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Description of key information

Reddish Blue administered daily by oral gavage for 28 days in Wistar rats did not result in mortality, clinical signs, changes in body weight, food consumption, coagulation parameters, clinical chemistry parameters or oestrus cycle at dose levels of 62.5, 250, or 1000 mg/kg bw/day during either the treatment or 14-day recovery/observation periods. 
Haematological changes considered related to treatment and suggesting a regenerative anaemia were observed in the 1000 mg/kg bw/day animals and possibly at the 250 mg/kg bw/day 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.
In conclusion, under the conditions of this study, the no observed effect level (NOEL) for Reddish Blue is considered to be 62.5 mg/kg bw/day the no observed adverse effect level (NOAEL) is considered to be 250 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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

Additional information

The toxicity of Reddish Blue when administered daily for 28 days by oral gavage to CRL:(WI)BR rats was investigated. 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 evaluating the induction of micronuclei in bone marrow erythrocytes of treated and control animals.

 

Reddish Blue, 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

8

5

5

5

5

2

Low Dose

62.5

7.81

5

5

-

-

3

Mid Dose

250

31.25

5

5

-

-

4

High Dose

1000

125

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

30

3

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 (1:1 PEG 400 and Sterile Distilled Water mixture), at the same volume as the high dose animals. No correction for purity ofReddish Bluewas 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 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.

 

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), on all organs with macroscopic findings from the low and mid dose groups (62.5 and 250 mg/kg bw/day, respectively), on all testes and epididymides from the mid dose 250 mg/kg bw/day malesand on all spleens from the 62.5 and 250 mg/kg bw/day male and female animals.

 

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 CiToxLAB Hungary Ltd. 

 

Stability tests (CiToxLAB study code 09/128-316AN) at concentrations from approximately 1 to 125 mg/mL in the vehicle(1:1 PEG 400 and Sterile Distilled Water mixture)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 98% and 103%, at 1 and 125 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 102% and 106% 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 Reddish Blue at dose levels up to and including 1000 mg/kg bw/day, no test item related mortality or systemic adverse effects were noted in CRL:(WI)BR rats. Black discoloration of the faeces was noted at all the dose levels tested from Day 1 (1000 and 250 mg/kg bw/day dose level, males and females) or Day 2 onwards (62.5 mg/kg bw/day dose level, males and females). The discoloration of the faeces persisted in the 1000 mg/kg bw/day recovery animals for 2 days after the last dose administration (on Days 28 and 29); thereafter no test item-related effects were noted until completion of the 14-day recovery period. In addition, grey urine was noted during the treatment period at 250 and 1000 mg/kg bw/day between Days 1 and 28. These changes were ascribed to elimination of Reddish Blue or its metabolites through faeces and/or urine (cage side observations) and an expected staining effect. 

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. 

There were no changes that could be ascribed to Reddish Blue administration noted in coagulation or clinical chemistry parameters.

Haematological changes including statistically lower red blood cell count, haemoglobin concentration and mean corpuscular (erythrocyte) haemoglobin concentration and statistically higher mean corpuscular (erythrocyte) volume, red cell (erythrocyte) volume, platelet and reticulocyte count mean values were observed in the 1000 mg/kg bw/day males and females and were considered related to treatment, suggesting a regenerative anaemia, possibly with a similar trend at the 250 mg/kg bw/day at the end of the treatment period. No test item related effects were observed after 14 days recovery. 

On macroscopic evaluation, small testes and/or epididymides were observed at 1000 mg/kg bw/day, noted in 4/5and 5/5 Recovery males. In addition, spleen enlargement was observed in 8/10 Main animals at 1000 mg/kg bw/day, but not in Recovery animals. These findings were considered related to test item administration and correlated with the organ weight and microscopic changes.

On histological examination of testes, tubular atrophy/degeneration and reduction of the sperm content combined with presence of ductal cell debris and apoptotic bodies in the epididymides was observed in all 1000 mg/kg bw/day males, with a slightly reduced severity in recovery animals. At additional histopathological examination of the testes and epididymides from all 250 mg/kg bw/day males, no test item-related changes were noted in these organs.

 

Slightly increased severity of extramedullary haematopoiesis in the spleen with an apparent dose response, associated with haematological and/or organ weight changes, was observed in 1000 mg/kg bw/day and 250 mg/kg bw/day rats when compared to controls (mild versus minimal). The effects observed in the spleen were considered to reflect an adaptive response to the slight regenerative anaemia noted in these animals and be potentially ascribed to the test item administration. At additional histopathological examination of the spleen from all 62.5 mg/kg bw/day animals, no test item-related changes were noted. In Recovery rats, extramedullary haematopoiesis in high-dose animals had a lower incidence than in control-group rats (9/10 Control and 6/10 High Dose Recovery rats) with minimal severity, similar to controls, indicating complete reversibility after 14 days of recovery.

 

In summary, Reddish Blue administered daily by oral gavage for 28 days in Wistar rats did not result in mortality, clinical signs, changes in body weight, food consumption, coagulation parameters, clinical chemistry parameters or oestrus cycle at dose levels of 62.5, 250, or 1000 mg/kg bw/day during either the treatment or 14-day recovery/observation periods.

Haematological changes considered related to treatment and suggesting a regenerative anaemia were observed in the 1000 mg/kg bw/day animals and possibly at the 250 mg/kg bw/day 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.

 

In conclusion, under the conditions of this study, the no observed effect level (NOEL) for Reddish Blue is considered to be 62.5 mg/kg bw/day the no observed adverse effect level (NOAEL) is considered to be 250 mg/kg bw/day.

Justification for classification or non-classification

no classification necessary