3-methyl-6-(p-toluidino)-3H-dibenz[f,ij]isoquinoline-2,7-dione

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: 24 May 2017. Experimental Completion Date: 2 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No deviations that affected the purpose and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-6-[(4-methylphenyl)amino]-3H-naphthol[1,2,3-de]quinolone-2,7-dione

Cas Number:

81-39-0

Molecular formula:

C24H18N2O2

IUPAC Name:

3-methyl-6-[(4-methylphenyl)amino]-3H-naphthol[1,2,3-de]quinolone-2,7-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Macrolex Rot 5B
Physical State/Appearance: Red Solid
IUPAC Name: 3-methyl-6-[(4-methylphenyl)amino]-3H-naphtho[1,2,3-de]quinoline-2,7-dione
Storage Conditions: Room temperature in the dark, used/formulated in the light
Expiry Date: 19 August 2021

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

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-health or injury. The animals initially allocated to the study were acclimatized for eight days during which time their health status was assessed. A total of forty-six animals (twenty-five males and twenty-one females) were accepted into the study. At the start of treatment the males weighed 190 to 215g, the females weighed 141 to 169g, and were approximately six to eight weeks old. An additional female was added to the study on Day 5 to replace a female that was found dead on Day 3. Procedures were the same for this animal as the others on the study but study timings were based on its own start of treatment date (Day 1) rather than the start date for the study.

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. A certificate of analysis of the batch of diet used is given in Annex 6. 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. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
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:

The dose levels were chosen in collaboration with the Sponsor, and based on the results of previous toxicity work including a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study Number MS88CX). 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.
A purity adjustment (for 90% purity) was made when preparing the dosing formulations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least 18 days when stored refrigerated in the dark. Formulations were therefore prepared three times during the treatment period and stored at approximately 4 ºC in the dark.
Samples of each test item formulations for Groups 1 to 4 were taken on two occasions and analyzed for concentration of Macrolex Rot 5B at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within
94-104% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 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 each dose

Control animals:

yes, concurrent vehicle

other: A positive control for the comet assay was also included

Details on study design:

The test item was administered to Group 1 to 4 (see table in section any other information on materials and methods) animals daily, for twenty-eight consecutive days (females) and twenty-nine consecutive days (males), by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 6 mL/kg of Polyethylene glycol 400. Group 5 males, used as positive controls for comet assay assessment remained untreated until Day 28 when they were dosed with N-Nitroso-N-methylurea on Days 28 and 29 alone.
All males (including positive controls for the comet assay) were dosed on Day 28 approximately 27 hours before scheduled euthanasia and Day 29 approximately 3 hours before scheduled euthanasia.
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. The volume of reference item administered to positive control animals was based on the scheduled body weight on Day 28.

Positive control:

N-Nitroso-N-methylurea (Comet assay)

Examinations

Observations and examinations performed and frequency:

Clinical Observations
Groups 1 to 4: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing one hour after dosing. For males dosed on Day 29, individual clinical observations were restricted to immediately before dosing and up to thirty minutes post dosing (due to the requirement to synchronize dosing and necropsy times). All observations were recorded.
Group 5 (positive controls for comet assay): Animals were monitored on a daily basis but formal recording of clinical signs did not begin until Day 28. On Day 28, individual clinical observations were performed immediately before dosing, up to thirty minutes post dosing and one hour after dosing. On Day 29, individual clinical observations were restricted to immediately before dosing and up to thirty minutes post dosing (due to the requirement to synchronize dosing and necropsy times).

Body Weight
Groups 1 to 4: Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.
Group 5 (positive controls for comet assay): During the pre-treatment period, individual body weights were recorded on a weekly basis to coincide with the scheduled weighing of Group 1 to 4 animals. Individual body weights were recorded on Day 28 (prior to start of dosing) and also recorded prior to terminal kill.

Food Consumption
Group 1 to 4: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.
Group 5 (positive controls for comet assay): No formal measurement of food consumption was required for these animals.

Water Consumption
Groups 1 to 4: 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.
Group 5 (positive controls for comet assay): No formal measurement of water consumption was required for these animals.

Special Evaluations

Functional Observations
Groups 1 to 4: Prior to the start of treatment 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.
Group 5 (positive controls for comet assay): No functional observations were required for these animals.

Behavioral Assessment
Groups 1 to 4: 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.
Group 5 (positive controls for comet assay): No behavioral assessments required for these animals.

Functional Performance Tests
Groups 1 to 4: 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).
Groups 1 to 4: 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).

Group 5 (positive controls for comet assay): No functional performance tests were required for these animals.

Sensory Reactivity
Groups 1 to 4: 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
Group 5 (positive controls for comet assay): No sensory reactivity assessments were required for these animals.

In-Life Sampling and Analysis
Groups 1 to 4: Hematological and blood chemical investigations were performed on all surviving 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.
Group 5 (positive controls for comet assay): No hematology or blood chemistry investigations were required for these animals.

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 surviving 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 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.


Comet Assay
Full details of the method are provided in the genetic toxicity endpoint.
Tissue Sampling
For the males from Groups 1 to 4 as well as all positive control males, samples of the liver (after recording of liver weight), glandular stomach and jejunum were processed appropriately by Envigo Research Ltd., Shardlow, UK Cell and Molecular Sciences Department to provide single cell suspensions with sufficient numbers of cells for the Comet Assay. The procedure was performed under subdued lighting and the Comet Assay tissues/cells were processed as quickly as possible, using ice-cold buffers to maintain the tissues and cell preparations at low temperature.
Liver - A small piece of liver was excised (approximately 1 cm2) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.
Glandular Stomach and Jejunum - The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides.
A section of jejunum (approximately 2 cm) was removed, cleaned and then immersed in stomach buffer for approximately 15 minutes on ice. A cell suspension was obtained by scraping the tissue of the jejunum into stomach buffer and filtering it through gauze.
Slide Preparation
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Once the cell suspensions were obtained, approximately 30 µL of the cell suspension was added to 270 µL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass coverslip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5) to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind, after which electrophoresis proceeded at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were then carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry, after which they were stored prior to staining and scoring. For each tissue, two of the four processed slide gels were scored and the remaining two slides were stored as backup slides.
Scoring
The processed Comet slides were coded to allow “blind” scoring using a computer generated code and stained just prior to analysis for comets. To each dry slide gel, 75 µL of propidium iodide (20 µg/mL) was placed on top of the slide and overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA, the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program (Comet IV version 4.3.1).
Two slide gels for each tissue for each animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparison between the vehicle control group response and that of the test item dose groups was made. The primary end-point was percentage DNA in the tail (percentage Tail intensity), although other endpoints such as tail moment and length may also be utilized.
Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

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 (weighed prior to tissue sampling for comet assay)
Brain
Epididymides
Ovaries
Heart
Spleen
Kidneys
Testes
Pituitary (weighed post-fixation)
Thymus
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Thyroid/Parathyroid (weighed post fixation)
Uterus with Cervix (and oviducts)

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 glands and fluids)
+Duodenum
+Epididymides ♦
Skin
Esophagus
+Spinal cord (cervical, mid thoracic and lumbar)
+Eyes *
+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 (with oviducts)
+Mammary gland
+Vagina
+Muscle (skeletal)


* 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 indicated with + 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.

Statistics:

Groups 1 to 4: 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.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.

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 for surviving animals that was considered to indicate any systemic effect of exposure to the test item at dosages of 100, 300 or 1000 mg/kg bw/day.
Fur staining was noted for all surviving animals (both sexes) at 300 and 1000 mg/kg bw/day; this finding was considered to be consistent with the colored nature of the test item.
At 1000 mg/kg bw/day, isolated incidences of increased post-dosing salivation were observed for all males and three of the surviving females. Similar increased post-dosing salivation was apparent for one male at 100 mg/kg bw/day and one male at 300 mg/kg bw/day, each on a single isolated occasion. Increased post-dosing salivation is frequently observed when test item is dosed via the oral gavage route and is generally considered to reflect distaste or slight irritancy of the test item formulations.
Additionally, at 1000 mg/kg bw/day, noisy respiration was observed for three males and three of the surviving females at 1000 mg/kg bw/day. At lower dosages, this finding was restricted to one female at 100 mg/kg bw/day on a single isolated occasion (Day 17) of the study. The higher incidence of noisy respiration apparent during this study at 1000 mg/kg bw/day was considered to reflect difficulties of dosing particular animals on occasions, particularly during the early stages of the study, and may also have been influenced by distaste or slight irritancy of the test item formulations.

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one unscheduled death on the study, occurring at 1000 mg/kg bw/day. Female number 40 was found dead and cannibalized on the morning of Day 3, having received two consecutive daily doses. No clinical signs had been apparent for this animal the previous day (Day 2) but increased post-dosing salivation and noisy respiration had been apparent on the first day of dosing. The extent of cannibalization prevented any meaningful necropsy examination and the cause of this death could not be established.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on body weight or body weight gain for either sex throughout the study at dosages of 100, 300 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on food consumption for either sex throughout the study at dosages 100, 300 or 1000 mg/kg bw/day.
Lower food consumption, compared to control, was observed for females at 1000 mg/kg bw/day during Week 1, however this was probably influenced by the rats at this dosage cannibalizing their decedent cage mate during this phase of the study.

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 100, 300 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 for males did not indicate any clear effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was considered to be no effect of treatment on hematology parameters for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.
For both sexes at all dosage, higher mean numbers of reticulocytes attained statistical significance when compared with control, however, mean values for females showed no dosage relationship and all individual values for both sexes were within the historical control range. In the absence of any supporting effects for hematology or blood chemistry parameters or evidence of histopathological change, particularly for the bone marrow and spleen, this finding was considered to be incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was considered to be no adverse effect of treatment on blood chemistry parameters for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.
For females at 1000 mg/kg bw/day, mean total protein and albumin level and mean albumin/globulin (A/G) ratio were lower than control, with differences attaining statistical significance. At 1000 mg/kg bw/day, two individual values for each parameter were below the historical control, however it was not always the same two animals affected. For females, at 300 mg/kg bw/day, statistically significant lower mean albumin/globulin ratio was also apparent but there were no corresponding statistically significant differences from control apparent for either total protein or albumin levels. Two individual values for mean albumin/globulin ratio were below the historical control range at 300 mg/kg bw/day. These findings, in the absence of any supporting histopathological findings, were considered insufficient to be regarded as an adverse effect of treatment.
For males at 100 and 300 mg/kg bw/day and both sexes at 1000 mg/kg bw/day, higher mean chloride levels attained statistical significance when compared with control, but all individual values were within the historical control range. Although the highest mean values occurred at 1000 mg/kg bw/day, mean values at lower dosages showed no dosage relationship. In the absence of any supporting histopathological change, this finding was considered to be incidental and of no toxicological significance.
For females at 1000 mg/kg bw/day, lower mean calcium levels attained statistical significance when compared with control, however all individual values for all animals, including controls, were below the historical control range. As the mean value at 1000 mg/kg bw/day was adversely influenced by a particular low value for one animal, an effect of treatment was considered unlikely and this finding, in the absence of supporting histopathological change, was considered incidental and unrelated to treatment.
For males at 1000 mg/kg bw/day, higher mean glucose levels attained statistical significance when compared with control, however only one individual value exceeded the historical 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):

not specified

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 100, 300 or 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Necropsy findings for surviving animals did not indicate any systemic effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Colored contents and/or discoloration were apparent for the stomach for the majority of males at 100 mg/kg bw/day, all males at 300 mg/kg bw/day and all males and one female at 1000 mg/kg bw/day. Colored contents were apparent in the cecum for one male and two females at 300 mg/kg bw/day and all males and the majority of females at 1000 mg/kg bw/day. Additionally, two males at 1000 mg/kg bw/day showed discoloration of the cecum. These findings were consistent with the colored nature of the test item and, as they were not associated with any evidence of histopathological change, they were considered not to indicate any systemic effect of the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At 1000 mg/kg bw/day, there were no histopathological changes considered to be related to treatment with Macrolex Rot 5B.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral Assessments
Assessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at dosages of 100, 300 or 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 of 100, 300 or 1000 mg/kg bw/day.
For males at 1000 mg/kg bw/day, lower hind limb grip strength at trial three attained statistical significance when compared to control. In the absence of any statistically significant differences from control in the previous two trials of hind limb grip strength or in any trial of forelimb grip strength, this finding was considered to be incidental and unrelated to treatment.

Sensory Reactivity Assessments
Scores during assessment of sensory reactivity did not indicate any obvious effect of treatment for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.

Comet Assay
The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver, and was considered not to induce DNA damage in these tissues.

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:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

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:

A dosage of 1000 mg/kg bw/day (the highest dosage tested) is considered to be the No Observed Adverse Effect Level (NOAEL) for this twenty-eight day toxicity study.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the testitem and to detect DNA strand breaks in cells or nuclei 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).

·        OECD Guidelines for Testing of Chemicals No. 489 “In vivoMammalian Alkaline Comet Assay” (Adopted 29 July 2016)

Methods

The test item was administered by gavage to three groups(Groups 2, 3 and 4),each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days (females) and twenty-nine consecutive days (males) at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group (Group 1) of five males and five females was dosed with vehicle alone (Polyethylene glycol 400). A group of five males (Group 5) used as a positive control for comet assay assessment, remained untreated until Day 28 when they were dosed with 25 mg/kg bw/day N-Nitroso-N-methylurea on Days 28 and 29.

All males were dosed on Day 28 approximately 27 hours before scheduled euthanasia, and dosed on Day 29 approximately 3 hours before scheduled euthanasia, and selected tissues from these animals were used for comet assay assessment.

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 Group 1 to 4 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. 

Additionally, samples of the liver, glandular stomach and jejunum were taken from male animals and processed to provide single cell suspensions with sufficient numbers of cells for the Comet Assay to detect DNA strand breaks in cells or nuclei.

Results

Mortality

At 1000 mg/kg bw/day, one female was found dead and cannibalized on the morning of Day 3, having received two consecutive daily doses. Increased post-dosing salivation and noisy respiration had been apparent on Day 1 but no clinical signs had been apparent on Day 2. The cause of this death could not be established, as the extent of cannibalization prevented any meaningful necropsy examination and microscopic evaluation of the tissues. However, this death appeared atypical and was considered to be unrelated to treatment. 

Clinical Observations

At 300 and 1000 mg/kg bw/day, fur staining, consistent with the colored nature of the test item was noted for all surviving animals during the study. 

At 1000 mg/kg bw/day, isolated incidences of increased post-dosing salivation were observed for all males and three of the surviving females. Similar increased post-dosing salivation was apparent for one male at 100 mg/kg bw/day and one male at 300 mg/kg bw/day, each on a single isolated occasion. Increased post-dosing salivation is frequently observed when test item is dosed via the oral gavage route and is generally considered to reflect distaste or slight irritancy of the test item formulations.

At 1000 mg/kg bw/day, noisy respiration was observed for three males and three of the surviving females at 1000 mg/kg bw/day. At lower dosage, this finding was restricted to one female at 100 mg/kg bw/day on a single isolated occasion (Day 17) of the study. The higher incidence of noisy respiration apparent during this study at 1000 mg/kg bw/day was considered to reflect difficulties of dosing particular animals on occasions, particularly during the early stages of the study, and may also have been influenced by distaste or slight irritancy of the test item formulations.  

Behavioral Assessment

Behavioral observations were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests

Functional performance was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day. 

Sensory Reactivity Assessments

Sensory reactivity was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Body Weight

Body weight gain was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day. 

Food Consumption

Food consumption was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Food Conversion Efficiency

Food conversion efficiency was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Water Consumption

Water consumption was unaffected by treatment at 100, 300 or 1000 mg/kg bw/day. 

Hematology

Hematology parameters were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day.

Blood Chemistry

There was no adverse effect of treatment on blood chemistry parameters for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.

Comet Assay

The test item did not demonstrate any significant increases in the percentage tail intensity or median percentage tail intensity of the jejunum, glandular stomach and liver, and was considered not to induce DNA damage in these tissues.

Necropsy

Necropsy findings observed on the study were consistent with the colored nature of the test item and did not indicate any systemic effect of the test item. Colored contents and/or discoloration were apparent for the stomach for the majority of males at 100 mg/kg bw/day, all males at 300 mg/kg bw/day and all males and one female at 1000 mg/kg bw/day.  Colored contents were also apparent in the cecum for one male and two females at 300 mg/kg bw/day and all males and the majority of females at 1000 mg/kg bw/day. Additionally, two males at 1000 mg/kg bw/day showed discoloration of the cecum. 

Organ Weights

Organ weights were unaffected by treatment at 100, 300 or 1000 mg/kg bw/day. 

Histopathology

At 1000 mg/kg bw/day, there were no histopathological changes considered to be related to treatment with Macrolex Rot 5B.

Conclusion

A dosage of 1000 mg/kg bw/day (the highest dosage tested) is considered to be the No Observed Adverse Effect Level (NOAEL) for this twenty-eight day repeat dose toxicity study.