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Administrative data

Description of key information

No adverse effects in subacute oral toxicity study up to highest dose tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

The toxicity of Thiazol Blau when administered daily for 28 days by oral gavage to CRL:(WI)BR rats was assessed in a subacute toxicity study. 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 examining the induction of micronuclei 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

 

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. 

 

Justification for classification or non-classification

no classification necessary