaluated by examination of vaginal smears. In addition, blood was collected prior to terminal necropsy, for clinical pathology (haematology, coagulation and clinical chemistry) assessment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives. 

Histopathology evaluation was conducted on tissues and organs retained in fixative and processed to slides from the control and high dose animals (1000 mg/kg bw/day), and on all organs with macroscopic findings from the low and mid dose groups (62.5 and 250 mg/kg bw/day, respectively).

 

Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle in the conditions employed on the study was performed in the Analytical Laboratory of LAB Research Ltd. 

 

Stability tests (LAB study code 09/068-316AN) at concentrations from approximately 1 to 250 mg/mL in PEG 400 indicated an up to 24 hour stability at room temperature. In addition, under refrigerated conditions (5±3°C), the solutions were stable for up to 72 hours, with a recovery of 94% and 100%, at 1 and 250 mg/mL, respectively (acceptance range: 100 ± 10%). Concentration and homogeneity of formulations were evaluated by UV-HPLC method on duplicate samples collected from the top, middle and bottom of test item solutions, and one sample from the control taken and analyzed fresh during the first and last weeks of treatment. Dose formulations were homogenous. The measured (actual) concentrations varied between 100% and 105% of the nominal concentrations.No test item was detected in the control solution samples.

 

These results were considered suitable for the study purposes.  

 

Following 28-days of treatment with Thiazol Blau at dose levels up to and including 1000 mg/kg bw/day did not result in test item related mortality or systemic adverse effects in CRL:(WI)BR rats. Black discoloration of the faeces was noted at all dose levels tested from Day 2 (1000 mg/kg bw/day dose level, males and females), Day 3 (250 mg/kg bw/day dose level, males and females) or Day 5 onwards (62.5 mg/kg bw/day dose level, males and females). Black discolored faeces were still visible 3 days after the last dose administration in the 1000 mg/kg bw/day recovery animals, thereafter no test item-related effects were noted until completion of the 14-day recovery period. In addition, blue urine was noted during the treatment period at 250 and 1000 mg/kg bw/day from Day 5 or Day 4 onwards, respectively, and up to and/or including Day 28. These changes were ascribed to elimination of Thiazol Blau or its metabolites through faeces and/or urine (cage side observations) and an expected staining effect. In the absence of any clinical pathology alterations or pathology findings, they were not considered to be adverse effects.

 

The behaviour and general condition of the animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed. 

 

Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats. 

 

There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups. 

 

Minor variations, on occasion attaining statistical significance, were noted in the clinical pathology parameters (haematology, coagulation, or clinical chemistry) in both main and recovery animals. However, no dose or gender-response was observed, and/or the results were within the historical range. These changes were not considered toxicologically significant.

 

There were no macroscopic or microscopic findings, or changes in the absolute or relative organ weights that could be ascribed to Thiazol Blau-administration. No induction of micronuclei in bone marrow erythrocytes was observed, thus, there was no evidence of any genotoxic activity of the test item.

 

Under the conditions of this study, the no observed adverse effect level (NOAEL) for Thiazol Blau is considered to be 1000 mg/kg bw/day.