Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

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: 30 September 2016 Experimental Completion Date: 15 December 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

EC Number:

277-146-9

EC Name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Cas Number:

72968-71-9

Molecular formula:

C23H29N7O4S

IUPAC Name:

methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

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 Rot B, batch CHA009
Identity and content reported in study 2015/0125/01 (CURRENTA GmbH & Co. OHG; March 3, 2016)
Purity: 99.7 %

Specific details on test material used for the study:

Identification : Macrolex Rot B
CAS-Number : 72968-71-9
Empirical Formula : C23H29N7O4S
Molecular Mass : 499.59 g/mol
Appearance : Red powder
The Test Item will be used as supplied
Storage Conditions : At room temperature
Purpose of Use : Industrial Chemical
Expiry Date : 13 November 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 originally from Envigo RMS (UK) Limited, Oxon, UK were transferred to the study from stock. On receipt the animals had been 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 forty animals (twenty males and twenty females) were formally accepted into the study. At the start of treatment the males weighed 191 to 220g, the females weighed 138 to 164g, and were approximately six to eight weeks old.

Animal Care 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% respectively; there were no deviations from these targets.
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:

polyethylene glycol

Details on oral exposure:

Dose levels were based on available toxicity data, including the results of the Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number PV42VP), where a dosage of 1000 mg/kg bw/day was well tolerated. The test item was formulated in the same vehicle (Polyethylene glycol 400) as that used in the aforementioned preliminary study. Dosages of 0 (control), 250, 500 and 1000 mg/kg bw/day were chosen, in collaboration with the Sponsor, using a treatment volume of 4 mL/kg body weight, for investigation in this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Representative samples of two sets of dosing formulations used during the study were analyzed for concentration of Macrolex Rot B at Envigo Research Limited Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 97-100% of nominal concentration.

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 single peak.

Reagents
Control Vehicle: PEG 400
Tetrahydrofuran; gradient HPLC grade
Acetonitrile: Gradient HPLC grade
Water: Reverse Osmosis
Extract Solvent: Tetrahydrofuran
Dilution solvent: Acetonitrile

Preperation of calibration standard
Stock solutions of test item in extract solvent were prepared for external standard calibration. An aliquot, approx. 0.05g of test item was exactly weighed into a 100 mL volumetric falsk and brought to volume with extract solvent to yeild a solution with a concentration of 0.5 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.002 mg/mL.
Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence aas a minimum.

Preparation of test sample
the formulations received were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brough to volume with extract solvent this was then ultra-sonicated for 2 minutes. Where necessary, sample solutions were further diluted with extract solvent before further dilution with dilution solvent to achieve the working concentration.

Conclusion
The mean concentration of the tets item in formulations analyzed for the study were within ± 10 % of nominal concentrations confirming accurate formulation

Duration of treatment / exposure:

28 consecutive 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
5 males and 5 females at 500 mg/kg
5 males and 5 females at 1000 mg/kg

Control animals:

yes, concurrent vehicle

Details on study design:

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 Polyethylene glycol 400.
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.

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. Any staining of the cage/bedding and/or discoloration of the faeces was also recorded. All observations were recorded.

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

Functional Observations
Prior to the start of treatment and on Days 6, 13, 20 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 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++) Triglycerides (Tri)

Sacrifice and pathology:

Necropsy
On completion of the dosing 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, which included a check for any tissue discoloration to judge local and systemic bioavailability, 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 -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:
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~
Muscle (skeletal)~
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 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 in the low and intermediate groups.

* 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

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The low incidence and distribution of clinical signs observed during the study did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.
Dark faeces were observed on the cage floors for treated animals throughout the majority of the treatment period. This finding was not unexpected considering the colored 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 adverse effect of treatment on body weight or body weight gain for either sex throughout the study at dosages of 250, 500 or 1000 mg/kg bw/day. For males at 1000 mg/kg bw/day lower mean body weight gain during the third week of treatment attained statistical significance when compared with control, however, in the absence of any statistically significant difference in overall mean body weight gain, this difference was considered to be of no toxicological significance and not to represent an adverse effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

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 clear effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.
There was a tendency for slightly lower water consumption for males at 1000 mg/kg bw/day, however, the intergroup differences in water intake were not convincing; the difference observed probably reflected normal biological variation and at the level observed did not represent an adverse effect of treatment.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious adverse effect of treatment on blood chemistry parameters for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.
For males at 1000 mg/kg bw/day, higher albumin/globulin ratio attained statistical significance compared with control, but all individual values were within the historical control range. There were no statistically significant differences from control observed for total protein or albumin and this isolated finding, in the absence of any supporting histopathological change, was considered to be incidental and of no toxicological significance.
For females at 1000 mg/kg bw/day, statistically significant higher sodium levels were apparent compared to control, with two individual values exceeding the historical control range. In the absence of any supporting histopathological change, this isolated finding was considered to be incidental and of no toxicological significance.
For females at 1000 mg/kg bw/day, lower mean glucose levels attained statistical significance when compared with control, but all values were within the historical control range. In the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious adverse effect of treatment on blood chemistry parameters for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.
For males at 1000 mg/kg bw/day, higher albumin/globulin ratio attained statistical significance compared with control, but all individual values were within the historical control range. There were no statistically significant differences from control observed for total protein or albumin and this isolated finding, in the absence of any supporting histopathological change, was considered to be incidental and of no toxicological significance.
For females at 1000 mg/kg bw/day, statistically significant higher sodium levels were apparent compared to control, with two individual values exceeding the historical control range. In the absence of any supporting histopathological change, this isolated finding was considered to be incidental and of no toxicological significance.
For females at 1000 mg/kg bw/day, lower mean glucose levels attained statistical significance when compared with control, but all values were within the historical control range. In the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.

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:

no effects observed

Description (incidence and severity):

Intergroup differences in absolute and body weight relative organ weights did not indicate any obvious effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy findings did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.
One male at 500 mg/kg bw/day and one male at 1000 mg/kg bw/day showed a raised limiting ridge within the stomach. While this finding is often suggestive of some local irritancy of the test item, as microscopic evaluation did not reveal any obvious effect of treatment, it was considered to be incidental and of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

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.

Sensory Reactivity Assessments
Scores during assessment of sensory reactivity did not indicate any obvious effect of treatment for either sex at dosages of 250, 500 or 1000 mg/kg bw/day.

Details on results:

Treatment of rats with Macrolex Rot B at dosages of 250, 500 or 1000 mg/kg bw/day for twenty-eight consecutive days was well tolerated and clinical signs, functional observations (as assessed by arena and cage observations, grip strength or motor activity and sensory reactivity to various stimuli), body weight performance, food consumption, food conversion efficiency and water consumption did not indicate any obvious adverse toxicity for either sex.
Occasional statistically significant differences from control for hematological and blood chemistry parameters, in the absence of any supporting histopathological change, were considered to be of no toxicological significance and insufficient to indicate an adverse effect. Necropsy findings for animals at 250, 500 or 1000 mg/kg bw/day did not indicate any adverse effect of treatment and there were no treatment related histopathological findings for either sex at 1000 mg/kg bw/day. Based on these results, the No Observed Adverse Effect (NOAEL) for the oral administration of Macrolex Rot B was considered to be 1000 mg/kg bw/day.

Effect levels

Key result

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 (NOAEL) for the oral (gavage) administration of Macrolex Rot 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 compatiblewith 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)

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 250, 500 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400) over the same treatment period.

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

Clinical signs did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Behavioral Assessment

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

Water Consumption

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

Hematology

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

Blood Chemistry

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

Necropsy

Necropsy findings did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day.

Organ Weights

There was no obvious adverse effect of treatment on absolute and body weight relative 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 Rot B did not result in any treatmentrelated histopathological changes.

Conclusion

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