Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, sulfo [[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]sulfonyl derivs.

Endpoint summary

Description of key information

No adverse effects were observed up to the limit dose tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 01 May 2014; Experimental Completion Date: 07 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Remarks:

None considered to impact the quality or validity of study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Remarks:

None considered to impact the quality or validity of study

Qualifier:

according to guideline

Guideline:

other: USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Deviations:

no

Remarks:

None considered to impact the quality or validity of study

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study

Deviations:

no

Remarks:

None considered to impact the quality or validity of study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 174 to 198g, the females weighed 144 to 170g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from the target range for temperature and short term deviations from the target range for relative humidity were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

TEST ITEM PREPARATION:
The test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined as part of this study. Results show the formulations to be stable for at least seven days when stored at 4 ºC in the dark. Formulations were prepared daily and used on the day of preparation throughout the treatment period.

PROCEDURE:
See any other information on materials and methods section for table of treatment groups.
The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Distilled water. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of each test item formulation were taken and analyzed for concentration of Reactive Blue 21.

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

The results indicate that the prepared formulations were within 101-107% of the nominal concentration confirming the accuracy of the formulation procedure.

The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.

Duration of treatment / exposure:

Twenty-eight consecutive days.

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
62.5, 250, 1000 mg/kg bw/day
Basis:
other: nominal

No. of animals per sex per dose:

5 males and 5 females per group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen by the sponsor based on on available toxicity data.

Positive control:

None

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing during the working week, weekends and public holidays. During the treatment-free period, animals were observed daily. All observations were recorded.

FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment (Day -1) and on Days 7, 14, 21 and 27, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

BEHAVIORAL ASSESSMENT:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behavior
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper/Hypothermia
Skin color
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

FUNCTIONAL PERFORMANCE TESTS:
Motor activity:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength:
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
The following parameters were observed:
Grasp response
Vocalization
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Blink reflex
Startle reflex

BODY WEIGHT:
Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weight measurements were also performed prior to terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Weeks 3 and 4 where water intake was measured gravimetrically.

LABORATORY INVESTIGATIONS:
Hematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.

Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

HEMATOLOGY:
The following parameters were measured:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Gamma glutamy/transpeptidase

URINALYSIS:
The following parameters were measured on collected urine:
Volume
Specific Gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

Sacrifice and pathology:

NECROPSY:
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

THYROID HORMONE ASSESSMENT:
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

ORGAN WEIGHTS:
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Pituitary (post-fixation)
Prostate and Seminal Vesicles
(with coagulating glands and fluids)
Spleen
Testes
Thymus
Thyroid/Parathyroid (post fixation)
Uterus with Cervix

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and
pons)
Caecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Gross lesions
Heart
Ileum (including Peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Skin (hind limb)
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina

All tissues were dispatched to the histology processing test site for processing

Following the results of the initial histopathological examinations, examination of the liver, spleen and bone marrow was extended to intermediate group females. Additionally the mesenteric lymph nodes were examined for recovery group animals.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were generally analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method.

Urinalysis (Volume and Specific Gravity) data were assessed separately using the R Environment for Statistical Computing.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis 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

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY:
There were no unscheduled deaths on the study.

CLINICAL OBSERVATIONS:
There were no clinical signs observed on the study that were considered to indicate an adverse effect of the Test Item.

FUNCTIONAL OBSERVATIONS:
Behavioral Assessments: Behavioral assessment of the animals in a standard arena did not reveal any obvious effects of treatment for either sex at dosages up to and including 1000 mg/kg bw/day.

Functional Performance Tests: Assessment of functional performance using grip strength and motor activity did not indicate any obvious effects of treatment for either sex at dosages up to and including 1000 mg/kg bw/day.

Sensory Reactivity Assessments:
Sensory reactivity scores were the same in the control and all treatment groups.

BODY WEIGHT:
There was no obvious effect of treatment on body weight gain for either sex at dosages up to and including 1000 mg/kg bw/day.

FOOD CONSUMPTION:
There was no obvious effect of treatment on food consumption for either sex at dosage up to and including 1000 mg/kg bw/day.

Food conversion efficiency for either sex was also considered to be unaffected by treatment at dosages up to and including 1000 mg/kg bw/day.

WATER CONSUMPTION:
There was no obvious effect of treatment on water consumption for either sex at dosages up to and including 1000 mg/kg bw/day.

LABORATORY INVESTIGATIONS:
HEMATOLOGY:
There was no effect of treatment on hematology parameters for either sex at dosages up to and including 1000 mg/kg bw/day.

BLOOD CHEMISTRY:
There was no effect of treatment on blood chemistry parameters for either sex at dosages up to and including 1000 mg/kg bw/day.

URINALYSIS:
Urinalysis assessments did not indicate any obvious effect of treatment for either sex at dosages up to and including 1000 mg/kg bw/day.

PATHOLOGY:
NECROPSY:
Necropsy examination did not reveal any findings that were considered to represent an adverse effect of treatment.

ORGAN WEIGHTS:
Assessment of organ weights did not reveal any findings that were considered to represent an adverse effect of treatment.

HISTOPATHOLOGY:
Histopathology examination did not reveal any findings that were considered to represent an adverse effect of treatment.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

DISCUSSION:

Treatment with Reactive Blue 21 over a period of twenty-eight consecutive days was well tolerated with no adverse effects of treatment being indicated by clinical signs, behavioral assessments, body weight gain, food and water consumption, hematology, blood chemistry and urinalysis parameters, necropsy findings or subsequent microscopic examination of the tissues. As anticipated, given the nature of the test item, internal and external staining/discoloration by the item test was apparent at all dosages although at 62.5 mg/kg bw/day, the staining/discoloration of tissues was not apparent microscopically. At higher dosages of 250 and 1000 mg/kg bw/day staining/discoloration was apparent for numerous tissues but, in the absence of any histopathological change, this was considered not to represent an adverse effect of treatment. The blue content and discoloration in the gastro-intestinal tract, lymph nodes and kidneys were considered to reflect normal physiological processes related to the absorption and distribution of food and the excretion of waste products. Blue discoloration of the lungs was considered to be a consequence of aspiration of test item formulation during the oral gavage dosing procedure (probably as the dosing canula was being withdrawn) and was considered to be of no toxicological significance.

Conclusions:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Reactive Blue 21 was considered to be 1000 mg/kg bw/day.

Executive summary:

Introduction:

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines: ·        Commission Directive 96/54/EC (Method B7). Commission Directive 96/54/EC (Method B7).

·        The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

·        The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

·        USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Methods:

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 62.5, 250 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Distilled water) over the same treatment period. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

 

Clinical signs, behavioral assessments, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

Results:

Mortality:

There were no unscheduled deaths on the study.

 

Clinical Observations:

There were no clinical signs observed on the study that indicated an adverse effect of the Test Item.

 

Behavioral Assessment:

Behavior was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Functional Performance Tests:

Functional performance was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Sensory Reactivity Assessments:

Sensory reactivity scores were unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Body Weight:

Body weight gain was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Food Consumption:

Food consumption and food conversion efficiency were unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Water Consumption:

Water consumption was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Hematology:

There was no effect of treatment on hematology parameters at dosages up to 1000 mg/kg bw/day.

 

Blood Chemistry:

There were no adverse effects of treatment on blood chemistry parameters at dosages up to 1000 mg/kg bw/day.

 

Urinalysis:

There were no adverse effects of treatment on urinalysis parameters at dosages up to 1000 mg/kg bw/day.

 

Necropsy:

Necropsy examination did not reveal any findings considered to represent an adverse effect of treatment.

 

Organ Weights:

Assessment of organ weights did not reveal any findings that were considered to represent an adverse effect of treatment.

 

Histopathology:

Histopathology examination did not reveal any findings that were considered to represent an adverse effect of treatment.

 

Conclusion:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Reactive Blue 21 was considered to be 1000 mg/kg bw/day. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Introduction:

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines: ·        Commission Directive 96/54/EC (Method B7). Commission Directive 96/54/EC (Method B7).

·        The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

·        The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

·        USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Methods:

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 62.5, 250 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Distilled water) over the same treatment period. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

 

Clinical signs, behavioral assessments, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

Results:

Mortality:

There were no unscheduled deaths on the study.

 

Clinical Observations:

There were no clinical signs observed on the study that indicated an adverse effect of the Test Item.

 

Behavioral Assessment:

Behavior was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Functional Performance Tests:

Functional performance was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Sensory Reactivity Assessments:

Sensory reactivity scores were unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Body Weight:

Body weight gain was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Food Consumption:

Food consumption and food conversion efficiency were unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Water Consumption:

Water consumption was unaffected by treatment at dosages up to 1000 mg/kg bw/day.

 

Hematology:

There was no effect of treatment on hematology parameters at dosages up to 1000 mg/kg bw/day.

 

Blood Chemistry:

There were no adverse effects of treatment on blood chemistry parameters at dosages up to 1000 mg/kg bw/day.

 

Urinalysis:

There were no adverse effects of treatment on urinalysis parameters at dosages up to 1000 mg/kg bw/day.

 

Necropsy:

Necropsy examination did not reveal any findings considered to represent an adverse effect of treatment.

 

Organ Weights:

Assessment of organ weights did not reveal any findings that were considered to represent an adverse effect of treatment.

 

Histopathology:

Histopathology examination did not reveal any findings that were considered to represent an adverse effect of treatment.

 

Conclusion:

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of Reactive Blue 21 was considered to be 1000 mg/kg bw/day.