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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US Food and Drug Administration, Red Book 2000, ‘Toxicological Principles for the Safety Assessment of Food Ingredients’ (November 2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately 5 to 8 weeks old
- Weight at study initiation: at the start of treatment the males weighed 134 to 254 g, the females weighed 126 to 205 g
- Fasting period before study: NDA
- Housing: polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum - a ground diet (Rat and Mouse SQC Ground Diet No. 1 Diet, Special Diets Services, Witham, Essex) was used
- Water (e.g. ad libitum): ad libitum (mains drinking water was supplied from polycarbonate bottles attached to the cage)
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose of integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK) and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target of 21 ± 2ºC
- Humidity (%): target of 55 ± 15%
Occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 18 November 2005 To: 11 August 2006

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: dietary admixture
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixtures were prepared prior to treatment and at regular intervals during the three month study period (ie at approximately twice monthly intervals).
- Mixing appropriate amounts with (Type of food): A known amount of test material was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a Hobart QE200 mixer. 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 mixer.
The stability and homogeneity of the test materials in the diet were determined by Safepharm Analytical Laboratory. Results show the dietary admixtures to be stable for a period of at least five weeks.
- Storage temperature of food: The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use. Samples were taken of each dietary admixture and were analysed for homogeneity and concentration at Safepharm Analytical Laboratory prior to use. The results indicate that the mean prepared dietary admixture concentrations were within acceptable limits for the purpose of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Experimental 12894-58 in the dietary admixtures was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.
Samples
The dietary admixtures were extracted with 5% formic acid to give a final, theoretical test material concentration of approximately 50 ppm or 25 ppm.
Standards
Standard solutions of test material were prepared in 5% formic acid at a nominal concentration of 50 ppm or 25 ppm. Diet was added to the standards in the same ratio as the relevant samples.
Procedure
The standards and samples were analysed by HPLC using the following conditions:
HPLC System : Agilent Technologies 1100 MSD, incorporating autosampler and workstation
Mass selective detector
Source : electrospray:API-ES
Fragmentation energy : 70 volts
Polarity : negative
Mode : single ion mode with 171 amu
Gas temperature : 350ºC
Drying gas : 12 litre/minute
Nebuliser pressure : 35 psig
Capillary voltage : 3000 volts
Gain : 1.0
Column : Hypersil SAX 5μ (50 x 2.1 mm id)
Column temperature : ambient
Mobile phase : 0.1 % Formic acid
Flow rate : 0.5 ml/min
Injection volume : 25 μl
Retention time : approximately 0.8 minutes
Homogeneity Determinations
The dietary admixtures were sampled from the middle and two opposite sides in triplicate and analysed.
Stability Determinations
The dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for forty one days.
Duration of treatment / exposure:
Test duration: 90 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
2500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
20000 (reduced to 10000 ppm)
Basis:
nominal in diet
No. of animals per sex per dose:
Control: Male: 20 animals at 0 ppm (mean achieved dose level = 0 mg/kg bw/day)
Low: Male: 20 animals at 500 ppm (mean achieved dose level = 37 mg/kg bw/day)
Intermediate: Male: 20 animals at 5000 ppm (mean achieved dose level = 370 mg/kg bw/day)
High: Male: 20 animals at 20000 / 10000 ppm (mean achieved dose level = 1343 mg/kg bw/day)*
Control (II): Male: 20 animals at 0 ppm (mean achieved dose level = 0 mg/kg bw/day)
Low (II): Male: 20 animals at 250 ppm (mean achieved dose level = 20 mg/kg bw/day)
Intermediate (II): Male: 20 animals at 2500 ppm (mean achieved dose level = 207 mg/kg bw/day)

Control: Female: 20 animals at 0 ppm (mean achieved dose level = 0 mg/kg bw/day)
Low: Female: 20 animals at 500 ppm (mean achieved dose level = 37 mg/kg bw/day)
Intermediate: Female: 20 animals at 5000 ppm (mean achieved dose level = 370 mg/kg bw/day)
High: Female: 20 animals at 20000 / 10000 ppm (mean achieved dose level = 1343 mg/kg bw/day)*
Control (II): Female: 20 animals at 0 ppm (mean achieved dose level = 0 mg/kg bw/day)
Low (II): Female: 20 animals at 250 ppm (mean achieved dose level = 20 mg/kg bw/day)
Intermediate (II): Female: 20 animals at 2500 ppm (mean achieved dose level = 207 mg/kg bw/day)

* Due to the deterioration in health and deaths at 20000 ppm, the highest dose level was reduced to 10000 ppm from Day 22. In view of the longer administration period at the reduced high dose level, and for the purpose of clarity, 10000 ppm will be used throughout the report and all Tables, Figures and Appendices as appropriate.
Due to the progressive decline in physical health, the high dose male group was terminated on Day 37/38 and additional dose levels were introduced onto the study (250 and 2500 ppm).
A further group of twenty males and twenty females were exposed to basal laboratory diet to serve as a second control group for comparative purposes.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations were chosen based on the results of a range-finding/palatability study at dose levels of 0 ppm and 20000 ppm. No deaths occurred, and no treament-related effects were observed. Therefore the above detailed dose levels for the main study were chosen after consultation with the study sponsor.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: NDA
- Post-exposure recovery period in satellite groups: NDA
- Section schedule rationale (if not random): random
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
All animals were examined for overt signs of toxicity, ill-health or behavioural change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As above

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to the start of the treatment) and at weekly intervals thereafter. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of ten selected male and female animals from the control, low, intermediate and high dose animals (Groups 1 – 4) were examined pre-treatment. Due to the early termination of high dose males, ten high dose females and ten intermediate dose males were examined before termination of treatment, together with the ten control males and females (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex and, following pupil dilation with a mydriatic (Mydriacyl - Alcon Laboratories (UK) Ltd., Imperial Way, Watford, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope.
- Dose groups that were examined: As above

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on ten male and ten females animals from each test and control group during Week 2, Week 7/8 and at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.
- Anaesthetic used for blood collection: No data
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Het), Erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulucyte count (Retic), Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ 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
- Time schedule for collection of blood: As above
- Parameters examined: Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), γ-glutamyl-transpeptidase (γ-GT), Sorbitol dehydrogenase (SDH), Triglycerides (Tri)

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on five males and five females from the control, low, intermediate and high dose groups (Groups 1-4) during Week 3 and for ten males and ten females from all surviving dose groups during the final week of treatment. 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.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, Bilirubin, Specific gravity, Urobilinogen, pH, Reducing substances, Protein, Blood - erythrocytes, Glucose, Blood - haemoglobin, Ketones, Microscopic examination of sediment


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On one occasion prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on ten selected males and females from each dose group during Week 12, together with an assessment of sensory reactivity to different stimuli. Observations were carried out at a similar time on each occasion.
- Dose groups that were examined: As above
- Battery of functions tested:
Detailed individual clinical observations were performed for each animal using a purpose built arena.
The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation, Posture.

Functional Performance Tests:
Motor Activity:
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Ten selected males and females per dose group were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

Forelimb/Hindlimb Grip Strength:
An automated grip strength meter was used. Each selected 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. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
The following parameters were observed:
Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Startle reflex, Tail pinch, Blink reflex, Finger approach

OTHER: No data
Sacrifice and pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS PATHOLOGY: Yes
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Thyroid/parathyroid (weighed after fixation)
Uterus

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:

Adrenals
Aorta (thoracic)
Bone & Bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides
Eyes*
Gross lesions
Heart
Illeum (including peyer's patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Nasal turbinates
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spinal cord (cervical, mid-thoracic and lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid/parathyroid
Tongue
Trachea
Urinary bladder
Uterus (corpus and cervix)
Vagina
Zymbal's gland

* Eyes were fixed in Davidson's fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to Propath UK Ltd for processing (Principal Investigator: N Candy). Initially, all tissues from control and 20000 ppm dose group animals, and all tissues from the 5000 ppm males were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of suspected treatment-related changes in liver, spleen, kidneys, urinary bladder, bone marrow, stomach, prostate, seminal vesicles, vagina, uterus and bone (femur), examination was subsequently extended to include similarly prepared sections from all animals in the other treatment groups. Furthermore, the kidney and urinary bladder for five control (II) male and females, two males and females treated with 250 ppm and three males and two females treated with 2500 ppm were also processed.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Other examinations:
None
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed 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 showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes.
1. Chi squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One male treated with 20000 ppm was found dead on Day 13. Another male from the high dose group was killed in extremis on Day 16 and another male was terminated on Day 19. Following the reduction in the highest dose level from 20000 ppm to 10000 ppm from Week 3, further high dose males were killed in extremis on Days 28, 29, 34, 35 and Day 36. At this point, it was considered necessary to terminate this dose group, which was undertaken on Day 37 and Day 38.
One control male was killed in extremis on Day 56, following a severe physical injury to the tail, which was unrelated to treatment. There were no further unscheduled deaths.

Red/dark/pink staining was evident on the cage tray liners from males treated with 20000 ppm from Day 7. No clinical observations were evident for the 20000 ppm male prior to death on Day 13. One male treated with 20000 ppm showed signs of loss of muscle mass, decreased respiration, pilo-erection, lethargy, hunched posture and dehydration on Day 16, resulting in the sacrifice of this animal on the same day. Another male showed signs of hunched posture and tip-toe gait on Day 19. This animal was subsequently terminated following observations of blood in the urine seen during the weekly behavioural assessments.
The high dose level was reduced from Week 3 to 10000 ppm, however, clinical signs were still evident for males. One male was terminated following signs of lethargy, decreased and laboured respiration and malfunction of the hind limbs on Day 28, and another male was terminated on Day 29 after signs of laboured and decreased respiration, ptosis, pilo-erection, lethargy and hunched posture. On Day 34, a further male was terminated following signs of tip-toe gait, hunched posture and pallor of extremities. Due to the persistent decline in physical health of the male dose group, it was considered necessary to terminate the remaining males on Day 37 and Day 38.
Clinical signs for high dose females were confined to transient incidents of red staining on the cage tray-liners on five occasions during treatment period, and hunched posture was noticed on Day 18 only. The presence of a mass was also noted for one female.
An incident of red coloured staining on the cage tray-liners was noted for one cage housing males treated with 5000 ppm, but this was confined to Day 10 only.

The following clinical signs were considered not to represent an adverse effect of treatment:
Red staining was detected on the cage tray liners for one cage housing males treated with 2500 ppm on a number of occasions during the study. In addition, red staining was also noted around the ano-genital region for one male from this cage and this male was also observed to have a bleeding wound on the claw, therefore, these observations were considered to be related to a physical injury. Another male treated with 2500 ppm showed an increase in activity (rapid chewing / opening and closing of the mouth) on one occasion during the course of the study, however, this was an isolated incident and as such, was considered unrelated to test material toxicity. Red staining was also noted on the cage tray-liner housing males treated with 500 and 250 ppm, although this was confined to only one occasion in each case. The origin of the staining was not obvious, therefore the incidental observations were considered to be of no toxicological importance.

One control male was killed in extremis on Day 56, following a severe physical injury to the tail, which was unrelated to treatment. An incident of hunched posture was also noted in the control group, however the spurious finding was unrelated to treatment. All remaining clinical signs detected in the control and treatment groups (generalised fur loss, scab formation, red/brown staining of the external body surface and corneal opacity) were low incident findings in laboratory maintained animals and not considered to represent test material toxicity.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on bodyweight change was detected.

Males treated with 20000 ppm showed a statistically significant reduction in bodyweight gain when compared to the concurrent male control values, during the first week of treatment (p<0.05). This isolated incident was considered to be an initial adaptive response to treatment and there were no such reductions thereafter. Prior to termination, males treated with the reduced high dose (10000 ppm) showed a statistically significant increase in bodyweight gain, although once again, the significance achieved was minimal (p<0.05). A convincing trend in bodyweight change was not detected for this group and as such, these minor intergroup differences were considered not to represent a toxicologically significant effect of treatment.

A reduction in bodyweight gain was detected for females treated at the highest dose level during Week 5 (p<0.05), however an increase in bodyweight gain when compared to controls was noticed in this dose group during Week 7 and also during Week 11 (p<0.01). Percentage bodyweight gain for the female high dose group over the duration of the study was comparable to controls, and as such, these intergroup differences were considered not to represent an adverse effect of treatment.

The remaining statistically significant intergroup differences in bodyweight change were confined to an increase for males treated with 5000 ppm during Week 5, and increases in bodyweight gain for males treated with 2500 ppm during Week 8, females treated with 5000 ppm during Week 7 and Week 11 and an increase in bodyweight gain for females treated with 250 ppm during Week 11. These were isolated findings and as such, these intergroup differences were considered to be unrelated to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake was detected for treated animals in comparison to controls throughout the treatment period.

FOOD EFFICIENCY
Food efficiency for treated animals was comparable to controls throughout the treatment period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Daily visual inspection of water bottles did ot reveal any overt intergroup differences in water intake for treated animals when compared to controls.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment related ocular effects detected for high dose females and intermediate (5000 ppm) dose males prior to termination, however, it was noticed that males from the 5000 ppm dose group were more readily excited in comparison to control males.

HAEMATOLOGY
A reduction in haematocrit count was detected for animals of either sex treated with the highest dose level during the Week 2 assessments (p<0.05). Reductions in erythrocyte counts was also evident but statistical significance was also achieved for males (p<0.05) and reductions in haemoglobin was also noted but statistical significance was only obtained for females (p<0.05). Males treated with the highest dose also showed statistically significant increases in mean cell haemoglobin concentration. Furthermore, elevated neutophil counts were also evident at the highest dose, but once again, statistically significant increases were only evident for males (p<0.01).

The Week 8 assessments revealed a slight but statistically significant increase for mean cell haemoglobin concentration and mean cell haemoglobin for females treated with the highest dose (p<0.05). Elevated mean cell haemoglobin concentration was still evident for high dose females during the Week 13 assessments when compared to concurrent controls.

There were no treatment-related effects detected for animals of either sex treated with 5000, 2500, 500 or 250 ppm during the Week 2, 7, 8 or 13 assessments. Females treated with the highest dose level showed a slight but statistically significant increase in prothrombin clotting times (p<0.05),

although males from this treatment group showed a statistically significant reduction, which was also evident for males treated with 5000 ppm (p<0.05). A similar effect was not observed during Week 8 or Week 13 and a convincing dose-related response was not detected. As such, these slight intergroup differences were considered to be any toxicological importance. Males treated with 250 ppm showed a reduction in haemoglobin and haematocrit during Week 7. These reductions were not observed during the Week 2 assessments or prior to termination during Week 13. These findings were also not evident for females treated at this dose and therefore, in the absence of a clear dose-related response, these slight reductions were considered to have arisen incidentally and of no toxicological importance. Furthermore, the statistically significant increase in leucocyte counts detected for males treated with 2500 ppm was a spurious result and in isolation, was considered to be of no toxicological importance.

CLINICAL CHEMISTRY
The Week 2 biochemical profile revealed elevated creatinine for animals of either sex treated with the high dose (p<0.05). Increases in blood urea were also evident for either sex, although statistical significance was only achieved for males (p<0.05). In addition, calcium levels were substantially elevated, although this was confined to females (p<0.01). Inorganic phosphorus levels were reduced for males treated with 20000 and 5000 ppm (p<0.01). In addition to this, males treated with the highest dose level showed a slight but statistically significant reduction in ASAT (p<0.05) during Week 2.

Week 7 assessments showed a reduction in inorganic phosphate and sodium ions for males treated with 5000 ppm, together with an increase in triglycerides.

Week 13 assessments revealed increases in calcium ions for females treated with the highest dose. A slight but statistically significant increase in aspartate aminotransferase (ASAT) was also detected for females treated with 10000 ppm (p<0.05).

There were no toxicologically important changes detected for females treated with 5000 ppm or for animals of either sex treated with 2500, 500 and 250 ppm. Increases in alkaline phosphatase were detected for animals of either sex treated with 2500 and 250 ppm during Week 2. An increase in ASAT was also evident for males treated with 2500 ppm during Week 2. Increases in AP were also observed for females treated with 2500 and 250 ppm during Week 7, and ASAT was also elevated for females treated with 5000 and 500 ppm during Week 13. Changes in these enzymes are often considered to represent an adaptive response to the administration of xenobiotics, and in the absence of any histopathological correlates, these findings were considered not to represent an adverse effect of treatment.

Males treated with 500 ppm showed a statistically significant reduction in inorganic phosphate (p<0.01) during Week 2. A reduction in sodium ions was noted for males from this dose group during Week 8 (p<0.05). During the pre-terminal assessments, a reduction in ASAT was noted for this dose group. These were incidental findings in the male 500 mg/kg/day dose group and in the absence of any supporting evidence, these findings were considered to be of no toxicological importance. Additionally, males treated with 2500 and 2500 showed a statistically significant increase in triglycerides during the Week 13 assessment. The significance achieved was minimal (p<0.05) and in isolation, these effects were considered entirely incidental and of no toxicological significance.

URINALYSIS
The interim assessments introduced during Week 3 (prior to the reduction of the high dose level to 10000 ppm) revealed the presence of haemoglobin for three of the five 20000 ppm males examined. One male from this group also showed the presence of bilirubin. A substantial amount of haemoglobin was also evident for one female treated with 20000 ppm. Sediment analysis revealed the presence of markedly high number of erythrocyte cells from four of the five 20000 ppm males examined. Three females treated with 20000 ppm showed a higher number of red blood cells in comparison to controls. Furthermore, males treated with 20000 ppm showed a reduction in micturated urine volume of increased specific gravity (p<0.05).

Pre-terminal urinalytical assessments still showed the presence of haemoglobin in five of the ten 5000 ppm males examined. This was also seen for two females treated with the highest dose, and two females treated with 5000 ppm, however, it was also observed for one control female therefore minimising the toxicological significance of this finding.

Pre-terminal sediment analysis revealed a higher number of erythrocytes for one male treated with 5000 ppm. One female treated with 10000 ppm also showed a high higher number of erythrocyte cells.

No toxicologically important effects were evident for animals of either sex treated with 2500, 500 or 250 ppm. During the interim assessments during Week 3 undertaken on selected animals of either sex treated with 20000, 5000 and 500 ppm, haemoglobin was present for one female treated with 5000 ppm and one female treated with 500 ppm. Another female treated with 5000 ppm also showed the presence of erythrocytes. One female treated with 5000 ppm displayed a very high number of erythrocyte cells, and red blood cells were also present for another female treated with the intermediate dose level, but at a much lower number. During the pre-terminal assessments, one male treated with 500 ppm displayed haemoglobin. Low incidence findings of haemoglobin in the urine are occasionally reported for control animals and isolated finding was considered not to represent a toxic effect of treatment. One female treated with 2500 ppm and one male and one female treated with 250 ppm also showed a higher number of erythrocytes. Additionally, one female treated with 2500 ppm and three males treated with 250 ppm showed haemoglobin in the urine. These were individual and isolated findings. Findings such as these are occasionally observed in laboratory maintained rats and histopathological examinations did not reveal any adverse renal effects. These findings were therefore considered not to represent an adverse effect of treatment. Sediment analysis also revealed a higher number of erythrocytes for two females treated with 5000 ppm in comparison to controls, however there were no histopathological correlates to suggest a toxicological effect of treatment.

NEUROBEHAVIOUR
Behavioural Assessments

Weekly open field arena observations revealed the following treatment-related changes: One male treated with 20000 ppm was observed to be micturating blood in the urine during the Week 2 assessments. This animal was subsequently terminated. Tip-toe gait was evident for two high dose females and hunched posture was observed for high dose females during the Week 3 assessments. One male showed increased respiration and pallor of the extremities during the Week 5 assessments.

All remaining inter and intra group differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

One female treated with 2500 ppm showed hunched posture and tiptoe gait during the Week 11. In addition, fur loss was observed for one male treated with 2500 ppm during Week 13. These were isolated findings and considered not to represent toxic effects of treatment.

There were no further observations detected during the study.

Functional Performance Tests

There were no treatment-related effects detected for treated animals in comparison to controls.

Males treated with 2500 and 250 ppm showed statistically significant reductions in forelimb and hindlimb grip strength and males treated with 5000 ppm showed a statistically significant reduction in forelimb grip strength in comparison to concurrent controls. The significance achieved was minimal and evident for one out of three tests on each occasion (p<0.05). In the absence of supporting evidence and no convincing dose-related response, these intergroup differences were considered to have arisen incidentally and were of no toxicological importance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.


ORGAN WEIGHTS
Elevated kidney weights, both absolute and relative to terminal bodyweights, was evident for female treated at the highest dose level (p<0.01), but not at any other dose level.

No such effects were evident for males treated with 5000, 500 or 250 ppm.

Males treated with 2500 ppm also showed a statistically significant increase in spleen weight, both absolute and relative to terminal bodyweight (p<0.01). In the absence of any histopathological correlates, or a dose-related response, this finding was considered not to represent a toxic effect of treatment. Additionally, females treated with 2500 and 250 ppm showed a slight but statistically significant reduction in absolute and relative brain weight (p<0.05). Furthermore, females treated with 10000, 5000 and 500 ppm showed increases in absolute and relative adrenal weights. A clear dose-related response was not evident and the increases were considered attributed to lower than expected control values. There were no histopathological correlates to suggest an effect of treatment.

GROSS PATHOLOGY
The 20000 ppm male found dead on Day 13 showed enlarged and reddened kidneys, a dark liver, reddened lungs and black contents in the stomach. The urinary bladder was distended, reddened and filled with red coloured contents. The male killed on Day 16 also displayed a dark, discoloured and distended bladder, containing dark coloured contents. The kidneys were also enlarged and the stomach was thickened with sloughing of the glandular gastric epithelium. Dark discolouration of the prostate and seminal vesicles was also apparent and the lungs were reddened. The most peculiar finding was the presence of a clear fluid which filled the abdominal cavity. The termination of another high dose male on Day 19 revealed enlarged, pale kidneys displaying hydronephrosis and a dark discoloured and distended urinary bladder filled with dark coloured contents. The male killed on Day 28 displayed a fluid filled abdominal cavity and enlarged, hydronephrotic and pale kidneys. Dark discolouration of the prostate, seminal vesicles and urinary bladder was also observed. The urinary bladder was distended and filled with dark contents. The ureter showed the presence of multiple solid masses. Similar observations were evident for the male terminated the following day. The male terminated on Day 34 showed pallor of the lungs and the male terminated on Day 35 showed pale, hydronephrotic and enlarged kidneys, dark discolouration of the seminal vesicles and urinary bladder. The bladder was also distended and filled with dark contents and multiple solid masses, and the ureter was fluid filled. Similar observations were detected for the male killed on Day 36 with the exception of solid masses, which were not evident for this animal.

The remaining high dose males were terminated on Day 37 / Day 38 of the study. One of these animals showed a hydronephrotic left kidney and another male showed hydronephrosis of the right kidney. Treatment-related effects for females treated with the highest dose level were confined to one animal, which showed enlarged, hydronephrotic and pale kidneys. In addition, multiple solid masses were evident in the urinary bladder and ureter, which was also fluid filled.

No treatment-related macroscopic abnormalities were detected for animals treated with 5000, 2500, 500 or 250 ppm at terminal sacrifice. The following macroscopic abnormalities detected throughout the control and treatment groups were considered unrelated to treatment:

The control male killed in extremis displayed a dark and swollen tail. This was attributed to a physical injury and was unrelated to test material toxicity. One control female displayed a small left eye. Corneal opacity of this eye was also noted. Another control female displayed reddened

lungs. Pallor of the right kidney was seen for one female treated with 250 ppm and a subcutaneous fibrous mass was found in the lower abdominal region of one female treated with 500 ppm. Reddened lungs were observed for one female treated with 2500 ppm and a male treated with 2500 ppm showed a small and flaccid right testis together with a small epididymis. Reddened lungs were also noted for one female treated with 5000 ppm. These incidental observations were considered to be without toxicological importance. In addition to these findings, damage to the trachea for one female treated with the highest dose and damage to the left epididymis was recorded for one male treated with 5000 ppm, were accidents incurred during the post-mortem procedure and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment-related changes were observed:

BONE (Femur): A greater incidence of higher grades of severity of trabecular bone formation was observed for males treated with 5000 ppm. A similar effect was not seen for high dose males for a shorter duration, or at 2500, 500 or 250 ppm nor among females at any treatment level.

LIVER: Generalised hepatocyte enlargement and hepatocyte basophilia were observed among high dose males. These findings were positively correlated with animals having urinary bladder changes and are probably not a primary effect of treatment. Similar changes are often seen in generally debilitated rats.

Hepatic changes were not seen for high dose females or among males treated with 5000 or 500 ppm.

KIDNEYS: Focal papillary degeneration/necrosis, tubular basophilia and dilatation, pelvic dilatation (hydronephrosis), and associated focal haemorrhage/congestion were observed among high dose males. Renal pelvic calculi were seen in two males. Mineralisation was not otherwise observed in the renal tissue of males.

For high dose females focal hyperplasia of the pelvic/papillary epithelium was observed, frequently in association with renal pelvic calculi.

Treatment-related renal changes were not observed for animals of either sex treated with 5000 or 500 ppm.

In addition to five control (II) animals of either sex, kidneys were examined from two male and from two females from Group 6 (250 ppm), and from three male and from two females from Group 7 (2500 ppm). Significant renal pathology was not observed for any of these animals.

URINARY BLADDER: Epithelial hyperplasia, with or without associated subepithelial and peripheral inflammation or haemorrhage, was observed for animals of either sex treated with the highest dose level, the changes being more severe for males. Calculus formation was observed in two males and in two females.

In addition to five control rats of either sex urinary bladder was examined from two male and from two female rats from Group 6 (250 ppm), and from three male and from two female rats from Group 7 (2500 ppm). No urinary bladder pathology was observed for any of these animals.

STOMACH: Acanthosis and hyperkeratosis were seen in the forestomach of four high dose males. A similar effect was not seen for high dose females or males treated with 5000 ppm.

LYMPHOID TISSUE: Lymphoid atrophy of the thymus was more severe among high dose males but this was more likely a consequence of the debilitated condition of these rats rather than a primary effect of treatment, just four rats being affected. Mesenteric lymph nodes were similarly affected.

PROSTATE: Inflammatory cell infiltrates, oedema, and focal haemorrhage were all seen among high dose males but not at any other dose level. Generally these changes were associated with and secondary to urinary bladder changes.

SEMINAL VESICLES: Interstitial and peripheral inflammatory cell infiltrates, were all seen among high dose males but not at any other dose level. Generally these changes were associated with and secondary to urinary bladder changes. Reduced secretory content of the vesicles was also seen for several high dose animals and this was probably secondary to the generally debilitated condition of the affected rats.

OTHER HISTOPATHOLOGY
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed. Although group differences in the incidence or severity of lesions occasionally attained statistical significance, none was considered to be related to treatment. The following conditions warrant specific mention:

BONE MARROW: Generally lower grades of severity of adipose infiltration of the bone marrow were seen in relation to treatment for high dose males. This is more likely a consequence of the age difference between control and high dose animals, the latter group being terminated early. A difference was not seen between control male rats and those dosed at 5000 or 500 ppm or for female rats at any treatment level.

EYES: Retinal disruption, considered to be an incidental congenital lesion was observed in a few rats in the study.

HEART: Focal myocarditis was observed in several control and treated rats and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

LIVER: Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encountered.

SPLEEN: Extramedullary haemopoiesis is a normal background condition in the rat spleen and the severities observed were considered to be within normal limits. Generally lower grades of severity of haemosiderin accumulation were seen for high dose males but this may be related to the age difference between control and high dose animals. Males treated with 5000 ppm were similarly affected, the difference attaining statistical significance, however, this was based upon just three animals having one grade of severity less than in the control group and the toxicological significance cannot be regarded as reliable. A similar effect was not seen among female rats.

KIDNEYS: Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the frequencies reported in this study. Similarly focal corticomedullary mineralisation is a commonly observed background condition amongst female rats, although in this study the incidence was statistically lower among high dose and intermediate dose female rats compared with the control group. Corticomedullary mineralisation is a highly variable condition in female rats and is commonly observed to be absent when other renal changes are present. The group distribution and apparent relationship to treatment in this study is of doubtful toxicological significance.

LUNGS: A minimal severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.

THYROID: Follicular cell hypertrophy is a highly variable condition and frequently observed as a spontaneous entity among control rats. The group distribution of the condition in this study is not indicative of a treatment-related effect.

PANCREAS: Focal exocrine atrophy is a commonly observed spontaneous change in laboratory maintained rats of this age.

PITUITARY: Vacuolation of pars anterior cells is a commonly observed background condition in laboratory maintained rats, especially males. There were no indications of an effect of treatment in this study.

UTERUS: A higher incidence of dilatation of the uterine horns was observed in relation to treatment for high and intermediate dose females with statistical significance at both treatment levels. Uterine dilatation is a highly variable condition in laboratory maintained female rats and commonly seen among control animals. The condition is usually associated with oestrus cyclical changes and on this occasion the group difference is due entirely to a lower than might normally be expected incidence among the control group. The condition of the vaginal epithelium is, however, a more reliable indicator of oestrus cycle staging.

Contrary to the results obtained for uterine dilatation from the main study, there was no toxicologically significant differential group distribution of this condition and in particular it was present among control (II) rats with and incidence of 2/20 which was the same as that for females treated with 2500 ppm. The variability of the incidence of this condition was however emphasised by a relatively high incidence of 6/20 among females treated with 250 ppm compared with the control group or among rats treated with 2500 ppm.

VAGINA: Keratinisation of the vaginal epithelium was observed with a higher incidence for females treated with 10000, 5000 and 500 ppm and achieved statistical significance in each treatment group. The incidence of keratinisation in control (II) and 2500 ppm group animals is more or less what would normally be expected and in particular the control group illustrates the spurious nature of the abnormally low incidence reported in the main group animals. The fact that the highest group incidence in this supplementary study occurred in the 500 ppm dose group further illustrates the highly variable nature of the prevalence of different stages of the oestrus cycle in laboratory maintained female rats.

Although the group distribution of keratinisation of the vaginal epithelium in this study suggests an influence of treatment leading to a greater number of treated female rats being at the oestrus – met-oestrus stages of the oestrus cycle, this is far more likely to be a spurious result as a consequence of a much lower than expected incidence of rats in oestrus – met-oestrus in the control group. Keratinisation of the vaginal epithelium (at various stages of development and regression) is related to the stage of the ovarian cycle and is usually indicative of oestrus – met¬oestrus in the rat. Allowing for group cage housing of female rats and the possibility of synchronisation of the oestrus cycling one would normally expect 40-50% of animals to be at the oestrus - met-oestrus stages of the oestrus cycle at any given time. In this investigation keratinisation of the vaginal epithelium appeared in just two out of twenty control group female rats, which is considered to be well below the normal incidence based upon experience with animals at this laboratory. This conclusion is substantiated by the results from an almost concurrent population of control female rats used in a supplementary 90-day study with the same test material with an additional two lower dose levels. In this control group the incidence of keratinisation of the vaginal epithelium was 45% which is within the normally expected range of incidence.

A range of other minor histopathological lesions was observed for the above or other tissues in the study but all were considered to be of no toxicological significance.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
2 500 ppm
Sex:
male
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Effect level:
5 000 ppm
Sex:
female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The oral administration of Experimental 12894-58 by continuous dietary admixture for a period of up to ninety consecutive days at dietary concentrations on up to 20000 ppm (reduced to 10000 ppm), resulted in treatment-related effects at the highest dose level.

 

Effects were evident for males treated with 20000 ppm, which resulted in severe clinical signs, deaths and eventually a reduction in the highest dose level to 10000 ppm. Unfortunately, the reduction in dietary concentration did not result in a regression in clinical signs and concluded in the early termination of the remaining high dose males.

 

The primary treatment-related lesions among these animals were in the urinary tract, affecting both the kidneys and the urinary bladder. These changes included elevated kidney weights, changes in the biochemical, haematological and urinalytical profiles. Clinical signs of red staining detected on the cage tray liners, later confirmed as blood from the urinalytical assessments were most probably attributed to the presence of renal calculi. These stones were confirmed to be composed of calcium phosphate. The most plausible reason for the formation of these calculi was the high calcium content of the test material which resulted in a state of hypercalcaemia. An excess of calcium build-up in the kidneys and urine, combined with other waste products to form the calculi resulted in the hypercalcuria seen in this study. Several other conditions, many of which were probably secondary to the urinary tract pathology were observed in the liver, stomach,thymus, mesenteric lymph nodes, seminal vesicles, and prostate.

 

Although there were no unscheduled deaths, treatment-related renal and urinary bladder pathology was also seen in females treated with 20000 ppm, including slight haemopoietic effects, increases in plasma calcium, and the presence of haemoglobin and erythrocytes in the urine. Post­mortem findings were confined to effects only in one female, which displayed the presence of renal calculi.

 

In addition to these changes, increased trabecular bone formation was observed as a consequence of treatment for males treated with 5000 ppm, together with slight biochemical changes, haemoglobin/erythrocyte cells in the urine. Interestingly, these males were noted to be more readily excitable than control males, which although the toxicological significance of this is dubious, it cannot be discarded. No such effects were evident for females treated with 5000 ppm or animals of either sex treated at the lower dose levels.

 

Isolated changes in the urinalysis profile at the lower dose levels did not show any concomitant histopathological changes in the kidneys or urinary bladder which were representative of the findings observed at the highest dose levels. The changes in haemoglobin and erythrocytes were not consistent for sex or dose level, and therefore a dose-related response was not established. These are also low incident findings in laboratory maintained rats of the age and strain employed and the numbers affected in each of the lower dose groups were very small. In conclusion, these changes at the lower levels were considered unrelated to treatment.

Applicant's summary and conclusion

Conclusions:
The oral administration of Experimental 12894-58 to rats by admixture with the diet for a period of up to ninety days at a maximum dose level of 20000 ppm resulted in treatment-related histopathological changes at 20000 ppm (reduced to 10000 ppm) for animals of either sex, and for males treated with 5000 ppm.
Toxicity of the test material contributed to the premature termination of all males treated with 10000 ppm. Treatment-related renal and urinary bladder pathology was also seen in females treated with 10000 ppm. In addition, treatment-related effects were also detected for males treated with 5000 ppm.
Based on the effects observed in this study, a ‘No Observed Effect Level’ (NOEL) for males was considered to be 2500 ppm and the NOEL for females was considered to be 5000 ppm.
Executive summary:

The oral administration of Experimental 12894-58 to rats by admixture with the diet for a period of up to ninety days at a maximum dose level of 20000 ppm resulted in treatment-related histopathological changes at 20000 ppm (reduced to 10000 ppm) for animals of either sex, and for males treated with 5000 ppm.

Based on the effects observed in this study, a ‘No Observed Effect Level’ (NOEL) for males was considered to be 2500 ppm and the NOEL for females was considered to be 5000 ppm.