1-hydroxy-4-(p-toluidino)anthraquinone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 12 September 2016. Experimental Completion Date: 29 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-4-(p-toluidino)anthraquinone

EC Number:

201-353-5

EC Name:

1-hydroxy-4-(p-toluidino)anthraquinone

Cas Number:

81-48-1

Molecular formula:

C21H15NO3

IUPAC Name:

1-hydroxy-4-[(4-methylphenyl)amino]-9,10-anthraquinone

Constituent 2

Reference substance name:

1,4-bis(p-tolylamino)anthraquinone

EC Number:

204-909-5

EC Name:

1,4-bis(p-tolylamino)anthraquinone

Cas Number:

128-80-3

Molecular formula:

C28-H22-N2-O2

IUPAC Name:

1,4-bis[(4-methylphenyl)amino]-9,10-anthraquinone

impurity 1

Reference substance name:

Unknown impurity

Molecular formula:

not available

IUPAC Name:

Unknown impurity

Test material form:

solid: particulate/powder

Details on test material:

Macrolex Violet B, batch CHA23079

Specific details on test material used for the study:

Description: Dark blue/violet powder
Storage/Usage Conditions: Ambient temperature in the dark (formulated in the light)
Expiry Date: 02 June 2020

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-heal
th or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study.
At the start of treatment the males weighed 223 to 273g, the females weighed 163 to 193g, and were approximately six to eight weeks old.

Animal Car and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) 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 (Datesand Ltd., Cheshire, UK). 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 Envigo Research Limited, Shardlow, UK 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 Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20%. There were no deviations from the target range observed for temperature but the target range for relative humidity was exceeded for quite prolonged periods on a number of occasions during the study, the majority of these were within the acclimatization phase of the study. These deviations were considered not to have affected the purpose or validity 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.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

arachis oil

Details on oral exposure:

The dose levels were chosen based on the results of previous toxicity work (Envigo study number XD69DQ).
The dose levels were selected as 250, 500 or 1000 mg/kg bw/day.
The test item was administered daily, for 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 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analyzed for concentration of Macrolex Violett B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the formulations used for dosing were within 93-109% of the nominal concentration and were acceptable for use on the study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females at 250 mg/kg bw/day
5 males and 5 females at 500 mg/kg bw/day
5 males and 5 females at 1000 mg/kg be/day

Control animals:

yes, concurrent vehicle

Examinations

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.
All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weightswere 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 Week 3 where water intake was measured gravimetrically.

Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 25, 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 Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

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, Reiter and Macphail 1979).
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. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Hematology
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 Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)

Sacrifice and pathology:

Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone 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 each animal via the exsanguination procedure, allowed to clot, centrifuged, and the serum from each blood sample was stored frozen at lower than -60 °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 at the end of the dosing period, were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles Uterus with Cervix
(with coagulating glands and fluids)

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adipose tissue~ Muscle (skeletal)~
Adrenals~ Ovaries~
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary~
Bone & bone marrow (sternum)~ Prostate~
Brain (including cerebrum, cerebellum and Rectum~
pons)~ Salivary glands (submaxillary)
Caecum~ Sciatic nerve~
Colon~ Seminal vesicles (with coagulating
Duodenum~ glands and fluids)~
Epididymides ♦~ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes *~ and lumbar)~
Gross lesions~ Spleen~
Heart~ Stomach~
Ileum~ Testes ♦~
Jejunum~ Thymus~
Kidneys~ Thyroid/Parathyroid~
Liver~ Trachea~
Lungs (with bronchi)#~ Urinary bladder~
Lymph nodes (mandibular and mesenteric)~ Uterus & Cervix~
Mammary gland~ Vagina~
♦ Preserved in modified Davidson’s fluid
* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues maked with a ~ from all control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were no indications of treatment-related changes, examination was not extended to include animals from the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (Wendy Henderson). A peer review of the findings observed was conducted by Vasanthi Mowat (Envigo CRS Limited) at the histopathology peer review test site.

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, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were 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. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed that indicated any systemic effect of the test item.
Fur staining by the test item was apparent for all treated males and for 1, 2 and all females at 250, 500 and 1000 mg/kg bw/day respectively. Additionally, dark/blue staining of the faeces was frequently observed from Day 5 to termination for both sexes at 250, 500 and 1000 mg/kg bw/day. These findings were not unexpected considering the nature of the test item

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no obvious effect on body weight or body weight gain for either sex throughout the study at dosages of 250, 500 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no obvious effect on food consumption for either sex throughout the study at dosages of 250, 500 or 1000 mg/kg bw/day.
Food consumption for treated males was generally higher than control throughout the study but differences showed no dosage relationship and were considered to reflect normal biological variation.

Food efficiency:

no effects observed

Description (incidence and severity):

Intergroup differences in food conversion efficiency did not indicate any obvious effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

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

no effects observed

Description (incidence and severity):

Intergroup differences in water consumption did not indicate any obvious consistent effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At all dosages, mean reticulocyte count were statistically significantly higher than control for both males (p<0.01) and females (p<0.05), however all individual values were within the historical control range.
For males, statistically significant lower mean corpuscular hemoglobin concentration was observed at all dosages (p<0.01) but all individual values were within the historical control range. Statistically significant lower mean corpuscular hemoglobin was also apparent for males at 500 and 1000 mg/kg bw/day (p<0.05); values for all treated animals were within the historical control range but 2/5 control values exceeded this historical control range.
For females at 1000 mg/kg bw/day, mean erythrocyte count, hemoglobin and hematocrit values were statistically significant lower than control (p<0.05). For these treated females, 3/5 individual values for erythrocyte count and for hemoglobin were below the historical control range and 2/5 individual hematocrit values were below the historical control range; one control hematocrit value exceeded the historical control range.
Overall these finding suggest a possible slight treatment related effect on erythrocytes but, in the absence of any supporting histopathological change for the bone marrow or spleen, this finding was considered insufficient to represent an adverse effect of treatment.
For females at 1000 mg/kg bw/day, lower platelet count also attained statistical significance (p<0.05), however only one individual value for these treated animals was below the historical control range. In the absence of any supporting histopathological change for the bone marrow, this finding was considered likely to be incidental and would be insufficient to represent an adverse effect of treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

For females at 1000 mg/kg bw/day, statistically significant lower total protein (p<0.05) and albumin (p<0.01) levels were apparent compared to control but there was no accompanying statistically significant difference from control observed for albumin/globulin ratio. For treated females at this dosage, 1/5 individual values for total protein and 2/5 individual values for albumin were below the historical control range. There was no similar decrease for males at this dosage and, in the absence of any supporting histopathological change, this finding was considered likely to be incidental and would be insufficient to represent an adverse effect of treatment.
For males at all dosages, higher calcium and lower inorganic phosphorus levels attained statistical significance when compared to control (p<0.05) but mean values showed no dosage relationship. For calcium, 1/5 individual values at 500 mg/kg bw/day and 1/5 individual value at 1000 mg/kg bw/day were below the historical control range compared to 4/5 control values; this finding was, therefore, considered to reflect atypically low control values rather than any effect of treatment. For inorganic phosphorus, 2/5 individual values at 500 mg/kg bw/day and 1/5 individual value at 1000 mg/kg bw/day were below the historical control range. In view of the lack of dosage response and the absence of any supporting histopathological change, this finding was considered to be incidental and unrelated to treatment.
For males at 500 mg/kg bw/day, higher albumin/globulin ratio attained statistical significance (p<0.05) compared to control, but all individual values were within the historical control range. For females at 250 and 500 mg/kg bw/day, higher triglycerides levels attained statistical significance compared to control; two individual values at 250 mg/kg bw/day exceeded the historical control range. In the absence of similar statistically significant differences from control for high dosage animals, these findings were considered incidental and unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Assessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at dosages of 250, 500 or 1000 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, statistically significant lower absolute and body weight relative adrenal weights were apparent for females compared to control. For these treated females, 2/5 individual absolute and 2/5 individual body weight relative adrenal weights were below the historical control range, however for the control group, 1/5 individual absolute and 2/5 individual body weight relative adrenal weights exceeded the historical control range. In the absence of any supporting histopathological change, this finding was considered not to represent an adverse effect of treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy of treated animals revealed a number of organ/tissues showing blue discoloration or blue coloured contents including the aorta, bone and bone marrow (sternum), adipose tissue, mammary gland, mesenteric lymph nodes, mandibular lymph nodes, skin, stomach, ileum, caecum and thyroid/parathyroid. Given the nature of the test item, these findings are not unexpected and, in the absence of any resulting histopathological change being detected during microscopic evaluation of the tissues, they were considered to be of no toxicological significance and did not represent an adverse effect.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At 1000 mg/kg bw/day, administration of Macrolex Violett B did not result in any histopathological changes.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

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 of 250, 500 or 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, lower forelimb grip strength during test 2 attained statistical significance compared to control but, in the absence of any significant differences in the other tests of grip strength, this finding was considered to be incidental and unrelated to treatment

Sensory Reactivity Assessments
Scores during assessment of sensory reactivity were identical to control for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) for the oral administration of Macrolex Violett B over twenty eight consecutive days was 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).

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

·        Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Method

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 250, 500 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

Clinical signs,functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Incidences of fur staining and dark/blue staining of the faeces were apparent for both sexes at 250, 500 or 1000 mg/kg bw/day but there were no clinical signs observed that indicated any systemic effect of the test item.

Behavioral Assessment

There was no obvious effect on behavioral assessments for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Functional Performance Tests

There was no obvious effect on functional performance tests results for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Sensory Reactivity Assessments

There was no obvious effect on sensory reactivity assessments results for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Body Weight

There was no obvious effect on body weight or body weight gain for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Food Consumption

There was no obvious effect on food consumption for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Food Conversion Efficiency

There was no obvious effect on food conversion efficiency for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Water Consumption

There was no obvious effect on water consumption for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Hematology

There was no adverse effect on hematology parameters for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Blood Chemistry

There was no adverse effect on blood chemistry parameters for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Necropsy

Necropsy revealed blue discoloration or blue coloured contents for a number of organ/tissues including the aorta, bone and bone marrow (sternum), adipose tissue, mammary gland, mesenteric lymph nodes, mandibular lymph nodes, skin, stomach, ileum, caecum and thyroid/parathyroid at dosages of 250, 500 or 1000 mg/kg bw/day. Given the nature of the test item, these findings are not unexpected and due to an absence of histopathological change they were considered not to represent an adverse effect.

Organ Weights

There was no adverse effect on organ weights for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Histopathology

At 1000 mg/kg bw/day, administration of Macrolex Violett B did not result in any histopathological changes.

Conclusion

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) for the oral administration of Macrolex Violett B over twenty eight consecutive days was 1000 mg/kg bw/day.