Ethyl 4-methyl-2-oxocyclohexanecarboxylate

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:

The study was conducted between 1 November 2016 and 5 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD guidelines and in compliance with GLP, without deviations that influence the results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

EC Number:

236-910-1

EC Name:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Cas Number:

13537-82-1

Molecular formula:

C10H16O3

IUPAC Name:

ethyl 4-methyl-2-oxocyclohexane-1-carboxylate

Test material form:

liquid

Details on test material:

Physical appearance: Clear colourless liquid
Storage conditions: In refrigerator (2-8°C) in clear glass bottle

Specific details on test material used for the study:

Test item identity (including alternative names): FRET 13-0545
4-methyl-2-oxo-cyclohexanecarboxylic acid, ethyl ester.
Appearance: Clear colourless liquid.
Storage conditions: Refrigerated (2 to 8ºC) in the dark.

Test animals

Species:

rat

Strain:

other: RccHan™:WIST rat.

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Envigo RMS Limited.
Number of animals: 35 males and 35 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization: 13 days before commencement of treatment.
Age of animals at start of treatment: 49 to 55 days.
Weight range of animals at the start of treatment
Males: 169 to 224 g
Females: 132 to 167 g

Allocation and Identification
Allocation: Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ± 20% of the mean for the appropriate sex. No replacements were required.

Animal Care and Husbandry
Environmental Control
Animal facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex (main study and recovery).
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet: Rat and Mouse No. 1 Maintenance, powdered diet
Availability: Non-restricted (removed overnight during the period of urine collection which preceded clinical pathology blood sampling procedures).

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (except during urine collection).

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance was administered orally, via the diet.
Control animals received untreated diet of the same batch.

Each animal received constant concentrations in ppm.

Animals were treated continuously, except during periods of overnight food deprivation during, or prior to, routine clinical pathology investigations. During the recovery period, all animals were given untreated diet.

Prior to dosing, each formulation was mixed using a Turbula mixer for 100 cycles.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly and divided into daily aliquots.
- Mixing appropriate amounts with (Type of food): Plain diet was pre-frozen prior to formulation procedures. The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix at a concentration of 57675.9 ppm. The amount of test substance required for the premix was
added to an equal amount of plain diet and stirred. An amount of plain diet equal to the weight of the mixture was added and the mixture was stirred again until visibly
homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 100 cycles. An appropriate amount of the first premix was used to prepare a second premix (by adding a suitable quantity of plain diet and using a similar doubling up process) at a concentration of 6608.8 ppm. The first premix was used to prepare final dietary preparations for the intermediate and high dose concentrations and the second premix was used to prepare
final dietary preparations for the low dose concentration. In all cases, the required amounts of premix diet were diluted with further quantities of plain diet using the
doubling up process to prepare the final test formulations. Each formulation was mixed using a Turbula mixer for 100 cycles.
- Storage temperature of food: Frozen (-10ºC to -30ºC) on completion of preparation/aliquoting procedures. Daily aliquots of diet were allowed to thaw at room temperature (+15ºC to +25ºC) prior to the commencement of feeding procedures. Fed diet was discarded within approximately 24 hours (±4 hours) from the time of withdrawal from frozen storage conditions.


A record of the usage of the diets was maintained on all occasions when food consumption was measured. This was performed using the initial weight of the diet container and an on-line data check on completion of the feeding procedure to ensure that all cages were fed the correct amount of diet. No significant discrepancy was found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation Analysis

Stability and homogeneity
Results from the pre-study formulation analysis demonstrated adequate homogeneity of the test item within powdered diet at concentrations of 500 to 20000 ppm (536.5 to 21460 ppm as supplied). These diets were shown to be stable for up to 15 days following frozen storage ( 10ºC to 30ºC) followed by 28 hours at ambient temperature (+15ºC to +25ºC).

Achieved concentration
GC-FID was used to analyse the test formulations.
The analytical procedure was successfully validated for FRET 13-0545 in Rat and Mouse No.1 Maintenance powdered diet with respect to the specificity of chromatographic analysis, limits of detections and quantification, the linearity of detector response, repeatability, method accuracy and precision.
Samples of each formulation prepared for administration in Weeks 1, 2, 3 and 4 of treatment were analyzed for achieved concentration of the test item. All formulation samples were initially stored frozen ( 10ºC to 30ºC) for a period not exceeding 15 days and were subsequently transferred to ambient storage (+15ºC to +25ºC) for a minimum period of 28 hours prior to being analyzed for achieved concentration.

Duration of treatment / exposure:

Minimum period: Four weeks followed by a two week recovery period
Cessation of treatment for the recovery phase animals started on the first day of the necropsy of animals allocated to the main study. Serial observations were recorded at appropriate intervals.

Frequency of treatment:

Continuously, except during periods of overnight food deprivation during, or prior to, routine clinical pathology investigations. During the recovery period, all animals were given untreated diet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 400 ppm

Doses / concentrations 2

Dose / conc.:

4 200 ppm

Doses / concentrations 3

Dose / conc.:

12 500 ppm

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The high dosage was selected on the basis of available toxicity data and a preliminary oral toxicity investigation performed at this laboratory Other levels were selected on the basis of the key dosages relative to EEC labelling requirements.

Examinations

Observations and examinations performed and frequency:

For group identification see tables in section 'any other information on materials and methods'

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed on all main study animals in Groups 2 and 3 and all recovery animals in Groups 1 and 4 during Week 4 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 4 of treatment, the motor activity of each main study animal in Groups 2 and 3 and all recovery animals in Groups 1 and 4 was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study (last scheduled body weight recorded on the day preceding clinical pathology investigations) and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and daily throughout the treatment and recovery periods.

Water Consumption
Fluid intake was assessed by daily visual observation during the treatment and recovery periods. No test item-related effect was observed (animals which had previously received 12500 ppm appeared to consume more water than the controls on one occasion during the second week of recovery, Day R9, but this was considered an incidental finding due to its isolated nature) and consequently quantitative measurements were not performed.

Estrous cycles - vaginal smears
Dry smears were taken and assessed as follows:
Last 14 days of treatment period (during weeks 3 and 4 of treatment)* - All main study and recovery study animals
*Vaginal smears were not taken on Day 26 of the treatment phase

Hematology, Peripheral Blood
Blood samples were collected at the following occasions:
Main study day 29 - All main study animals
Recovery day 15 - All recovery study animals

Blood samples were collected after overnight withdrawal of food and on the morning after overnight collection of urine so the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected at the following occasions:
Main study day 29 - All main study animals
Recovery day 15 - All recovery study animals

Blood samples were collected after overnight withdrawal of food and on the morning after overnight collection of urine so the animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Urinalysis
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasions:
Main study day 29 - All main study animals
Recovery day 15 - All recovery study animals

The individual samples were examined for the following characteristics:
Using manual methods:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
Ketones (Keto)
Bile pigments (Bili)
Blood pigments (UBld)

Using a Roche P Modular Analyzer:
Protein (T-Prot)
Glucose (T-Gluc)
Creatinine (T-Creat)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

Biomarkers
Blood samples were collected at the following occasions.
Main study day 29 - All main study animals
Recovery day 15 - All recovery study animals

Sampling procedures were performed at the same time (and under the same conditions) as blood collection procedures for routine clinical pathology investigations and occurred at approximately the same time of day, in the morning, at each sampling occasion.
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein and collected into tubes containing EDTA as anticoagulant. After separation, one 100 µL aliquot of plasma was retained and labelled as T3 and T4; all remaining plasma was retained and labelled as TSH. All plasma samples were retained frozen (-60 to -80°C) but no analysis was performed as none of the results from this study indicated any potential test item-related effect on the thyroid and/or pituitary-thyroid axis. These samples will be discarded upon issue of the final report.

Sacrifice and pathology:

An example is presented below, select what is applicable and add what is needed
GROSS PATHOLOGY: Yes
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: Main study animals were killed following four weeks of treatment.
Recovery animals were killed at the end of the scheduled two week recovery period.
Sequence: To allow satisfactory inter-group comparison.


Organ weight
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals. The following organs from each animal were dissected free of fat and weighed:
adrenals
brain
kidneys
Liver (section from two lobes)
ovaries
spleen
testes
epididymides
Heart (including auricular and ventricular regions)
Pituitary
Prostate
Seminal vesicles (with coagulating glands)
Thymus
Uterus with cervix

Macroscopic pathology
The histology and pathology of the following tissue samples was carried out:

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Eyes
Femur and marrow (femorotibial joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Ovaries
Peyer’s patches
Pituitary
Prostate
Rectum
Sciatic nerves
Seminal vesicles (with coagulating glands)
Skeletal muscle
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Skin with mammary glands (inguinal area)
Spleen
Sternum (and bone marrow)
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina


Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes - In modified Davidson’s fluid.
Eyes - In Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full list - Main study animals of Groups 1 and 4.
Abnormalities (males and females) and kidneys (males only) - All main study animals of Groups 2 and 3 and all recovery phase animals.
Routine staining - Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:
Terminal sacrifice: All main study animals of Groups 1 and 4. - All specified in the table in section 'any other information on materials and methods'
Terminal sacrifice: All main study animals of Groups 2 and 3. - Abnormalities (males and females) and kidneys (males only).
Recovery sacrifice: All recovery study animals from all groups - Abnormalities (males and females) and kidneys (males only).

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

All statistical analyses were carried out separately for males and females.
The following were analyzed at each timepoint separately: Grip strength, motor activity, Body weight,
Hematology, Blood chemistry, Urinalysis, Organ weights, absolute, and adjusted for terminal body weight, Estrous cycles
The following sequence of tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting a non monotone dose response, Dunnett's test was performed instead. If there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at 1% level following logarithmic and square-root transformations. The H1 approximate testwas applied. If the H1 approximate test was not significant at 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, Steel's test was performed instead. If there were only two groups, comparisons were made using Wilcoxon rank sum tests.
For grip strength, motor activity and clinical pathology data, if 75% of the data were the same value Fisher’s exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against control.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Significant differences were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See results

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See results

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See results

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, non-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:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycles

Details on results:

Detailed Physical Examination and Arena Observations
There were no deaths and the appearance and behaviour of the animals were unaffected by treatment.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength were unaffected by treatment.

Motor Activity
Motor activity was unaffected by treatment.

Body Weight
Body weight gain during the first week of treatment (Weeks 0 to 1) was slightly low, when compared with the controls, for males and females receiving 12500 ppm although gains were generally similar to controls from Week 2. This resulted in a slightly low overall body weight gain (Weeks 0 to 4) in these animals with the difference attaining statistical significance.
During the first week of recovery (Week R0 to R1) the body weight gain was slightly high, when compared to the controls, in males and females which had previously received 12500 ppm with the difference attaining statistical significance in males and resulting in slightly high overall body weight gain (Weeks R0 to R2) in this sex.

Food Consumption
Food consumption was low for the first day of treatment, when compared with the controls, for males and females receiving 12500 ppm and remained marginally lower than the controls up to Day 4. This resulted in lower than control overall mean values for the first week of treatment (Day 1-7). For the remainder of the treatment period, daily food consumption values were similar between control and treated animals.
During the recovery period, daily food consumption values were slightly high (especially during the first day of recovery), when compared to the treatment period and controls, for males which had previously received 12500 ppm. Daily food consumption values for females which had previously received 12500 ppm were similar to the controls throughout the recovery period.

Water consumption
A visual assessment of water intake did not reveal any test-item related effects.

Estrous cycles
There was no effect of treatment on estrous cycles.

Hematology, Peripheral Blood
The hematological examination after 4 weeks of treatment revealed slightly high neutrophil counts (1.4 X Control) in males which received 12500 ppm, with 3/5 individual values being just above the background control range (90-percentile range of 0.53 to 1.39; n=39 values). No similar effect was evident in females but there was an increase in total white blood cell count (1.3 X Control) due to increases in lymphocyte (1.3 X Control) and monocyte (1.7 X Control) counts in this sex at 12500 ppm; all or the vast majority of individual values were, however, within the background control ranges (90-percentile ranges of 2.76 to 7.81 x109/L for white blood cell count, 2.17 to 6.69 x109/L for lymphocytes and 0.04 to 0.17 x109/L for monocytes; n=40 values in all cases). The findings in females showed complete recovery but neutrophil counts remained slightly high (1.6 X Control) in males which had previously received 12500 ppm.
Reticulocyte counts were slightly low (down to 0.8 X Control) at the end of the treatment period for all treated groups of males. This occurred in absence of any other indicators of altered erythrocyte turnover or formation (total red blood cell counts, mean cell volume and red cell distribution width were all normal), however, and there was no dose-relationship or any similar effect in females. Taking all of these factors into consideration, this finding was considered to have a doubtful/uncertain relationship to treatment. Similarly, the slightly high (1.1 X Control) red cell distribution width seen at the end of the treatment period in females which received 12500 ppm (which occurred in the absence of any significant increase in either total red blood cell count or mean cell volume and was not affected in males) was also considered to be of doubtful/uncertain relationship to treatment.
All other inter-group differences from controls seen at the end of the treatment period, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation. Such changes included the slightly low platelet counts for all treated groups of females, where there was no dose-response and all individual values were within the background control range (90-percentile range of 743 to 1119 x109/L; n=40 values). They also included the marginally high haemoglobin concentration in males which received 12500 ppm which was due to a slightly lower than expected control mean value; 3/5 control males (Nos.12, 13 and 15) had values just below the background control range (90-percentile range of 14.7 to 16.7 g/dL n=39 values) but all values for the high dose males were within this range.
Some differences from control (higher haematocrit, erythrocyte count and mean cell haemoglobin concentration and lower mean cell haemoglobin) attained statistical significance at the end of the recovery period for females which had previously received 12500 ppm. These were slight, non-adverse, differences that were considered unlikely to have been due to the previous treatment of the test item; all individual values were within the background control ranges (90-percentile ranges of 0.385 to 0.464 L/L for haematocrit, 7.16 to 8.58 x1012/L for erythrocyte counts, 33.3 to 36.6 g/dL for mean cell haemoglobin concentration and 17.8 to 19.6 pg for mean cell haemoglobin; n=40 values in all cases and using the same background control data as that applied to the findings seen at the end of the treatment period).

Blood Chemistry
The biochemical examination of the blood plasma after 4 weeks of treatment revealed a dose- related reduction of glucose concentration (down to 0.6 X Controls) in all treated groups of males, but all individual values were within the background control range (90-percentile range of 4.81 to 10.19 mmol/L; n=40 values) with the exception of a low concentration (4.46 mmol/L) for an intermediate group male (No. 19) that was just below this range. It should also be acknowledged that one control value (10.85 mmol/L for animal No. 14) was just above the background control range. This finding showed complete recovery.
Slightly high albumin concentration (1.06 X Control) was evident at the end of the treatment period in all treated groups of males, with a slight increase in albumin to globulin ratio in males which received 12500 ppm; all individual albumin values for the treated groups were within the background control range (90 percentile range of 35 to 39 g/L; n=40 values) whereas 2/5 control values (for animal Nos. 12 and 15) were just below this range. Taking this factor into consideration, along with the absence of any dose-response, or similar effect in females, these findings were considered to have a doubtful/uncertain relationship to treatment.
Slightly high phosphorus concentration (up to 1.3 X Control) was apparent at the end of the treatment period in all treated groups of females but there was no dose-relationship, no similar effect in males and all individual values from treated animals were within the background control range (90-percentile range of 1.32 to 2.49 mmol/L; n=25 values). Consequently, this finding was considered to have a doubtful/uncertain relationship to treatment.
All other inter-group differences from controls seen at the end of the treatment period, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation. Such differences included the slightly low alanine amino transferase activity in males which received 12500 ppm, where the majority of individual values were within the concurrent control range (23 to 33 U/L) and all values were within the background control range (90-percentile range of 22 to 60 U/L; n=40 values), and the slightly high calcium concentration in males which received 1400 ppm, where no similar finding was evident at the higher levels (≥4200 ppm). They also included the slightly low urea concentrations seen in all treated groups of males, since these differences lacked any dose-response, were not evident in females and all individual values were within the background control range (90 percentile range of 5.70 to 10.34 mmol/L; n=40 values).

Urinalysis
The analysis of the urine after 4 weeks of treatment revealed slightly low urinary volume (0.5 X Control) for males which received 4200 or 12500 ppm; all individual values were within the background control range (90-percentil range of 1.8 to 9.2 mL; n=25 values) with the exception of two slightly low volumes (1.7 mL) for one intermediate group male (No. 16) and one high dose male (No. 9). No similar effect was evident in females. Urine volume was unaffected at the end of the recovery period.

Organ Weights
Analysis of organ weights for animals killed after 4 weeks of treatment revealed slightly high adjusted kidney weight (maximum 1.1 X Control but without dose-relationship) in all treated groups of males. The individual absolute values for the treated groups were variable in relation to the concurrent control range (1.596 to 2.013 g) and showed no clear pattern in terms of dose-relationship. Some individual absolute values for treated animals were just above this range (2.020 g for low dose male No. 5 and 2.049 and 2.090 g for high dose male Nos. 17 and 18, respectively) whilst some were below it (1.530 g and 1.539 g for intermediate dose male Nos. 16 and 20, respectively, and 1.572 g for high dose male No. 8); all other individual absolute kidney weights were within the concurrent control range. There was no evidence of recovery, since adjusted kidney weights remained slightly high (1.1 X Control) at the end of the recovery period in males which had previously received 12500 ppm.
Adjusted liver weights were slightly high (1.1 X Control) at the end of the treatment period in females which had received 12500 ppm; all individual absolute values were within the concurrent control range (5.002 to 7.912 g). There was no statistically significant difference in adjusted liver weight at the end of the recovery period between controls and females which had previously received 12500 ppm.
Adjusted ovary and uterus weights were slightly low (0.7 and 0.6 X Control, respectively, but statistical significance was not attained for the differences in uterus weights) at the end of the treatment period in females which had received 12500 ppm; the difference in mean weights between the control and treated animals was partly exacerbated by a high absolute ovary or uterus weight amongst the controls (female No. 115 had high ovary weights and female No. 114 had a high uterus weight, both of which were just above the upper range of the background control range; 90-percentile ranges of 0.061 to 0.105 g and 0.334 to 1.023 g for absolute ovary and uterus weights, respectively, n=40 values in both cases). These findings showed complete or at least partial recovery.

Macropathology
The macroscopic examination performed after 4 weeks of treatment and 2 weeks of recovery revealed no test item-related lesions.
The incidence and distribution of all findings was considered incidental and unrelated to treatment.

Histopathology
Animals killed after 4 weeks of treatment
Treatment Related Findings
Changes related to treatment with FRET 13-0545 were seen in the kidneys.
Kidneys
An increased incidence and severity of hyaline droplet accumulation was seen in males of all treatment groups with a clear dose-relationship. This finding was associated with tubular basophilia in one male given 4200 ppm and one male given 12500 ppm.
Incidental Findings

Animals killed after 2 weeks of recovery
Treatment Related Findings
Evidence of recovery was seen in the kidneys after 4 weeks of treatment followed by a 2 week recovery period.
Kidneys
An increased incidence of hyaline droplet accumulation and an associated tubular basophilia were seen in males previously given 12500 ppm. Partial recovery was considered to have occurred with a reduction in severity of hyaline droplet accumulation after the 2 week recovery period but the incidence and severity of tubular basophilia was considered to represent a progression of this change seen at termination in this dose group.
Incidental Findings
All other histological changes were considered incidental and unrelated to the test item.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: All dietary concentrations were well tolerated with no adverse signs of toxicity. The kidney was identified as a potential target organ in males but there was no evidence of any adverse/degenerative renal pathology.

Remarks on result:

other: approximately 917 and 869 mg/kg/day in males and females, respectively

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Formulation Analysis

The mean achieved concentrations of FRET 13-0545 in test diets analysed for the study were within the applied limits of +10%/-15% of the nominal concentrations. 

Table of mean achieved concentration results

Occasion	Group	Nominal concentration (ppm)	Analysed concentration (ppm)	RME (%)
Week 1	1	0	ND	-
	2	1400	1230*	-12.1
	3	4200	3750*	-10.7
	4	12500	11700	-6.4
Week 2	1	0	ND	-
	2	1400	1270	-9.3
	3	4200	3990	-5.0
	4	12500	12000	-4.0
Week 3	1	0	ND	-
	2	1400	1240*	-11.4
	3	4200	3900	-7.1
	4	12500	11700	-6.4
Week 4	1	0	ND	-
	2	1400	1240*	-11.4
	3	4200	3770*	-10.2
	4	12500	11700	-6.4

RME     Relative mean error, representing the deviation from nominal.

ND         Not detected.

*            Analysed concentration was greater than -10% of nominal so the achieved concentration value was used for              the achieved dosage calculation.

Achieved Dose

The overall mean achieved dosages for FRET 13-0545 at nominal dietary concentrations of 1400, 4200 and 12500 ppm were 96, 307 and 917 mg/kg/day for males and 94, 280 and 869 mg/kg/day for females, respectively.

Summary of treatment related findings in the kidneys of animals killed after 4 weeks of treatment

Group/sex	1M	2M	3M	4M
Level (ppm)	0	1400	4200	12500
Accumulation, Hyaline Droplets
Minimal	1	4	2	1
Slight	0	0	3	4
Total	1	4	5	5
Basophilia, Tubular
Minimal	0	0	1	1
Total	0	0	1	1
Number of tissues examined	5	5	5	5

Summary of treatment-related changes in the kidneys of animals killed after 2 weeks of recovery

Group/sex	1M	4M
Level (ppm)	0	12500
Accumulation, Hyaline Droplets
Minimal	2	3
Total	2	3
Basophilia, Tubular
Minimal	0	3
Slight	0	2
Total	0	5
Number of tissues examined	5	5

 

Discussion

The continuous administration of the test item, FRET 13-0545, to Han Wistar(RccHan™:WIST) rats via the diet over a period of four consecutive weeks was well tolerated with no adverse signs of toxicity.

At the highest dietary concentration of 12500 ppm, there was a reduction of food intake during the first four days of treatment (with the overall gains for weeks 1 to 4 also being lower than controls), with the greatest effect occurring on Day 1, seen in association with slightly lower body weight gain during the first week. These findings did not associate with any clinical signs or mortality and were likely attributable to inpalatability of the test diet but the animals showed clear accommodation to this.

Changes in renal parameters in this study included a slight increase in adjusted kidney weight (in all treated groups of males which showed no evidence of recovery) and a slight reduction in urinary volume (in males at 4200 or 12500 ppm where recovery was evident). In the absence of any degenerative renal pathology, these were considered non-adverse findings at the slight degree of change observed. 

An increased incidence and severity (minimal to slight) of hyaline droplet accumulation was observed within the kidney in males at all dietary concentrations (≥1400 ppm) and occurred with minimal tubular basophilia in one male at each of the 4200 and 12500 ppm levels. Hyaline droplet accumulation in male rats is generally due to accumulation of alpha_2u-globulin within the proximal tubules. Alpha_2u-globulin is androgen regulated and synthesized in the liver of male rats and hyaline droplets are more commonly seen in this sex due to a lower capacity for renal protein filtration than in female rats (Greaves, 2012). An increase in renal alpha_2u-globulin is a fairly common response following administration of a wide range of drugs and chemicals, such as sodium barbital (Kurata, 1994) and d‑limonene (Lehman‑MckeenmanandCaudill, 1994) and is considered to be a male rat‑specific (KhanandAlden, 2002) response. There was evidence of partial recovery for the hyaline droplet accumulation, since the overall severity (minimal) at the end of the recovery period was lower than that seen at the end of treatment. It should also be acknowledged that this finding was also seen at minimal severity in two control males at the end of the recovery period. There was some evidence of slight progression of the tubular basophilia finding, in terms of incidence and/or severity, at the end of the recovery period but this finding and the accumulation of hyaline droplets occurred in the absence of any adverse degenerative tubular findings. Furthermore, there were no indicators of kidney damage from biochemical and urine investigations performed at the end of the treatment period. Consequently, the histopathology findings in the kidneys were considered non-adverse in the context of this study.        

The histopathological examination (which included evaluation of a range of lymphoid tissues such as lymph nodes, spleen, thymus and bone marrow from the sternum) did not identify the cause of theslight increases in some white blood cell parameters (increased neutrophils in males and increased lymphocyte and monocyte counts in females) seen at 12500 ppm.  These findings showed full recovery except the neutrophil count in males. Variations in circulating leucocyte counts are, however, a known stress-related response in animal toxicity studies (Everds et al, 2013). Since these findings showed a limited degree of change, and were not indicaticative of an overt or adverse stimulatory effect on the immune system, they were considered non-adverse in the context of this study.

The reduction in plasma glucose seen at the end of the treatment period in all treated groups of males was considered a non-adverse change given the slight degree of change observed (with the majority of values being within the background control range), the abscence of any clinical consequence and/or corroberative histopathological change and that full recovery occurred.

The slightly high adjusted liver weight and low adjusted ovary and uterus weights in females which received 12500 ppm (the difference in uterus weight did not attain statistical significance) ocurred in the abcence of any corroberative histopathological changes and showed complete or at least partial recovery. Consequenty, these were considered non adverse findings.

Findings in this study which had a doubtful or uncertain relationship to treatment included slightly low reticulocte counts and high albumin concentration in all treated groups of males (with a slight increase in albumin to globulin ratio at 12500 ppm), slightly high red cell distribution width in females which received 12500 ppm and slightly high phosphorus concentration in all treated groups of females. None of these findings was considered to be adverse at the slight degree of change observed and in the abscence of any corroberative histopathology change.   

Applicant's summary and conclusion

Conclusions:

It was concluded that 12500 ppm (equivalent to approximately 917 and 869 mg/kg/day in males and females, respectively) represented the no-observed-adverse-effect level (NOAEL) for FRET 13-0545 when administered throught the diet for 28 consecutive days to the rat. The kidney was identified as a potential target organ in the male rat (where an increased incidence and severity of hyaline droplet accumulation occurred at all test concentrations and was seen with some incidences of tubular basophilia at 4200 and 12500 ppm, with the latter finding showing some progression in severity at the end of the 2 week recovery period) but there was no evidence of any adverse/degenerative renal pathology. None of the other test item-related findings identified was considered adverse.

Executive summary:

This study was performed to assess the systemic toxicity of Substance FRET 13-0545 to the rat according to OECD TG 407.

The substance was administered continually vie the diet to groups of five male and five female rats for twenty-eight consecutive days at fixed dosage levels of 1400, 4200 and 12500 ppm (96, 307 and 917 mg/kg/day for males and 94, 280 and 869 mg/kg/day for females, respectively). The test material was prepared using premixed test substance and plain diet whch were further diluted to the required test concentrations. A further group of rats (five males and five females) was held as a concurrent control receiving the vehicle alone.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), estrous cycles, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The general appearance and behaviour, sensory activity, grip strength and motor activity of the animals were not affected by treatment and there were no deaths.

Body weight: Body weight gain was slightly low during the first week of treatment in both sexes receiving 12500 ppm and was higher than the controls during the first week of recovery in both sexes which had previously received this dietary concentration. Food consumption was low during the first four days of treatment in both sexes receiving 12500 ppm and was slightly higher than controls during the recovery period (especially on the first day of recovery) in males which had previously received this dietary concentration. A visual assessment of water intake did not reveal any test-item related effects.

Haematology: The hematological examination revealed slightly high neutrophil counts (1.4 X Control) in males which received 12500 ppm and slightly high lymphocyte (1.3 X Control) and monocyte (1.7 X Control) counts in females which received 12500 ppm with a concomitant slight increase in total white blood cell counts in this sex (1.3 X Control).  The findings in females showed complete recovery but neutrophil counts remained slightly high (1.6 X Control) in males which had previously received 12500 ppm. Hematological findings which had a doubtful/uncertain relationship to the test item includedslightly low reticulocte counts (down to 0.8 X Control) in all treated groups of males and slightly high red cell distribution width (1.1 X Control) in females which received 12500 ppm; these were considered slight, non-adverse changes and were not present at the end of the recovery period

Biochemistry: The biochemical examination of the blood revealed a dose-related reduction of glucose concentration (down to 0.6 X Control) in all treated groups of males but this finding showed full recovery. Biochemical findings which had a doubtful/uncertain relationship to the test item includedhigh albumin concentration (1.06 X Control) in all treated groups of males (with a slight increase in albumin to globulin ratio at 12500 ppm) and slightly high phosphorus concentration (up to 1.3 X Control) in all treated groups of females; these were considered slight, non-adverse changes and were not present at the end of the recovery period.

Organ weights: Adjusted kidney weights were slightly high (maximum 1.1 X Control) in all treated groups of males. There was no apparent evidence of recovery, but no progression (in terms of the magnitude of change from controls) had occurred by the end of the 2-week recovery period. Adjusted liver weights were slightly high (1.1 X Control) in females which received 12500 ppm but there was evidence of recovery. Adjusted ovary and uterus weights were slightly low (0.7 and 0.6 X Control, respectively, but the difference in adjusted uterus weights did not attain statistical significance) for females which received 12500 ppm; complete or at least partial recovery was demonstrated. With the exception of the slight increase in kidney weights in males, none of the other changes in organ weight were associated with any test item-related histopathological change.

Macroscopic pathology: There were no test item-related macroscopic findings. 

Microscopic pathology: Test item-related histopathological findings were confined to the kidney in males; an increased incidence and severity (minimal/slight severity) of hyaline droplet accumulation was evident at ≥1400 ppm and occurred with tubular basophila (minimal severity) in two males (one at each of the 4200 and12500 ppmlevels). Partial recovery was considered to have occurred for the hyaline droplet accumulation, since the overall severity (minimal) was reduced by the end of the recovery period, but the incidence and severity of the basophilia finding had shown some progression (5/5 males which had previously received 12500 ppm showed minimal or slight severity at the end of the recovery period). There was no evidence of any degenerative renal pathology in this study.

Other findings: Estrous cycles were unaffected by treatment.

Urine investigations revealed slightly low urinary volume (0.5 X Control) in males which received 4200 or 12500 ppm but full recovery was demonstrated.

Conclusion: It was concluded that 12500 ppm (equivalent to approximately 917 and 869 mg/kg/day in males and females, respectively) mg/kg/day represented the no-observed-adverse-effect level (NOAEL) when administered in the diet for 28 consecutive days to the rat. The kidney was identified as a potential target organ in the male rat (where an increased incidence and severity of hyaline droplet accumulation occurred at all test concentrations and was seen with some incidences of tubular basophilia at 4200 and 12500 ppm, with the latter finding showing some progression in severity at the end of the 2 week recovery period) but there was no evidence of any adverse/degenerative renal pathology. None of the other test item-related findings identified was considered adverse.