[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 05 October 2016. Experimental Completion Date: 09 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 407, in compliance with GLP, without deviations that influence the quality of 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:

bis(cyclohexylmethyl) ether

Cas Number:

14315-63-0

Molecular formula:

C14H26O

IUPAC Name:

bis(cyclohexylmethyl) ether

Test material form:

liquid

Remarks:

Clear, colorless

Specific details on test material used for the study:

Identification: FRET 13-0460
Physical State/Appearance: Clear colorless liquid
Chemical Name: bis(cyclohexylmethyl) ether
Chemical Formula: C14H26O
Date Received: 18 July 2016
Storage Conditions: Approximately 4 °C in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 191 to 217g, the females weighed 147 to 171g, and were approximately six weeks old.

Animal Care and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A powdered diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services, Dietex International Ltd, Witham, Essex, UK was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cages were distributed in dose group columns within the holding rack to minimize the potential of cross contamination of the treated diet.The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical propert ies of the test item, and the results of the study are believed to be of value in predicting the likely toxi
city of the test item to man.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test substance was administered orally via the diet.
Control animals received basal laboratory diet at the same dose volume.

The test item was administered continuously, for twenty-eight consecutive days, by dietary admixture. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment

A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800/U200 mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and uniformity of distribution of the test item in the diet were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the dietary admixtures to be stable for twenty-one days at room temperature. Dietary admixtures were prepared prior to the first treatment, and fortnightly thereafter. The diet was stored in labelled, double plastic bags in labelled, covered plastic bins at room temperature. Samples were taken from the dietary admixtures and analysed for uniformity of distribution and concentration at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the mean prepared dietary admixture concentrations were within ± 6% of the nominal concentration.

The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated for the test item n dietary admixtures with respect to the specificity of chromatographic analysis, the linearity of detector response, method accuracy and precision.

Duration of treatment / exposure:

Twenty-eight consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Doses / concentrations 2

Dose / conc.:

4 500 ppm

Doses / concentrations 3

Dose / conc.:

12 000 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 dietary concentrations were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work (Envigo Study Number: NN03JY).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health or behavioral change daily from the
start of treatment. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded on Day 1 (prior to the start of treatment) and at weekly inter
vals thereafter. Body weights were also measured prior to terminal kill..

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency and mean achieved dosages were calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

Hematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all recovery group animals at the
end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29
and 43. Animals were not fasted prior to sampling.

HAEMATOLOGY: Yes
- Parameters examined
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT)
was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
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++)
Gamma glutamyltranspeptidase
Triglycerides (Tri)

URINALYSIS: Yes
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 4 and on all recovery group animals during Week 6. Urine samples were collected overnight
by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food. The following parameters were measured on collected urine:
Volume
Ketones
Specific Gravity
Bilirubin
pH
Urobilinogen
Protein
Blood
Glucose
Appearance

OTHER:
Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during
Week 4, together with an assessment of sensory reactivity to different stimuli.
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 fo
r each animal was made. Three consecutive trials were performed for each animal. The assessmentwas developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et
al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free period, all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.

Organ weight
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Liver
Brain
Ovaries
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Pituitary (post-fixation)
Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Uterus with Cervix

Macroscopic pathology
All animals were subjected to a full external and internal examination, and any macroscopic abnormali
ties were recorded.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% f
ormalin, except where stated:
glands and fluids)
~Adrenals
~Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
~Pituitary
~Bone & bone marrow (sternum)
~Prostate
~Brain (including cerebrum, cerebellum andpons)
~Rectum
~Caecum
Salivary glands (submaxillary)
~Sciatic nerve
~Colon
~Seminal vesicles (with coagulating glands and fluids)
~Duodenum
~Epididymides ♦
Skin
Esophagus
~Spinal cord (cervical, mid thoracic and lumbar)
~Eyes *
~Gross lesions
~Spleen
~Heart
~Stomach
~Ileum
~Testes ♦
~Jejunum
~Thymus
~Kidneys
~Thyroid/Parathyroid
~Liver
~Trachea
~Lungs (with bronchi)#
~Urinary bladder
~Lymph nodes (mandibular and mesenteric)
~Uterus & Cervix
~Mammary gland
~Vagina
~Muscle (skeletal)
All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffo lk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues marked with ~ from all
control and 12000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination.
Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 12000 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related liver and kidney (males only) changes, examination was subsequently extended to include similarly prepared sections of the liver and kidneys (males
only) from animals in the low, intermediate and recovery groups.
* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

OTHER:
Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored frozen at approximately -80 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Statistics:

Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Urinalysis, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module.Where appropriate, data transformations were performed. Homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using ANOVA, or if required, ANCOVA with appropriate covariates. Transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (nonparametric) test to determine significant difference from control. 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).
Data not analyzed by the Provantis data capture system was assessed separately using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was
followed by the Mann-Whitney "U" test. Dose response relationships may have also been investigated by linear regression.
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See results

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See results

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

no effects observed

Description (incidence and severity):

See results

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

See results

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

See results

Urinalysis findings:

no effects observed

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:

no effects observed

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

Details on results:

Mortality
There were no treatment-related deaths.
One recovery control male died during the warming procedure prior to necropsy on Day 43. Macroscopically, this animal had a hardened heart, reddened lungs and enlarged kidneys which were also fluid filled (light yellow in color). The significant microscopic findings included chronic progressive nephropathy in the kidney and chronic cardiomyopathy in the heart. The kidney lesion was considered to be the major factor contributing to the death of this animal.

Clinical Observations
No clinical signs of toxicity were detected.

Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 1500, 4500 and 12000 ppm.

Functional Performance Tests
There were no treatment-related changes in functional performance at 1500, 4500 and 12000 ppm.
Statistical analysis of the data did not reveal any significant intergroup differences

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 1500, 4500 and 12000 ppm.

Body Weight
Males treated with 12000 ppm showed a reduction in body weight gain throughout the treatment period, with statistical significance (p<0.01) being achieved during Weeks 1 and 4. Females treated with 12000 ppm also showed a reduction in body weight gain during the first two weeks of treatment. Improvement was evident during Week 3, however, a reduction in body weight gain was again evident for these females during Week 4. Statistical significance was not achieved in females. Consequently, overall body weight gain for either sex treated with 12000 ppm was reduced (26% males, 16% females). Recovery males that were previously fed 12000 ppm continued to show a reduced body weight gain during the first week of the treatment free period, however recovery was evident during the final week. Recovery females that were previously fed 12000 ppm showed full recovery in body weight gain during the treatment free period.
Males treated with 4500 and 1500 ppm showed a reduction in body weight gain during Weeks 2, 3 and 4 of treatment. Statistical significance (p<0.01) was achieved during Week 4. Overall body weight gain for these males was reduced (17% and 14% respectively).
No toxicologically significant effects on body weight gain were evident in females treated with 4500 or 1500 ppm.
Body weight gain for females treated with 4500 ppm was slightly reduced during Week 4 of treatment. Overall body weight gain in these females was comparable to controls and previously these females had showed superior body weight gains when compared to controls.
The intergroup difference was therefore considered to represent normal biological variation rather than an adverse effect of the test item.

Food Consumption
Males treated with 12000 ppm showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter.
No such effects were detected in females treated with 12000 ppm, in animals of either sex treated with 4500 or 1500 ppm or in recovery animals following fourteen days without treatment.
Fluctuations in food conversion efficiency were evident in treated animals, however, these generally followed the reductions evident in body weight gain.

Water Consumption
No adverse effect on water consumption was evident in treated animals.

Hematology
There were no toxicologically significant effects detected in the hematological parameters measured.
Non-recovery males from all treatment groups and recovery males that were previously given 12000 ppm showed a statistically significant increase (p<0.01) in reticulocyte count. Nonrecovery males treated with 12000 ppm also showed statistically significant reductions (p<0.05) in neutrophil count and eosinophil count. The effect on neutrophil count also extended to males treated with 4500 ppm. All of the individual values were within historical control ranges and in the absence of true dose related responses or any associated histopathological correlates, the intergroup differences were considered not to be of
toxicological significance.
Non-recovery females from all treatment groups and recovery females that were previously given 12000 ppm showed a statistically significant increase (p<0.05-0.01) in reticulocyte count. Non-recovery females from all treatment groups also showed a statistically significant reductions (p<0.05-0.01) in mean corpuscular hemoglobin concentration. The majority of individual values were within historical control ranges and in the absence of true dose related responses or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
At the end of the fourteen day treatment free period, males that were previously given 12000 ppm showed a statistically significant reduction (p<0.05) in mean corpuscular hemoglobin concentration and a statistically significant increase (p<0.01) in lymphocyte count whilst females from this treatment group showed statistically significant increases (p<0.05) in erythrocyte count and activated partial thromboplastin time. With the exception of one mean corpuscular hemoglobin concentration and one lymphocyte value, all individual values were within the normal background ranges for these parameters and in the absence of similar effect seen at the end of the treatment period the intergroup differences were considered to be of no toxicological importance.

Blood Chemistry
There were no toxicologically significant effects detected in the blood chemical parameters measured.
Non-recovery males treated with 12000 ppm showed statistically significant increases (p<0.05-0.01) in creatinine, inorganic phosphorus, calcium concentration and a statistically significant reduction (p<0.05) in chloride concentration. The effect on inorganic phosphorus also extended to males treated with 4500 ppm. The majority of individual values were within historical control ranges and in the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance. Males treated with 4500 ppm showed a statistically significant increase (p<0.05) in potassium concentration. Although the majority of individual values were outside of the historical control range, in the absence of a similar effect at 12000 ppm, the intergroup difference was considered not to be of toxicological significance.
At the end of the fourteen day treatment free period, males that were previously given 12000 ppm showed statistically significant increases (p<0.05-0.01) in alanine aminotransferase, total protein and chloride concentration. All individual values were within the normal background ranges for these parameters and in the absence of similar effect seen at the end of the treatment period the intergroup differences were considered to be of no toxicological importance.

Urinalysis
There were no treatment-related effects detected in the urinalytical parameters examined.

Necropsy
No toxicologically significant macroscopic abnormalities were detected.
One non-recovery female treated with 4500 ppm had an increased pelvic space in the left kidney. Histopathological examinations of this animal revealed slight pelvic dilatation. Observations of this nature in the kidneys are commonly observed in this strain of rat and at this incidence and in the absence of a similar effect at 12000 ppm, was considered to be unrelated to treatment. One recovery male that was previously given 12000 ppm had an enlarged spleen. In the absence of a similar effect seen at the end of the treatment period the macroscopic finding was considered not to be of toxicological importance.
The recovery control male that died during the warming procedure prior to necropsy on Day 43 had a hardened heart, reddened lungs and an enlarged left kidney which was also fluid filled (light yellow in colour). Microscopically, this animal had chronic progressive nephropathy in the kidney and chronic cardiomyopathy in the heart. The kidney lesion was considered to be the major factor contributing to the death of this animal.

Organ Weights
Non-recovery animals of either sex treated with 12000 and 4500 ppm showed a statistically significant increase (p<0.01) in liver weight; both absolute and relative to terminal body weight.
No such effects were detected in animals of either sex treated with 1500 ppm or in recovery animals following fourteen days without treatment.
Non-recovery females treated with 12000 ppm and recovery females that were previously given 12000 ppm showed a statistically significant increase (p<0.05) in kidney weight both absolute and relative to terminal body weight. The majority of individual values were within historical control range and in the absence of any associated histopathological correlates in this sex, the intergroup differences were considered not to be of toxicological importance.
Males treated with 1500 ppm showed a statistically significant increase (p<0.01) in kidney weight both absolute and relative to terminal body weight. No such effect on kidney weight was evident in males treated with 12000 or 4500 ppm and although there were microscopic kidney findings in males treated with 12000 and 4500 ppm, microscopic findings were not evident in males treated with 1500 ppm. As such the intergroup difference in kidney weight at 1500 ppm was considered not to represent an adverse effect of treatment.
At the end of the fourteen day treatment free period, females that were previously given 12000 ppm showed a statistically significant increase (p<0.05) in ovary weight both absolute and relative to terminal body weight. The majority of individual values were within the historical control range and in the absence of a similar effect at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological importance.

Histopathology
The following treatment-related microscopic abnormalities were detected:
Liver: minimal to slight centrilobular hypertrophy was evident in animals of either sex treated with 12000 ppm and in males treated with 4500 ppm.
Kidneys: minimal to slight accumulation of hyaline droplets was evident in males treated with 12000 and 4500 ppm.
The nature and incidence of all other microscopic lesions were considered to be unrelated to the test item.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The effect on body weight reflects the reluctance to eat the dietary formulation. Liver changes considered adaptive response. Hyaline droplets in renal tubules sex and species specfic.

2

Dose descriptor:

NOEL

Effect level:

4 500 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Treatment-related changes in animals of either sex treated where noted at 12000 ppm and in males treated at 4500 ppm. No toxicologically significant effects were evident in females treated with 4500 ppm

3

Dose descriptor:

NOEL

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Treatment-related changes in animals of either sex treated where noted at 12000 ppm and in males treated at 4500 ppm. No toxicologically significant effects were evident in females treated with 1500 ppm

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Achieved Intake

The mean achieved dosage of FRET 13-0460 in mg/kg bw/day during the study were calculated using nominal concentration for dietary formulations.

At 12000 ppm, mean achieved dosage for males was 942.7 mg/kg bw/day and for females was 970.1 mg/kg bw/day. Mean achieved intake was slightly higher for either sex during the first week of treatment, however, they became fairly consistent thereafter and generally maintained an 8 fold interval between this high dietary level and the low dietary level.

At 4500 ppm, mean achieved dosage for males was 358.1 mg/kg bw/day and for females was 363.6 mg/kg bw/day. Mean achieved intake was slightly higher for either sex during the first week of treatment, however, they became fairly consistent thereafter and generally maintained a 3 fold interval between this intermediate dietary level and low dietary level.

At 1500 ppm, mean achieved dosage for males was 121.9 mg/kg bw/day and for females was 120.9 mg/kg bw/day. Mean achieved intake was slightly higher for males during the first week of treatment, however, it became fairly consistent for males thereafter and was consistent throughout the treatment period for females.

DISCUSSION

The oral administration of FRET 13-0460 to rats for a period of twenty-eight consecutive days at dietary concentrations of 1500, 4500 and 12000 ppm resulted in treatment related effects detected in animals of either sex treated with 12000 ppm and in males treated with 4500 ppm.

There were no clinical signs of toxicity evident, however, the physical condition of animals of either sex treated with 12000 ppm was affected with lower body weight gains evident throughout the treatment period for males and during Weeks 1, 2 and 4 for females. A slight reduction was also evident in males treated with 4500 and 1500 ppm between Weeks 2 and 4. Males treated with 12000 ppm showed a slight reduction in food consumption during the first two weeks of treatment and food efficiency was slightly reduced in treated animals, generally following the fluctuations in body weight development. Reduced body weight gains and food consumptions are often reported when the dietary admixture is unpalatable and can therefore be considered a non-adverse event.

Histopathological examination of the liver revealed minimal to slight centrilobular hepatocyte hypertrophy in animals of either sex treated with 12000 ppm and in males treated with 4500 ppm. Organ weight data supported these findings with increased absolute and relative liver weights observed in animals of either sex treated with 12000 and 4500 ppm. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any degenerative or inflammatory changes is generally considered to be an adaptive change or considered to reflect a metabolic disturbance that is a result of hepatic enzyme induction.

Microscopic examination of the kidneys revealed hyaline droplets in males treated with 12000 and 4500 ppm. Hyaline droplets can be directly linked to the accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is not found in immature rats, female rats or humans and therefore is considered to be of no relevance to man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for males can be established based on the observed effects excluding those related to alpha 2u-globulin nephropathy.

There were no toxicologically significant effects observed during the weekly open field arena observations or the hematological, blood chemical and urinalytical parameters measured.

Applicant's summary and conclusion

Conclusions:

It was concluded that 12000 ppm represented the no-observed-adverse-effect level (NOAEL) for Substance FRET 13-0460 when administered orally for 28 consecutive days to the rat.

The effect on body weight gain and food consumption at 12000 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of
treatment.The microscopic liver changes evident in females treated with 12000 ppm wereconsidered to be an adaptive response to mixed function oxidase induction. Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 12000 ppm for females, equivalent to 970.1 mg/kg bw/day. The accumulation of hyaline droplets in the renal tubules of males treated with 12000 ppm and 4500 ppm was not observed with any associated pathological changes. Therefore, this increase was considered not to be toxicologically significant within the study and is consistent with the accumulation of alpha 2u-globulin which is an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, this finding would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 12000 ppm for males, equivalent to 942.7 mg/kg bw/day, because the finding does not reflect true systemic toxicity.Therefore, the No Observed Effect Level (NOEL) was considered to be 4500 ppm for females, equivalent to 363.6 mg/kg bw/day, and 1500 ppm for males, equivalent to 121.9 mg/kg bw/day.

Executive summary:

This study was performed to assess the systemic toxicity of Substance FRET 13-0460 to the rat according to OECD TG 407.

The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 1500, 4500 and 12000 ppm (equivalent to a mean achieved dosage of 121.9, 358.1 and 942.7 mg/kg bw/day for males and 120.9, 363.6 and 970.1 mg/kg bw/day for females respectively). A control group of five males and five females were treated with basal laboratory diet. Two recovery groups, each of five males and five females, were treated with the high dose (12000 ppm) or basal laboratory diet for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

Mortality: There were no treatment-related deaths.

Clinical Observations:No clinical signs of toxicity were detected.

Behavioral Assessment: There were no treatment-related changes in the behavioral parameters at 1500, 4500 and 12000 ppm.

Functional Performance Tests: There were no changes in functional performance considered to be related to treatment at 1500, 4500 and 12000 ppm.

Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 1500, 4500 and 12000 ppm.

Body Weight: Males treated with 12000 ppm showed a reduction in body weight gain throughout the treatment period and during the first week of the treatment-free period. Recovery was evident in these males during the final week of the treatment-free period. Females treated with 12000 ppm also showed a reduction in body weight gain during the first two weeks of treatment. Improvement was evident during Week 3. A reduction was again evident in body weight gain during Week 4. Recovery was evident in these females during the treatment-free period. Males treated with 4500 and 1500 ppm showed a reduction in body weight gain during Weeks 2, 3 and 4 of treatment. Overall body weight gain was reduced in males from all dietary groups and females treated with 12000 ppm. No adverse effect on body weight gain was evident in females treated with 4500 or 1500 ppm.

Food Consumption: Males treated with 12000 ppm showed a slight reduction in food consumption during the first two weeks of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 12000 ppm, in animals of either sex treated with 4500 or 1500 ppm or in recovery animals following fourteen days without treatment. Fluctuations in food conversion efficiency were evident in treated animals, however, these generally followed the reductions evident in body weight gain.

Water Consumption: No adverse effect on water consumption was evident in treated animals.

Blood Chemistry: There were no toxicologically significant effects detected in the blood chemical parameters measured.

Urinalysis: There were no treatment-related effects detected in the urinalytical parameters examined.

Hematology: There were no toxicologically significant effects detected in the hematological parameters measured.

Necropsy: No toxicologically significant macroscopic abnormalities were detected.

Organ Weights: Animals of either sex treated with 12000 and 4500 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. No such effects were detected in animals of either sex treated with 1500 ppm or in recovery animals following fourteen days without treatment.

Histopathology: The following treatment-related microscopic abnormalities were detected:

Liver: minimal to slight centrilobular hypertrophy was evident in animals of either sex treated with 12000 ppm and in males treated with 4500 ppm.

Kidneys: minimal to slight accumulation of hyaline droplets was evident in males treated with 12000 and 4500 ppm.

Conclusion: It was concluded that 12000 ppm represented the no-observed-adverse-effect level (NOAEL) when administered orally for 28 consecutive days to the rat.

The effect on body weight gain and food consumption at 12000 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment.The microscopic liver changes evident in females treated with 12000 ppm wereconsidered to be an adaptive response to mixed function oxidase induction. Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 12000 ppm for females, equivalent to 970.1 mg/kg bw/day. The accumulation of hyaline droplets in the renal tubules of males treated with 12000 ppm and 4500 ppm was not observed with any associated pathological changes. Therefore, this increase was considered not to be toxicologically significant within the study and is consistent with the accumulation of alpha 2u-globulin which is an effect recognized as being both species and sex specific and not relevant for humans. In terms of risk assessment, this finding would suggest that a No Observed Adverse Effect Level (NOAEL) can be established at 12000 ppm for males, equivalent to 942.7 mg/kg bw/day, because the finding does not reflect true systemic toxicity.Therefore, the No Observed Effect Level (NOEL) was considered to be 4500 ppm for females, equivalent to 363.6 mg/kg bw/day, and 1500 ppm for males, equivalent to 121.9 mg/kg bw/day.

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