Registration Dossier

Administrative data

Description of key information

1000 mg/kg bw/day this dose level can be considered the No Observed Adverse Effect Level (NOAEL). The No Observed Effect Level (NOEL) was indicated to be 100 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 13 April 2015 and 22 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
None
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
None
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of one hundred and forty animals (seventy males and seventy females) were accepted into the study. The weight ranges of animals allocated to the 90 days of treatment were males 227 to 275 g, and females 154 to 196 g, and were approximately six to eight weeks of age. Satellite groups were of similar weight and age.

The animals were housed in groups of three or four 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; 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.

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.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Test Item Preparation
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results from an earlier study (Harlan Study No. 41403247) showed the formulations to be stable for at least twenty-eight days. Formulations in this study were prepared fortnightly and stored at approximately +4 °C in the dark.

Samples of each test item formulation were taken and analyzed for concentration of FAT 40868/A TE at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Introduction
FAT 40868/A TE concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique.

Test item
The test item FAT 40868/A TE described in the main part of the study was also used as the analytical standard.

Analytical procedure
Preparation of standard solutions
Stock solutions of FAT 40868/A TE in water were prepared for external standard calibration. An aliquot, approximately 0.02 g of FAT 40868/A TE was accurately weighed into a 200 mL volumetric flask and brought to volume with water to yield a solution with a concentration of 0.1 mg/L.
On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were diluted with water. An aliquot of FAT 40868/A TE formulation was accurately weighed into a volumetric flask and brought to volume with water; this was then shaken to dissolve.

Preparation of accuracy samples
The accuracy determinations were performed under Envigo study number 41403247.

Preparation of linearity standards
The linearity determinations were performed under Envigo study number 41403247.

Instrumental setup
HPLC: Agilent Technologies 1200, incorporating auto-sampler and workstation
Column: Gemini C18 (100 x 4.6 mm id)
Column temp: 30 °C
Mobile phase:
Eluent A: 2 g TRAB in 100 mL acetonitrile + 90 mL water.
Eluent B: 2 g TRAB in 1000 mL acetonitrile.

Time % B
0 0
5 90
10 90

Flow rate: 1 mL/min
UV detector wavelength; 590 nm
Injection volume: 10 µL
Retention time: ~ 6 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.

Results
Validation of analytical method
Specificity
The control dose samples and an analysed solvent blank showed no significant interfering response at the retention time of FAT 40868/A TE. The standard solutions contained a peak specific for FAT 40868/A TE whose area changed accordingly with known concentration; hence the specificity of the method by retention time was confirmed.

Linearity
The linearity determinations were performed under Envigo study number 41403247.
Accuracy
The accuracy determinations were performed under Envigo study number 41403247.

Test item formulations
The test item FAT 40868/A TE was found to be stable in the formulations during Envigo study number 41403247 when kept for 28 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time zero mean.
In conclusion, the results obtained during Envigo study number 41403247 indicate the accurate use of FAT 40868/A TE and distilled water as a vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Discussion
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of FAT 40868/A TE in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
90 days for the main study,
29 days for the satellite groups.
Frequency of treatment:
Daily
No. of animals per sex per dose:
10 male and 10 female for all groups in the main study,
3 males and 3 females for the satellite control group,
9 males and 9 females for the satellite groups.
Control animals:
yes, concurrent vehicle
Details on study design:
Procedure
The test item was administered daily, for up to ninety consecutive days at dose levels of 10, 100 and 1000 mg/kg bw/day, controls received vehicle alone (distilled water). Administration was via gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of distilled water.

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.

Dose selection
The dose levels were chosen in consultation with the study sponsor following review of previous toxicity work. 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.
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals maintained for 90 days were observed for signs of functional/behavioral toxicity. These observations were performed outside the home cage, in a standard arena, at least two hours after treatment to ensure that any transient effects of treatment were identified. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

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.

Body Weight
Individual body weights were recorded for all animals on Day 1 (prior to dosing) and at weekly intervals thereafter. Individual body weights of animals maintained for 90 days were also recorded at terminal kill.

Food Consumption
Dietary intake was recorded weekly for each cage group of animals maintained for 90 days. Food efficiency (body weight gain/food intake) 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.

Ophthalmoscopic Examination
The eyes of all control and high dose animals (maintained for 90 days) were examined pretreatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.

Laboratory Investigations
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. 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) - blue stained slides were performed but reticulocytes were not assessed
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 90 day treatment period, the animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination. These animals were then subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were removed from all animals killed at the end of the 90 day treatment period. The organs were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus (with cervix), Liver.

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 pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides ♦, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes ♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue•, Lungs (with bronchi) #, Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary glands, Uterus (with cervix), Muscle (skeletal)•, Vagina.

• Retained only and not processed
* 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 Test Site (Envigo CRS Limited, Eye Research Centre, Eye, Suffolk, UK) for processing (Principal Investigator: J Schofield). All tissues from 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.

Since there were indications of possible treatment-related changes, examination was subsequently extended to include similarly prepared sections of the kidneys from each sex of animals in the low and intermediate groups.
Other examinations:
Toxicokinetics
Additional to the primary purpose of this study, satellite animal groups were included at the request of the study sponsor to provide supplementary toxicokinetic (TK) blood profile evaluation of the test item. Following completion of the live phase of the study the sponsor instructed that as the TK results were not available they would be reported at a later date independently of this study report. Furthermore, as the TK phase had no bearing on the outcome of the 90 day study; reference to the TK phase should be kept to a minimum in this 90 day study report. The operational procedures involved in the conduct of the TK phase of the study are referenced in detail in the Study Plan Appendix 19 of the study report and relevant animal data will be retained and archived with the study records.
For reference a total of 30 male and 30 female rodents were allocated to the TK satellite groups. Blood samples were taken for toxicokinetic determinations following dosing on Day 1 and on Day 29. For animal health purposes clinical observations, body weights and visual assessments of food and water consumption were monitored. Following the final series of TK blood sampling the animals were humanely sacrificed and the cadavers disposed of without further examination.

With the exception of body staining (originating from exposure to the test item) no abnormalities were detected in the satellite groups and on this basis further reference in this study report to the animal data has been confined to the clinical observations.

Following completion of the TK study phase, the frozen plasma samples were shipped to Envigo CRS Ltd, into the care of the appointed Principal Investigators.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

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

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric 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)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See results
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See results
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
Mortality
There were no unscheduled deaths.

Clinical Observations
Blue staining of body surfaces was observed in each sex treated at 1000 mg/kg bw/day from the second week of dosing onwards and accompanied in both sexes from Week 8 by dark eyes. This was accompanied by frequent episodes of blue colored faeces and occasional blue stained bedding. Instances of blue staining of the body surfaces and blue stained faeces were also evident in females treated at 100 mg/kg bw/day. Isolated instances of staining around the snout (one male treated at 100 mg/kg bw/day) and increased salivation (one male trated at 1000 mg/kg bw/day) were also observed. Clinical findings of this nature are commonly observed when a colored test item is administered orally and in isolation are considered to be of no toxicological importance.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests
There were no treatment-related changes in the functional performance parameters measured.
A statistically significant reduction in the second of three tests for forelimb grip strength (p<0.05) and for overall activity (p<0.1) was identified for females from each test group in comparison with controls. However, in the absence of a dose dependent trend or supporting evidence to indicate neurotoxicity these findings were considered to be due to natural biological variation and not test item toxicity.

Females at 10 mg/kg bw/day also showed a slight increase (p<0.05) for the first of three hind limb tests. In the absence of a similar trend in high dose (1000 mg/kg bw/day) females or any other supporting indication of neurotoxicity this isolated finding was considered fortuitous and not associated with treatment.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

Body Weight
There was no effect of treatment on body weight development of either sex during the study at 10, 100 or 1000 mg/kg bw/day.
Occasional intergroup differences in body weight gains were observed in females from all test groups attaining statistical significance at Weeks 6, 7 and 11 (p<0.05 or p<0.01) in comparison with controls. However, as group mean values were almost certainly influenced by isolated unusually high or low control group means. These isolated differences were considered not to be related to treatment.

Food Consumption
No treatment-related adverse effect on food consumption or food efficiency (the ratio of body weight gain to dietary intake) was detected in test animals of either sex in comparison with controls throughout the study.

Water Consumption
Daily visual inspection of water bottles did not reveal any intergroup differences.

Ophthalmoscopic Examination
No adverse ocular changes were detected in any control or high dose animal prior to start of treatment or prior to termination. Incidental findings involved traces of blue staining detected on the eyelids and sclera of high dose animals at the pre-terminal ocular examinations.

Laboratory Investigations
Hematology
No treatment related hematological abnormalities were detected.
Hematological determinations revealed slight but statistically significant increases (p<0.05) in group mean activated partial thromboplastin time (APTT) and platelet count in males treated at 1000 mg/kg bw/day and 100 mg/kg bw/day in comparison with controls. However, the intergroup difference was unconvincing and in the absence of histopathological correlates indicating impaired liver function, these isolated findings were considered incidental and not associated with test item toxicity.

100 mg/kg bw/day females displayed an increase (p<0.05) in APTT. However, there was no dose dependent trend or supporting evidence connecting this with treatment and this finding was therefore considered spurious.

Blood Chemistry
There were no treatment-related changes detected in the blood chemistry parameters examined.
Both sexes treated at 1000 mg/kg bw/day showed a reduction (p<0.01) in plasma bilirubin levels in comparison with controls. Females that received 1000 and 100 mg/kg bw/day displayed slight reductions (p<0.05) in potassium and creatinine levels. In each instance there was an absence of histopathological correlates of treatment-related changes and furthermore as individual values were almost all within the anticipated historical ranges these findings were considered fortuitous and not associated with test item toxicity.

Males from each test group showed a slight increase in (p<0.05) in chloride levels when compared to the concurrent control. However, the difference was marginal and individual values were all within the normally expected ranges. Therefore in the absence of supporting evidence of impaired kidney function, this finding was attributed to natural biological variation and not test item toxicity.

100 mg/kg bw/day females displayed elevated (p<0.05) plasma inorganic phosphate levels. In the absence of a dose dependent trend this finding was considered to have arisen fortuitously and not to be associated with test item toxicity.

Pathology
Necropsy
Blue or dark discoloration of the eyes, ears, skin, kidneys, stomach, spinal cord, urinary bladder, rectum, testes or vagina were prevalent in the majority of animals treated at 1000 mg/kg bw/day.
Incidental findings involved instances of reddened lungs observed in three control females, three females at 10 mg/kg bw/day, two females at 100 mg/kg bw/day and in one male at 1000 mg/kg bw/day. Such discoveries are consistent with normally expected low incidence findings in laboratory maintained rats particularly when associated with gavage administration and are therefore considered not to be related to test item toxicity.

Organ Weights
At 1000 mg/kg bw/day increases in group mean absolute and relative (to terminal bodyweight) kidney and liver weights (males and females respectively) were slightly higher than controls. As there was no microscopic evidence of impaired kidney or liver function these findings were considered not to be indicative of test item toxicity.

Histopathology
Treatment-related microscopic abnormalities were confined to the kidneys of each sex at 1000 mg/kg bw/day; characterised by an increase in hyaline droplets in the kidney tubules of all males with the majority of females displaying vacuoles in the tubular cells; these were of minimal severity and contained dark brown pigment. These changes were considered not to be indicative of test item toxicity but more plausibly associated with the renal excretion of the test item.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: kidney
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Treatment with 1000 mg/kg bw/day, resulted in minor findings, most notably histopathological changes considered associated with deposition and excretion of the test item precluding this dose level being classified as the No Observed Effect Level (NOEL).
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: Renal system
Organ:
kidney
Treatment related:
no
Dose response relationship:
yes
Relevant for humans:
no

None

Conclusions:
1000 mg/kg bw/day was determined to be the NOAEL, while 100 mg/kg bw/day was determined to be the NOEL.
Executive summary:

A 90-day oral toxicity study was conducted for evaluation of potential of the test item to incite adverse effects on repeated administration for a long term period. The study was conducted according to OECD Guideline 408, and in compliance with GLP. In this study, test item was administered by gavage to three groups, each containing ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). 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. Ophthalmoscopic examination was performed on control group and high dose animals before the start of treatment and during Week 12 of the study. Following ninety days of treatment all animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

There were no unscheduled deaths observed during the study. No clinical signs considered to be related to test item toxicity were seen. No treatment-related changes in the behavioral parameters, functional performance parameters and sensory reactivity scores related to test item toxicity were detected. Food consumption, food efficiency, water consumption and body weight gains were not affected. No adverse ocular changes were detected in any control or high dose animal prior to start of treatment or prior to study termination. No treatment related changes in hematological parameters or blood chemistry parameters were detected. Blue or dark discoloration of the eyes, body surfaces and internal organs was identified in the majority of animals treated at 1000 mg/kg bw/day. At 1000 mg/kg bw/day increases in group mean absolute and relative (to terminal bodyweight) kidney and liver weights (males and females respectively) were slightly higher than controls. As there was no microscopic evidence of impaired kidney or liver function, these findings were considered not to be indicative of test item toxicity. Treatment-related microscopic abnormalities were confined to the kidneys of each sex at 1000 mg/kg bw/day; characterised by an increase in hyaline droplets in the kidney tubules of all males with the majority of females displaying vacuoles in the tubular cells; these were of minimal severity and contained dark brown pigment. These changes were considered not to be indicative of test item toxicity but more plausibly associated with the renal excretion of the test item.

 

Hence, based on the findings of the study, it can be concluded that oral administration of the test item, FAT 40868/A TE, to rats for a period of ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day, resulted in minor findings confined to animals treated at 1000 mg/kg bw/day most notably histopathological changes considered associated with deposition and excretion of the test item precluding this dose level being classified as the No Observed Effect Level (NOEL). However, in the absence of toxicological significant findings at 1000 mg/kg bw/day this dose level can be considered the No Observed Adverse Effect Level (NOAEL). There were no convincing treatment-related findings identified in either sex treated at 10 and 100 mg/kg bw/day and on this basis the No Observed Effect Level (NOEL) was indicated to be 100 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
High quality study conducted in compliance with GLP
System:
integumentary

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

None

Additional information

Oral:

A 90-day oral toxicity study was conducted for evaluation of potential of the test item to incite adverse effects on repeated administration for a long-term period. The study was conducted according to OECD Guideline 408, and in compliance with GLP. In this study, test item was administered by gavage to three groups, each containing ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day. There were no unscheduled deaths observed during the study. No clinical signs considered to be related to test item toxicity were seen. No treatment-related changes in the behavioural parameters, functional performance parameters and sensory reactivity scores related to test item toxicity were detected. Food consumption, food efficiency, water consumption and body weight gains were not affected. No adverse ocular changes were detected in any control or high dose animal prior to start of treatment or prior to study termination. No treatment related changes in hematological parameters or blood chemistry parameters were detected. Blue or dark discoloration of the eyes, body surfaces and internal organs was identified in the majority of animals treated at 1000 mg/kg bw/day. At 1000 mg/kg bw/day increases in group mean absolute and relative (to terminal bodyweight) kidney and liver weights (males and females respectively) were slightly higher than controls. As there was no microscopic evidence of impaired kidney or liver function these findings were considered not to be indicative of test item toxicity. Treatment-related microscopic abnormalities were confined to the kidneys of each sex at 1000 mg/kg bw/day; characterised by an increase in hyaline droplets in the kidney tubules of all males with the majority of females displaying vacuoles in the tubular cells; these were of minimal severity and contained dark brown pigment. These changes were considered not to be indicative of test item toxicity but more plausibly associated with the renal excretion of the test item. Oral administration of the test item, FAT 40868/A TE, to rats for a period of ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day, resulted in minor findings confined to animals treated at 1000 mg/kg bw/day most notably histopathological changes considered associated with deposition and excretion of the test item precluding this dose level being classified as the No Observed Effect Level (NOEL). However, in the absence of toxicological significant findings at 1000 mg/kg bw/day this dose level can be considered the No Observed Adverse Effect Level (NOAEL). There were no convincing treatment-related findings identified in either sex treated at 10 and 100 mg/kg bw/day and on this basis the No Observed Effect Level (NOEL) was indicated to be 100 mg/kg bw/day.

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).