Tetrasodium {5-[(4-chloro-6-{[4-(alkenylsulfonyl)phenyl]amino}-heteromonocycl-2-yl)amino]-4-(hydroxy-κO)-3-{[2-(hydroxy-κO)phenyl]diazenyl-κN}naphthalene-disulfonato(4-)}[3-(hydroxy-κO)-4-{[2-(hydroxy-κO)naphthalen-1-yl]diazenyl-κN}naphthalene-sulfonato(3-)]chromate(4-)

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:

Testing was conducted between 24th February and 12th November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See "principles of method if other than guideline" field.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See "principles of method if other than guideline" field.

Principles of method if other than guideline:

Deviation No.1:
Clinical Observations

On Day 14, the one-hour post dose clinical observations were performed approximately 80 minutes late in error.

All animals showed no clinical signs of toxicity, therefore, the late observations were considered not to affect the purpose or integrity of the study.

Deviation No.2
Motor Activity:
Animals of either sex were assessed on 21 May 2014 using activity monitors for 60 minutes over which five data acquisitions were recorded for each animal.

The acquired data were printed out, signed and dated, they were also saved in excel format at the end of the assessment. When the printed data were checked at a later date it was discovered that the data acquisition points for animals of either sex treated with 1000 mg/kg bw/day were incomplete due to a printer fault.

The incomplete data was recovered by transferring the saved file to a PC at which point they were printed, signed and dated. The retention of the data and file transfer has not been validated so this is considered to be a GLP exception. These data have been reported although no claim of GLP compliance can be made for this part of the work. This deviation was considered not to affect the purpose or integrity of the study.


Deviation No.3
Histopathology Examination
Upon histopathological examination, representative sections of the parathyroid from female no. 10 and the mammary tissue from male nos. 32, 33 and 35 were not present on the slide.

Study data indicates that samples of these tissues were taken at necropsy, processed to paraffin wax, sectioned and stained as detailed in the Study Plan. As representative sections of these tissues were not present on the slides processed, this constitutes a deviation to the Study Plan as these tissues were not examined.

Whilst this is less than ideal, there were no microscopic abnormalities detected in these tissues for the remaining animals. With respect to the mammary glands, there was no reason to suspect an effect of treatment on the male mammary tissue based on the overall results for the study, therefore the histopathological assessment of this tissue from only two high dose male animals is considered adequate. Therefore, the data lost due to the absence of these tissues was considered not to affect the purpose or integrity of the study as sufficient data was available to form an accurate interpretation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification : FAT 20341/A TE
Description : Black powder
Purity : 80.1%
Batch Number : BOP 02-12 (Navy PLK 241, BS)
Date Received : 23 July 2013
Storage Conditions : Room temperature in the dark
Expiry Date : 21 November 2017

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight 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 201 to 234g, the females weighed 171 to 209g, and were approximately six to eight weeks old.

The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., 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 Harlan Laboratories Ltd., 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.

Justification:
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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of administration:
Gavage
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in distilled water.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable.

- Storage temperature of food:
Not applicable.


VEHICLE
- Justification for use and choice of vehicle(if other than water):
Not applicable (N/A)

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 FAT 20341/A TE at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.

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 formulations investigated during the study were found to have the test material in the range of 100 to 114%. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study. In conclusion the results indicate the accurate use of the test item and distilled water as vehicle during this study. The analytical procedure wasa found to have acceptable linearity and acceptable recoveries of the test item in the vehicle. The method of analysis was validated and proved to be suitable for use.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Male and Female: 5 animals per sex at 0 mg/kg/day
Male and Female: 5 animals per sex at 30 mg/kg/day
Male and Female: 5 animals per sex at 300 mg/kg/day
Male and Female: 5 animals per sex at 1000 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on available toxicity information and compliance with regulatory hazard classification.

- Rationale for animal assignment (if not random):
Random.

- Rationale for selecting satellite groups:
Not applicable

- Post-exposure recovery period in satellite groups:
Not applicable

- Section schedule rationale (if not random):
Not applicable

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual bodyweights were recorded on Day 1 (prior to start of treatment) and on Days 8, 15, 22 and 29. Terminal bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period (Day 28).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals:
Five males and five females
- Parameters checked: Please see below:

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: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).
- Animals fasted: No
- How many animals: 5 males and 5 females
- Parameters checked: Please see below:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Creatinine (Creat) Calcium (Ca++)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Albumin/Globulin (A/G) ratio (by calculation)

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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.
- Dose groups that were examined: all dose groups were examined
- Battery of functions tested: sensory activity, grip strength and motor activity were all performed.Details of these can be seen below:

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

Sacrifice and pathology:

NECROPSY: On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

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


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

HISTOPATHOLOGY: Yes
Details: All control and high dose animals were subjected to a full histological examination and low and intermediate group animals were routinely subjected to examination of liver and spleen.

1000 mg/kg dose groups: All tissues were dispatched to the histology processing Test Site (Propath UK Ltd.) for processing (Principal Investigator: N Fower). Due to the nature of the test item, macroscopic observations of discoloration were not considered as gross lesions. Where microscopic examination of the discolored tissues revealed associated changes, examination was extended to the affected tissues of animals treated with 30 or 300 mg/kg bw/day. Any other macroscopic lesions were processed accordingly. 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 indications of treatment-related kidney changes in both sexes and changes in the testes of males, examination was subsequently extended to include similarly prepared sections of kidney from both males and females, and the testes of males (including Periodic Acid Schiff (PAS) staining) from the low and intermediate groups. Special staining of the kidneys of one control animal (Animal No. 4) and two treated animals (Animal Nos. 33 and 34) with PAS, Schmorl’s and Martius Scarlet Blue (MSB) stains was also conducted to aid diagnosis.

Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the histopathology findings was conducted by P Millar at Peter Millar Associates Ltd.

Other examinations:

ORGAN WEIGHTS:
The following organs, were removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period. These 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)

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 Provantis 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 variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. 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:

no effects observed

Description (incidence and severity):

No unscheduled deaths. Neither the type, incidence or distribution of clinical signs indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths. Neither the type, incidence or distribution of clinical signs indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in the behavioral parameters measured.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no clear adverse effects of treatment on food consumption at dosages up to 1000 mg/kg bw/day.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

No obvious effect on water consumption was detected for any treated animal.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment related changes in any of the hematological parameters measured.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in the blood chemistry parameters measured.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in functional performance. There were no toxicologically significant changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 1000 mg/kg had an increase in kidney weight. No such effects were detected in the remaining treatment groups. In the absence of any histopathothological correlates, effects were considered to be of no toxicological importance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of the animals revealed blue coloured contents through much of the digestive system. Many other internal tissues in addition to those of the gastro-intestinal tract were stained blue.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Staining in kidneys and testes. Also an increase in severity of hyaline droplet deposition in the tubules of the kidneys in males treated with 1000 mg/kg bw/day when compared to controls was noted.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Discussion:
The oral administration of FAT 20341/A TE to rats by gavage for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day was well tolerated, however, treatment related microscopic abnormalities were apparent in animals of either sex treated with 1000 mg/kg bw/day and females treated with 300 mg/kg bw/day.
 
Pigmented macrophages were present in the testes of males treated with 1000 mg/kg bw/day only and did not appear to be associated with any other pathological process. The presence of the pigmented macrophages may be related to the clearance of the specific type of test item and, in the absence of a pathological change, was considered to be of no toxicological importance.
 
There was an increase in incidence and severity of hyaline droplet deposition in the tubules of the kidneys in males treated with 1000 mg/kg bw/day when compared to controls. Hyaline droplet formation within the tubules is consistent with the accumulation of alpha 2u-globulin, a common finding in untreated male rats. This finding is considered to be specific to the male rat and does not represent a risk to humans.
 
Animals of either sex treated with 1000 mg/kg bw/day had two forms of pigment deposit in the tubular cells. These pigment deposits consisted of fine brown pigment and a yellow/brown pigment. The fine brown pigment was confirmed as lipofuscin with Schmorl’s stain. Lipofuscin deposits in the kidney tubules indicate a minor degenerate change in the tubular cells and as such probably represent an adverse effect of treatment. This finding along with the increase in hyaline droplet formation in males treated with 1000 mg/kg bw/day correlates with the increased kidney weight apparent at this dose level. Obvious cellular degeneration and moderate tubular basophilia were present in one female treated with 1000 mg/kg bw/day. The yellow/brown pigment was present superficially within some cells and was not identified by special staining; therefore this pigment was considered likely to be test item or a metabolite and as such was considered to be of no toxicological significance. The yellow/brown pigment deposits were also evident in females treated with 300 mg/kg bw/day; however, there was no evidence of any degenerative changes associated with treatment. No such effects were detected in males treated with 300 mg/kg bw/day.
 
Based on the pigment deposition in the kidneys, the No Observable Effect Level (NOEL) was considered to be 300 mg/kg bw/day for males and 30 mg/kg bw/day for females. As the yellow/brown pigment deposition in females treated with 300 mg/kg bw/day was superficial and no evidence of a pathological change was apparent, 300 mg/kg bw/day may be considered as the No Observed Adverse Effect Level (NOAEL) for animals of either sex.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results:

Mortality:

There were no unscheduled deaths.

 

Clinical Observations:

Neither the type, incidence or distribution of clinical signs indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Behavioral Assessment:

There were no treatment-related changes in the behavioral parameters measured.

 

Functional Performance Tests:

There were no toxicologically significant changes in functional performance.

 

Sensory Reactivity Assessments:

There were no toxicologically significant changes in sensory reactivity.

 

Body Weight:

There were no adverse effects of treatment on body weight development at 30, 300 or 1000 mg/kg bw/day.

 

Food Consumption

There were no clear adverse effects of treatment on food consumption at dosages up to 1000 mg/kg bw/day.

 

Water Consumption:

No obvious effect on water consumption was detected for any treated animal.

 

Hematology:

There were no treatment related changes in any of the hematological parameters measured.

 

Blood Chemistry:

There were no toxicologically significant changes in the blood chemistry parameters measured.

 

Necropsy:

Macroscopic examination of the animals revealed blue coloured contents through much of the digestive system. Many other internal tissues in addition to those of the gastro-intestinal tractwere stained blue. These tissues included the liver, kidneys, urinary bladder, seminal vesicles, testes, ovaries and uterus.

 

Organ Weights:

Males treated with 1000 mg/kg bw/day had a statistically significant increase in kidney weight, both absolute and relative to terminal body weight.

 

No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 30 or 300 mg/kg bw/day.

 

Histopathology:

The following microscopic abnormalities were detected:

Kidneys:

Special staining with Martius Scarlet Blue (MSB); Schmorl’s Stain and Periodic Acid Schiff (PAS) was carried out on the kidneys of animals 4, 33 and 34 to aid diagnosis.

 

There was an increase in incidence and severity of hyaline droplet deposition in the tubules of the kidneys in males treated with 1000 mg/kg bw/day when compared to controls. Special staining with MSB confirmed this. All males treated with 1000 mg/kg bw/day had pigment deposits in the tubular cells. This consisted of fine brown pigment which was confirmed as lipofuscin with Schmorl’s stain and also a yellow/brown pigment present superficially within some cells. This pigment was not identified by special staining.

 

In females, pigment deposits, similar to those in males were present in all females treated with 1000 mg/kg bw/day. Moderate tubular basophilia and mild tubular degeneration was also present in one female treated with 1000 mg/kg bw/day. The yellow/brown pigment was also evident in two females treated with 300 mg/kg bw/day.

 

Testes:

Pigmented macrophages, particularly notable with PAS staining were present in increased amounts in males treated with 1000 mg/kg bw/day only.

Applicant's summary and conclusion

Conclusions:

The oral administration of FAT 20341/A TE to rats by gavage for a period of twenty eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day was well tolerated however, treatment related microscopic abnormalities were apparent in animals of either sex treated with 1000 mg/kg bw/day and females treated with 300 mg/kg bw/day. The No Observable Effect Level (NOEL) was considered to be 300 mg/kg bw/day for males and 30 mg/kg bw/day for females.

Lipofuscin pigment deposits indicating a degenerative change in the kidneys of animals of either sex treated with 1000 mg/kg bw/day were treatment related and considered to represent an adverse effect of treatment. Yellow/brown pigment deposition was evident in animals of either sex treated with 1000 mg/kg bw/day and females treated with 300 mg/kg bw/day. This was regarded as superficial and therefore of no toxicological significance.

For this reason, 300 mg/kg bw/day may be considered as the No Observed Adverse Effect Level (NOAEL) for animals of either sex.

Executive summary:

Introduction:

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

 

·        Commission Directive 96/54/EC (Method B7). Commission Directive 96/54/EC (Method B7).

·        The 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 30, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Distilled water) over the same treatment period.

 

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 was performed.

 

Results:

Mortality:

There were no unscheduled deaths.

 

Clinical Observations:

Neither the type, incidence or distribution of clinical signs indicated an adverse effect of treatment at 30, 300 or 1000 mg/kg bw/day.

Behavioral Assessment:

There were no treatment-related changes in the behavioral parameters measured.

 

Functional Performance Tests:

There were no toxicologically significant changes in functional performance.

 

Sensory Reactivity Assessments:

There were no toxicologically significant changes in sensory reactivity.

 

Body Weight:

There were no adverse effects of treatment on body weight development at 30, 300 or 1000 mg/kg bw/day.

 

Food Consumption

There were no clear adverse effects of treatment on food consumption at dosages up to 1000 mg/kg bw/day.

 

Water Consumption:

No obvious effect on water consumption was detected for any treated animal.

 

Hematology:

There were no treatment related changes in any of the hematological parameters measured.

 

Blood Chemistry:

There were no toxicologically significant changes in the blood chemistry parameters measured.

 

Necropsy:

Macroscopic examination of the animals revealed blue coloured contents through much of the digestive system. Many other internal tissues in addition to those of the gastro-intestinal tract were stained blue. These tissues included the liver, kidneys, urinary bladder, seminal vesicles, testes, ovaries and uterus.

 

Organ Weights:

Males treated with 1000 mg/kg bw/day had a statistically significant increase in kidney weight, both absolute and relative to terminal body weight.

 

No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 30 or 300 mg/kg bw/day.

 

Histopathology:

The following microscopic abnormalities were detected:

Kidneys:

Special staining with Martius Scarlet Blue (MSB); Schmorl’s Stain and Periodic Acid Schiff (PAS) was carried out on the kidneys of animals 4, 33 and 34 to aid diagnosis.

 

There was an increase in incidence and severity of hyaline droplet deposition in the tubules of the kidneys in males treated with 1000 mg/kg bw/day when compared to controls. Special staining with MSB confirmed this. All males treated with 1000 mg/kg bw/day had pigment deposits in the tubular cells. This consisted of fine brown pigment which was confirmed as lipofuscin with Schmorl’s stain and also a yellow/brown pigment present superficially within some cells. This pigment was not identified by special staining.

 

In females, pigment deposits, similar to those in males were present in all females treated with 1000 mg/kg bw/day. Moderate tubular basophilia and mild tubular degeneration was also present in one female treated with 1000 mg/kg bw/day. The yellow/brown pigment was also evident in two females treated with 300 mg/kg bw/day.

 

Testes:

Pigmented macrophages, particularly notable with PAS staining were present in increased amounts in males treated with 1000 mg/kg bw/day only.

 

Conclusion:

The oral administration of FAT 20341/A TE to rats by gavage for a period of twenty eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day was well tolerated however, treatment related microscopic abnormalities were apparent in animals of either sex treated with 1000 mg/kg bw/day and females treated with 300 mg/kg bw/day. The No Observable Effect Level (NOEL) was considered to be 300 mg/kg bw/day for males and 30 mg/kg bw/day for females.

Lipofuscin pigment deposits indicating a degenerative change in the kidneys of animals of either sex treated with 1000 mg/kg bw/day were treatment related and considered to represent an adverse effect of treatment. Yellow/brown pigment deposition was evident in animals of either sex treated with 1000 mg/kg bw/day and females treated with 300 mg/kg bw/day. This was regarded as superficial and therefore of no toxicological significance.

 

For this reason, 300 mg/kg bw/day may be considered as the No Observed Adverse Effect Level (NOAEL) for animals of either sex.