Registration Dossier

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Administrative data

Description of key information

OECD 407: It was concluded that 4500 ppm represented theNo Observed Adverse Effect Level (NOAEL) when administered orally for 28 consecutive days to the rat.

The substance was tested at dietary concentrations of 1500, 4500 and 9000 ppm (equivalent to a mean achieved dosage of 114.5, 340.5 and 676.1 mg/kg bw/day for males and 114.1, 311.9 and 615.4 mg/kg bw/day for females respectively).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 29 September 2016. Experimental Completion Date: 27 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Specific details on test material used for the study:
Identification: FRET 12-0492
Physical State/Appearance: Clear colorless liquid
Chemical Name: 4,6-dimethyl-2-(1-phenylethyl)-3,6-dihydro-2H-pyran
Chemical Formula: C15H20O
Date Received: 18 July 2016
Storage Conditions: Approximately 4 °C in the dark
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 nine 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 192 to 225 g, the females weighed 139 to 163 g, 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. C. 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.
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 properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test substance was administered by 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 sixty 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 21 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 method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the mean prepared dietary admixture concentrations were within ±11% 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 multiple peaks.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
Dose / conc.:
1 500 ppm
Dose / conc.:
4 500 ppm
Dose / conc.:
9 000 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: LH29PF).
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
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio
(by calculation)
Sodium (Na+)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)
Gamma glutamyltranspeptidase (γGT)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)

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 for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et
al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period, or in the case of recovery group animals, at the end of the t reatment-free period, all animals were killed by intravenous overdose of a suitable barbiturate agent
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
Uterus with Cervix
(with coagulating glands and fluids)

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

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, 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, Suffolk, IP23 7PX) for processing (Principal Investigator: J Schofield). The tissues marked with ~ from all
control and 9000 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 9000 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related liver, thyroid (males only) and caecum changes, examination was subsequently extended to include similarly prepared sections of the liver, thyroid (males only) and caecum from animals in the low, intermediate and recovery groups.

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 forthese 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)
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:
effects observed, treatment-related
Description (incidence and severity):
See results
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See results
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See results
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
Details on results:
Mortality
There were no unscheduled deaths.

Clinical signs
No clinical signs of toxicity were detected

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 1500, 4500 and 9000 ppm.
Functional Performance Tests
There were no changes in functional performance considered to be related to treatment at 1500, 4500 and 9000 ppm.
Females treated with 9000 ppm showed a statistically significant reduction (p<0.05) in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any supporting clinical observations to suggest a neurotoxic effect, the intergroup difference was considered to be of no toxicological significance.
Sensory Reactivity Assessments
There were no intergroup differences in sensory reactivity scores that were considered to be related to treatment at 1500, 4500 and 9000 ppm

Body Weight
Animals of either sex treated with 9000 or 4500 ppm showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Actual body weight losses were evident in animals of either sex treated with 9000 ppm. Females treated with 4500 ppm continued to show a statistically significant reduction (p<0.01) in body weight gain during the second week of treatment. Improvement was evident during Week 3
however, reduced gains were again evident in these females during the final week of treatment (statistical significance was not achieved). During Week 2, improvement was evident in animals of either sex treated with 9000 ppm (a statistical significant increase (p<0.05-0.01) was evident) and in males treated with 4500 ppm. However, animals of either sex treated with 9000 ppm showed lower gains during Week 3 (statistical significance was
achieved for females) and males continued to show lower gains during the final week of treatment.
No adverse effect on body weight gain was evident in animals of either sex treated with 1500 ppm.
During the fourteen day treatment free period, animals of either sex that were previously given 9000 ppm showed an increase in body weight gain throughout. Statistical significance (p<0.05) was achieved in either sex during Week 5.

Food Consumption
Males treated with 9000 and 4500 ppm showed a reduction in food consumption during the first week of treatment. Recovery was evident thereafter. No such effects were detected in males treated with 1500 ppm.
Females from all treatment groups showed a reduction in food consumption during the first week of treatment. Improvement was evident during Week 2, however, females treated with 9000 and 4500 ppm showed lower food consumptions during Weeks 3 and 4.
Fluctuations in food conversion efficiency were evident in treated animals, however, these generally followed the reductions evident in body weight gain.
Food consumption and food conversion efficiency for recovery animals during the treatment free period was comparable to controls.

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

In-Life Sampling and Analysis
Hematology
There were no toxicologically significant effects detected in the hematological parameters measured.
Non-recovery males from all treatment groups showed a statistically significant increase (p<0.01) in reticulocyte count and a statistically significant reduction (p<0.05) in activated partial thromboplastin time. Non-recovery males treated with 9000 ppm also showed a statistically significant reduction (p<0.05) in mean corpuscular hemoglobin. With the exception of one mean corpuscular hemoglobin value, all 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 showed a statistically significant increase (p<0.01) in reticulocyte count. Non-recovery females treated with 9000 and 4500 ppm also showed statistically significant reductions (p<0.05-0.01) in platelet count, mean corpuscular volume and mean corpuscular hemoglobin and non-recovery females treated with 9000 ppm also showed statistically significant increases in hematocrit and erythrocyte count. 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 9000 ppm showed a statistically significant reduction (p<0.05) in hemoglobin and mean corpuscular hemoglobin concentration whilst females from this treatment group showed
statistically significant reductions (p<0.05) in erythrocyte count, hematocrit, hemoglobin and statistically significant increases (p<0.05) in eosinophils and prothrombin time. With the exception of eosinophils the majority of individual values were within the normal background range for these parameters and in the absence of similar effects seen at the end of the treatment period the intergroup differences were considered to be of no toxicological importance.

Blood Chemistry
Non-recovery animals of either sex treated with 9000 ppm showed a statistically significant increase (p<0.05-0.01) in bilirubin. Non-recovery males treated with 9000 ppm also showed statistically significant increases (p<0.05-0.01) in cholesterol and urea whilst non-recovery females from this treatment group also showed a statistically significant increase (p<0.01) in gamma glutamyl transpeptidase. Non-recovery males treated with 9000 and 4500 ppm showed a statistically significant reduction (p<0.05-0.01) in triglycerides. The effect on triglycerides also extended to recovery 9000 ppm males following fourteen days without treatment. Although the majority of the individual values were within the normal expected ranges, the potential association with histopathological changes in the liver means that a relationship to treatment cannot be excluded.
No such effects were detected in females treated with 4500 ppm or in animals of either sex treated with 1500 ppm.
At the end of the fourteen day treatment free period, males that were previously given 9000 ppm showed statistically significant reductions (p<0.01) in urea and calcium concentration and a statistically significant increase (p<0.05) in alanine aminotransferase and females showed a statistically significant increase (p<0.05) in bile acids. With the exception of two calcium values, all of the individual values were within the normal background range for these parameters and in the absence of similar effects 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 male treated with 9000 ppm had increased pelvic space in the left kidney. Two non-recovery females treated with 1500 ppm also had increased pelvic space in either the left or right kidney. Histopathological examinations of these animals revealed pelvic dilatation in both or one of the kidneys. Observations of this nature in the kidneys are commonly observed in this strain of rat and at this incidence, were considered to be unrelated to treatment.

Organ Weights
Males from all treatment groups showed a statistically significant increase (p<0.05-0.01) in liver weight both absolute and relative to terminal body weight. Females treated with 9000 ppm showed a statistically significant reduction (p<0.05) in absolute liver weight, however, relative liver weight was statistically significantly increased (p<0.05). Following fourteen days without treatment, males that were previously given 9000 ppm showed a statistically significant increase (p<0.05) in liver weight both absolute and relative to terminal body weight.
No such effects were detected in females treated with 4500 or 1500 ppm.
Non-recovery males treated with 9000 and 4500 ppm showed a statistically significant reduction (p<0.05-0.01) in prostate and seminal vesicle weight both absolute and relative to terminal body weight. Non-recovery males treated with 4500 and 1500 ppm showed a statistically significant reduction (p<0.05) in kidney weight both absolute and relative to terminal body weight. All of the 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 importance.

Histopathology
The following microscopic abnormalities were detected:
Liver: Centrilobular hepatocyte hypertrophy, spreading into the mid-zonal area was evident in three males and four females treated with 9000 ppm. This was not present in animals of either sex treated with 4500 or 1500 ppm, or in recovery animals following fourteen days without treatment.
Thyroid: follicular epithelial hypertrophy was evident in one male treated with 1500 and 4500 ppm and in two males treated with 9000 ppm. Following fourteen days without treatment, this was evident in one control male and one male that was previously given
9000 ppm. The occurrence of hypertrophy in one male treated with 1500 and 4500 ppm is considered to be incidental and this is confirmed by the incidence in one control male in the recovery group.
Caecum: inflammation, mixed cellular, was evident in one male treated with 1500 ppm and all males and one female treated with 9000 ppm. Reactive hyperplasia of the caecal mucosa was evident in one male treated with 1500 ppm and in three males treated with 9000 ppm. This was not present in recovery animals following fourteen days without treatment. The occurrence in one male treated with 1500 ppm is considered not to be of significance as there was no dose-dependent trend.
Key result
Dose descriptor:
NOAEL
Effect level:
4 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 9000 ppm resulted in adverse microscopic changes in the caecum. The effect on bodyweight and food consumption at 4500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment
Key result
Critical effects observed:
no

Achieved Intake

The mean achieved dosage of FRET 12-0492 in mg/kg bw/day during the study was calculated using nominal concentration for dietary formulations.

At 9000 ppm, mean achieved dosage for males was 676.1 mg/kg bw/day and for females was 615.4 mg/kg bw/day. Mean achieved intake was slightly lower for either sex during the first week of treatment, however, they became fairly consistent thereafter and generally maintained a 6 fold interval between this high dietary level and the low dietary level.

At 4500 ppm, mean achieved dosage for males was 340.5 mg/kg bw/day and for females was 311.9 mg/kg bw/day. Mean achieved intake was slightly lower for either sex during the first week of treatment, however, they became fairly consistent thereafter and generally maintained a 3.0 fold interval between this intermediate dietary level and low dietary level.

At 1500 ppm, mean achieved dosage for males was 114.5 mg/kg bw/day and for females was 114.1 mg/kg bw/day. Mean achieved intake was slightly higher for either sex during the first week of treatment. It was fairly consistent during Weeks 2 and 3, however, during the final week of treatment it was slightly reduced.

DISCUSSION

The oral administration of FRET 12-0492 to rats for a period of twenty-eight consecutive days at dietary concentrations of 1500, 4500 and 9000 ppm resulted in adverse microscopic changes in the caecum in animals of either sex treated with 9000 ppm.

There were no clinical signs of toxicity evident, however, the physical condition of animals of either sex treated with 9000 and 4500 ppm was initially affected with lower body weight gains evident during the first and second (4500 ppm females only) week of treatment. A slight reduction was also evident in females treated with 9000 ppm during Week 3, in females treated with 4500 ppm during Week 4 and in males treated with 9000 ppm during Weeks 3 and 4. Animals of either sex treated with 9000 and 4500 ppm and females treated with 1500 ppm showed a reduction in food consumption during the first week of treatment. Females treated with 9000 and 4500 ppm continued to showed a reduction in food consumption during Weeks 3 and 4. Food conversion efficiency was reduced in treated animals and generally followed the fluctuations in body weight development.

Reduced body weight gains and food consumptions are often reported when the dietary admixture is unpalatable, however microscopic examination of the caecum revealed mixed cellular inflammation and reactive hyperplasia of the caecal mucosa in animals treated with 9000 ppm. These changes in the caecum, whilst unusual, are of unknown significance and may be related to a secondary change in the gut flora, pH or an irritant effect of the administration of the test item at the high concentration. As there is a minimal inflammatory change and resultant hyperplasia these are considered to be an adverse change in the animals affected.

Histopathological examination of the liver revealed minimal centrilobular hepatocyte hypertrophy, spreading into the mid-zonal area in animals of either sex treated with

9000 ppm. Organ weight data supported these findings with increased absolute (males only) and relative liver weights observed in animals of either sex treated with 9000 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. Blood chemical investigations in animals of either sex treated with 9000 ppm revealed changes in bilirubin. Males from this treatment group also showed changes in cholesterol, urea and triglycerides and females also showed changes in gamma glutamyl transpeptidase. Although the majority of the individual values were within the normal ranges for rats of the strain and age used the intergroup differences were most likely to be associated with altered metabolism as a result of the adaptive liver changes.

Microscopic examination of the thyroid revealed follicular epithelial hypertrophy in males treated with 9000 ppm. The morphological change seen in the thyroid was considered to be an adaptive physiologic response of the thyroid gland to hepatic enzyme induction and stimulation of the hypothalamic–pituitary–thyroid axis and represent a well understood and familiar response in rats, especially in males, treated with high doses of xenobiotics.

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

Conclusions:
It was concluded that 9000 ppm represented the no-observed-adverse-effect level (NOAEL) for Substance FRET 12-0492 when administered orally for 28 consecutive days to the rat. FRET 12-0492 at dietary concentrations of 1500, 4500 and 9000 ppm resulted in adverse microscopic changes in the caecum in animals of either sex treated with 9000 ppm. The effect on body weight and food consumption at 4500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment.
Executive summary:

This study was performed to assess the systemic toxicity of Substance FRET 12 -0492 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 9000 ppm (equivalent to a mean achieved dosage of 114.5, 340.5 and 676.1 mg/kg bw/day for males and 114.1, 311.9 and 615.4 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 (9000 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 unscheduled 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 9000 ppm.

Functional Performance Tests: There were no changes in functional performance considered to be related to treatment at 1500, 4500 and 9000 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 9000 ppm.

Body Weight: Animals of either sex treated with 9000 or 4500 ppm showed a reduction in body weight gain during the first week of treatment. Females treated with 4500 ppm continued to show a reduction in body weight gain during the second week of treatment. Improvement was evident during Week 3 however, reduced gains were again evident in these females during the final week of treatment. Improved weight gain was evident in animals of either sex treated with 9000 ppm and in males treated with 4500 ppm during Week 2. However, animals of either sex treated with 9000 ppm showed lower gains during Week 3 and males continued to show lower gains during the final week of treatment. No adverse effect on body weight gain was evident in animals of either sex treated with 1500 ppm.

Food Consumption: Males treated with 9000 and 4500 ppm showed a reduction in food consumption during the first week of treatment. Recovery was evident thereafter. No such effects were detected in males treated with 1500 ppm. Females from all treatment groups showed a reduction in food consumption during the first week of treatment. Improvement was evident during Week 2, however, females treated with 9000 and 4500 ppm showed lower food consumptions during Weeks 3 and 4.

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.

Blood Chemistry: Animals of either sex treated with 9000 ppm showed a statistically significant increase in bilirubin. Males treated with 9000 ppm also showed statistically significant increases in cholesterol and urea whilst females from this treatment group also showed a statistically significant increase in gamma glutamyl transpeptidase. Males treated with 9000 and 4500 ppm showed a statistically significant reduction in triglycerides. No such effects were detected in females treated with 4500 ppm or in animals of either sex treated with 1500 ppm.

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

Necropsy: No toxicologically significant macroscopic abnormalities were detected.

Organ Weights: Males from all treatment groups showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Females treated with 9000 ppm showed a statistically significant reduction in absolute liver weight, however, relative liver weight was statistically significantly increased. No such effects were detected in females treated with 4500 or 1500 ppm. Following fourteen days without treatment, males that were previously given 9000 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight.

Histopathology: The following microscopic abnormalities were detected:

Liver: centrilobular hepatocyte hypertrophy, spreading into the mid-zonal area was evident in three males and four females treated with 9000 ppm. This was not present in animals of either sex treated with 4500 or 1500 ppm or in recovery animals following fourteen days without treatment.

Thyroid: follicular epithelial hypertrophy was evident in one male treated with 1500 and 4500 ppm and in two males treated with 9000 ppm. Following fourteen days without treatment, this was evident in one control male and one male that was previously given 9000 ppm.

Caecum: inflammation, mixed cellular, was evident in one male treated with 1500 ppm and all males and one female treated with 9000 ppm. Reactive hyperplasia of the caecal mucosa was evident in one male treated with 1500 ppm and in three males treated with 9000 ppm. This was not present in recovery animals following fourteen days without treatment.

Conclusion: It was concluded that 9000 ppm represented the no-observed-adverse-effect level (NOAEL) for Substance FRET 12-0492 when administered orally for 28 consecutive days to the rat. FRET 12-0492 at dietary concentrations of 1500, 4500 and 9000 ppm resulted in adverse microscopic changes in the caecum in animals of either sex treated with 9000 ppm. The effect on body weight and food consumption at 4500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
311.9 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
For repeated dose toxicity one 28 study available, conducted according to OECD guideline 407 in compliance with GLP, resulting in adquate information for the endpoint

Additional information

This study was performed to assess the systemic toxicity of Substance FRET 12 -0492 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 9000 ppm (equivalent to a mean achieved dosage of 114.5, 340.5 and 676.1 mg/kg bw/day for males and 114.1, 311.9 and 615.4 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 (9000 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 unscheduled 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 9000 ppm.

Functional Performance Tests: There were no changes in functional performance considered to be related to treatment at 1500, 4500 and 9000 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 9000 ppm.

Body Weight: Animals of either sex treated with 9000 or 4500 ppm showed a reduction in body weight gain during the first week of treatment. Females treated with 4500 ppm continued to show a reduction in body weight gain during the second week of treatment. Improvement was evident during Week 3 however, reduced gains were again evident in these females during the final week of treatment. Improved weight gain was evident in animals of either sex treated with 9000 ppm and in males treated with 4500 ppm during Week 2. However, animals of either sex treated with 9000 ppm showed lower gains during Week 3 and males continued to show lower gains during the final week of treatment. No adverse effect on body weight gain was evident in animals of either sex treated with 1500 ppm.

Food Consumption: Males treated with 9000 and 4500 ppm showed a reduction in food consumption during the first week of treatment. Recovery was evident thereafter. No such effects were detected in males treated with 1500 ppm. Females from all treatment groups showed a reduction in food consumption during the first week of treatment. Improvement was evident during Week 2, however, females treated with 9000 and 4500 ppm showed lower food consumptions during Weeks 3 and 4.

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.

Blood Chemistry: Animals of either sex treated with 9000 ppm showed a statistically significant increase in bilirubin. Males treated with 9000 ppm also showed statistically significant increases in cholesterol and urea whilst females from this treatment group also showed a statistically significant increase in gamma glutamyl transpeptidase. Males treated with 9000 and 4500 ppm showed a statistically significant reduction in triglycerides. No such effects were detected in females treated with 4500 ppm or in animals of either sex treated with 1500 ppm.

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

Necropsy: No toxicologically significant macroscopic abnormalities were detected.

Organ Weights: Males from all treatment groups showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Females treated with 9000 ppm showed a statistically significant reduction in absolute liver weight, however, relative liver weight was statistically significantly increased. No such effects were detected in females treated with 4500 or 1500 ppm. Following fourteen days without treatment, males that were previously given 9000 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight.

Histopathology: The following microscopic abnormalities were detected:

Liver: centrilobular hepatocyte hypertrophy, spreading into the mid-zonal area was evident in three males and four females treated with 9000 ppm. This was not present in animals of either sex treated with 4500 or 1500 ppm or in recovery animals following fourteen days without treatment.

Thyroid: follicular epithelial hypertrophy was evident in one male treated with 1500 and 4500 ppm and in two males treated with 9000 ppm. Following fourteen days without treatment, this was evident in one control male and one male that was previously given 9000 ppm.

Caecum: inflammation, mixed cellular, was evident in one male treated with 1500 ppm and all males and one female treated with 9000 ppm. Reactive hyperplasia of the caecal mucosa was evident in one male treated with 1500 ppm and in three males treated with 9000 ppm. This was not present in recovery animals following fourteen days without treatment.

Conclusion: It was concluded that 9000 ppm represented the no-observed-adverse-effect level (NOAEL) for Substance FRET 12-0492 when administered orally for 28 consecutive days to the rat. FRET 12-0492 at dietary concentrations of 1500, 4500 and 9000 ppm resulted in adverse microscopic changes in the caecum in animals of either sex treated with 9000 ppm. The effect on body weight and food consumption at 4500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study is reliable and for covering the endpoint

Justification for classification or non-classification

The NOAEL of the substance is 100 -1000 mg/kg bw (4500 ppm equivalent to 340.5 mg/kg bw/day for males and 311.9 mg/kg bw/day for females) above the classification and labelling limits. In addition, the effect on body weight and food consumption at 4500 ppm was considered to reflect the reluctance to eat the dietary formulation and not to represent an adverse effect of treatment.

Therefore, classification of is not warranted according to Directive 67/548/EEC (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

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