Hydroxyl-2-pyridone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15 - April 17, 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 18, 1994- June 21, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: US EPA (TOSCA)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 93100020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in vehicle

Species:

rat

Strain:

other: CD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 28-35 days
- Weight at study initiation: males: 120-122 g; females: 116-121 g
- Fasting period before study: overnight before blood sampling
- Housing: Stainless steel grid dates, with mesh floors and lids. The cages were suspended in a battery capable of holding up to 21 cages, above absorbent paper. The paper was changed three times per week; cages, cage-trays and water bottles were changed when necessary.
- Diet (e.g. ad libitum): Commercially available pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): tap water from public supply, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C (19-25 °C)
- Humidity (%): 55 % relative humidity (range 40-70%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

0.5% w/v aqueous

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared fresh each day as a series of graded concentrations in 0.5% w/v methylcellulose in purified water. The required dosages were provided at a constant volume-dosage of 10 mL/kg bodyweight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Before treatment commenced, the suitability of the proposed formulations was determined by a trial preparation, made up as for day 1 of treatment. Homogeneity was determined by analysis of trial formulations for the high and low dosage groups immediately following their preparation, and stability was assessed after 24 and 48 hours storage at room temperature. Results indicated that the homogeneity and stability of Oxypyrion in 0.5% w/v aqeous methylcellulose were satisfactory.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation samples, supplied as unit doses, were diluted with HPLC mobile phase and dissolved Oxypyrion further diluted to a concentration within a nominal range. The concentration of Oxypyrion in the dilute solution was determined by high performance liquid chromatography using a spectrophotometric detector. The total unit sample of known weight was transferred quantitatively into a volumetric flask with HPLC mobile phase. After ensuring complete dissolution, the solution was made to volume and an aliquot further diluted to a known volume with HPLC mobile phase to give a nominal concentration of Oxypyrion in the final solution between 80 and 160 µg/mL. The concentration was then determined by high performance liquid chromatography.
Validation: linearity: calibration was linear over the concentration range 40 to 200 µg/mL. Limit of assay: the procedure was adequately sensitive for the assay of formulations at concentrations between 2.5 and 100 mg/mL employed in the toxicity studies. THe transfer and dilution of samples was quantitative and no recovery correction was applied to the analyses.
Results indicated that the homogeneity and stability of Oxypyrion in 0.5% w/v aqeous methylcellulose were satisfactory.
In addition, duplicate samples (2 mL each) of each formulation prepared for administration on the first day of treatment (day 1) and on one occasion in week 4 (day 25) of treatment were retrospectively analysed. Results indicated that the achieved concentrations were satisfactory on both occasions.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on preliminary study
- Rationale for animal assignment (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

SIGNS AND MORTALITY: Yes
All rats were inspected regularly for visible, or otherwise sensible, signs of ill-health or reaction to treatment. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition.
Although the various examinations were not specific, they were aimed at the following features:
- A preliminary daily check for deaths or morbidity
- At least two daily examinations for evidence of systemic toxicity or ill-health, the first immediately before dosing and the second shortly after dosing
- An additional final check for systemic toxicity, or ill-health, on all full work days
- A detailed weekly examination including palpation
Any abnormality in the cage trays was noted when they were cleaned.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day that treatment commenced and twice weekly throughout the study period.

FOOD CONSUMPTION:
The weight of food eaten by each cage of rats was calculated weekly by measurement of the amound of food given and that remaining in the food hoppers, together with an estimate of any food scattered.

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: No

WATER CONSUMPTION: Yes
Water consumption was assessed visually in the course of daily observation. Quantitative measurements were not undertaken.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 following overnight food withdrawal and before dosing
- Anaesthetic used for blood collection: Yes (regulated mixture of oxygen, nitrous oxide and Halothane)
- Animals fasted: Yes
- How many animals: 40
- Parameters examined: Packed cell volume, haemoglobin concentration, erythrocyte count, total and differential leucocyte count (lymphocytes, eosinophils, basophils, monocytes and a small proportion of large unstained cells), platelet count, blood film (Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types including normoblasts.
The following characteristics were calculated:
- Mean cell haemoglobin concentration
- Mean cell haemoglobin
- Mean cell volume

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29, a further blood sample
- Animals fasted: Yes
- How many animals: 40
- Parameters examined (in plasma): ALT activity, AST activity, urea, creatinine, glucose, total bilirubin, total protein concentration, electrophoretic protein fractions, sodium, potassium and chloride concentrations.

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, a review of history of each animal and a detailed examination of the external features and orifices, the neck and the subcutaneous structures and the cranial, thoracic, pelvic and abdominal cavities and their viscera. External and cut surfaces of the organs and tissues were examined before or after weighing, as appropriate. Abnormalities, interactions and changes were recorded, and the required tissues preserved in fixative.

HISTOPATHOLOGY: Yes, the following tissues were dehydrated and embedded in paraffin wax, sectioned at approximately five microns thickness and stained with haematoxylin and eosin:
Abnormalities, adrenals, heart, kidneys, liver, spleen, testes
Both auricular and ventricular sections of the heart and sections from two lobes of the liver were prepared. For paired organs, one section was prepared for the left and one for the right side.
The sections from all animals were examined for micropathological change.

Other examinations:

Organ weights:
Following organs taken from all animals were dissected free of adjacent fat and other contiguous tissue and the weights recorded: adrenals, kidneys, liver and testes
Organ weights relative to bodyweight were calculated for all animals.

Statistics:

Intergroup differences in group mean bodyweight change, haematology and blood chemistry were evaluated by Student's 't'-test using a pooled variance. For organ weights, homogeneity of variance was tested using Bartlett's test. If this was found to be statistically significant, a Fisher-Behrens test was used to perform pairwise comparison, otherwise Dunnett's test was used. Intergroup differences in the incidence of macro-or micropathological lesions were assessed by the Fisher Exact Probability test. Two-tailed analyses were untertaken unless otherwise indicated. Levels of statistical significance were chosen as p<0.05, p<0.01 and p<0.001 for Student's 't'-test and p<0.05 and p<0.01 for Dunnett's or Fisher-Behrens tests and the Fisher Exact Probability test. Inter-group differences that were not statistically significant (p>0.05) were not annotated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of reaction to treatment were confined to post dose salivation, particularly during week 3 and 4, in animals treated at 1000 mg/kg/day and, on days 27 or 28, in two animals treated at 150 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

There was no death

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of males dosed at 1000 mg/kg were inferior to control males. The body weight gain of female rats treated at 1000 mg/kg/day and of trats treated at 25 or 150 mg/kg/day was considered to have been unaffected.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The overall food utilization efficiency, as evidenced by food conversion ration, of male and female rats treated at 1000 mg/kg/day was slightly inferior to that of the control animals. The food utilization efficiency of rats treated at 25 and 150 mg/kg/day was considered to have been unaffected.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

All examined parameters in the rats dosed at 1000 mg/kg were lower than the Controls. In addition, the blood films of 2 male and 2 female rats at 1000 mg/kg showed slight or moderate polychromasia, associated in 1 male with low spherocytes and in 1 female with slight anisocytosis and poikilocytosis. Neutrophil numbers in males at the 1000 mg/kg dose were higher than Controls. Haemotology of rats treated at 25 or 150 mg/kg was considered to have been unaffected.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights of rats treated at 1000 mg/kg/day and of female rats treated at 150 mg/kg/day were higher than those of controls. Liver weights of female rats treated at 25 mg/kg/day were also slightly higher than those of female controls, but the difference was small and was considered largely reflect the slightly higher bodyweights of the treated animals.

The small differences in adrenal, kidney and testis weights for animals treated at 1000 mg/kg/day, when compared with the controls, could not be unequivocally attributed to an effect of Oxypyrion.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was no macroscopic finding which was attributed to treatment with Oxypyrion.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to treatment with Oxypyrion were seen in the liver of the treated animals. In the liver, slight periacinar hepatocyte hypertrophy and a minimal degree of necrosis of individual hepatocytes were seen in all treated animals given Oxypyrion at the highest dosage of 1000 mg/kg/day. There was also marginally or slightly increased mitotic activity in the liver of all females treated at 1000 mg/kg/day. Other findings were of the types commonly seen in rats of this age and occurred with the expected frequency.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

The no-effect level was considered to be 25 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

temporary deviations from relative humidity, organs missing at trimming, 3 animals not dosed on 30 and 31 January 2003 and on 9 April 2003, different limit of detection for spectrophotometric absorbance determination. No effect on study integrity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

temporary deviations from relative humidity, organs missing at trimming, 3 animals not dosed on 30 and 31 January 2003 and on 9 April 2003, different limit of detection for spectrophotometric absorbance determination. No effect on study integrity.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyl-2-pyridone

EC Number:

212-506-0

EC Name:

Hydroxyl-2-pyridone

Cas Number:

822-89-9

Molecular formula:

C5H5NO2

IUPAC Name:

1-hydroxy-1,2-dihydropyridin-2-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A2093070
- Expiration date of the lot/batch: 30 September 2005
- Purity: 99.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable
- Stability in vehicle: at least 4 hours (determined at NOTOX)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI) BR

Details on species / strain selection:

Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EEC)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Males-199-204 g; Females- 157-162 g
- Fasting period before study: the animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided.
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). During activity monitoring, animals were individually housed overnight in Macrolon plastic cages (TYpe III, height 15 cm), with sterilised sawdust provided as bedding.
- Diet (e.g. ad libitum): Pelleted lab animal diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 5 days before start of treatmnt under laboratoryt conditions

DETAILS OF FOOD AND WATER QUALITY: Analysis of food and water quality were archived and deemed acceptable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3-23.8°C actual range
- Humidity (%): 14-86% actual range
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 dark/ 12 light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage via stainless steel stomach tube.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Doses prepared daily within 4 hours prior to dosing, and were homogenised to visually acceptable levels. Adjustment was made for specific gravity of the vehicle (specific gravity of 1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Dose volume: 5 mL/kg body weight, acutal dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the analytical study was to determine accuracy of preparation and homogeneity of oxypyrion in formulations used in week 1, week 6 and week 13 and to determine stability of oxypyrion in formulations used in week 1.

Methods:
Conditions:
Spectrophotometer: Varian Cary 50 (Varian, Mulgrave, Victoria, Australia)
Detection wavelength: 305 nm
Slit: 1.5 nm
Probe: Ultra mini immersion probe, titanium tube, staiinless steel handle and quartz prism. Path length 10 mm.
Background correction: Methanol
Linear range: 1.0-10 mg/L; regression line: correlation coefficient (r) >=0.99
Accuracy: Mean recovery: 70-110%
Precision - repeatability: cofficient of variation <=20%
Limit of Quantification: the mean recovery should be in the range of 70-110% and the coefficient of variation should be <=20%
Limit of Detection: 0.195 mg/L
Specificy: group 1 formulation: interfering peaks should be <=30% LOQ

Results:
Linearity: linear reliationship in concentration range of 1.00 - 10.00 mg/L
Accuracy and precision: mean recoveries between 70% and 110% and coefficient of variation below 20% at both concentration levels. Analytical method considered applicable to formulations in the concentration range of 0.888 and 88.8 mg/g
LOQ of 0.889 mg/g but is expected to be much lower
LOD: 0.05 mg/g in formulations (assuming a sample amount of 500 mg in a 25 mL volumetric flask diluted by a factor of 5 prior to analysis
Specificity: no absorption at 305 nm
Stability of solutions: solutions were stable during a series of 92 measurements
Samples:
- Homogeneity: The relative standard deviation of concentrations measured in Group 2 and Group 4 formulations of week 3, 6 and post treatment was up to 11%. Based on the measured concentrations fomrulations were considered to be homogeneous
- Stability: Based on the result, the formulations at target concentrations of 0.889 and 89.9 mg/g oxypyrion in polyethylene glycol 400 were considered stable for at least 4 hours at room temperature.

Duration of treatment / exposure:

7 days per week for 90 days, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on an earlier 28 day study with oxypyrion by daily gavage in the rat (Pharmaco LSR Report No: 94/0584)
- Rationale for animal assignment (if not random): Randomized by weight

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily; once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. THe time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales: maximum grade 1: grade 0=absent, grade 1=present; maximum grade 3 or 4: grade 1=slight, grade 2=moderate, grade 3=severe, grade 4=very severe

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and prior to scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: subjective apprasal only, no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest: all animals; at week 13: group 1 and 4 animals
- Dose groups that were examined: Groups 1 and 4, since no treatment-related findings were noted, the eyes of the rats of groups 2 and 3 were not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided
- How many animals: All tested
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils; clotting potential: prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled necropsy
- Animals fasted: Yes, fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided
- How many animals: All tested
- Parameters checked: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin total, chloride, cholesterol total, creatinine, glucose, phosphorus, protein total, protein albumin, urea, calcium, potassium, sodium,

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12-13 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength (score 0=normal/present, score 1=abnormal/absent); motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes; all tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group; all tissues and organs from all animals of all dose groups which were sacrificed in extremis; all gross lesions of all animals; lungs, liver and kidneys of all animals (all dose groups). Based on treatment related morphologic changes, the spleen was also examined from all rats of the intermediate dose groups. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Other examinations:

Organ weights: from surviving animals on the day of necropsy: adrenal glands, brain epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus

Statistics:

Dunnett test was used to compare treated and control groups for each sex when normal distribution was assumed.
Steel test was used was used when the data could not be assumed to follow a normal distribution.
The Fisher-Exact test was applied to frequency data.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Groups means were calculated for continuous data and medians for discrete data (scores).
No statistical analysis was performed on motor activity.
Test statistics were calculated on the basis of exact values for means and pooled variances. individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different statistics values

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Lethargy, hunched posture, abnormal gait, laboured respiration, gasping, squeaking, swelling of abdomen, piloerection and chromatochryorrhoea were noted among males and females treated at 500 mg/kg.
Brown and/or yellow skin staining and rales were noted among both the control and treated animals. The signs were more frequently noted in this type of studies using Polyethylene glycol 400 as a vehicle and were generally considered not toxicologically relevant. However, the incidence of these effects was clearly increased in males and females treated at 500 mg/kg, and therefore it cannot be excluded that these effects were related to treatment with oxypyrion.
In one female treated at 50 mg/kg, hunched posture, laboured respiration and piloerection were noted once during week 3. At the incidence observed these findings were considered to have occurred by chance and not to be related to treatment with the test item.
Incidental findings that were noted included scabs, alopecia, rales, diarrhoea and salivation. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions of thist study. Therefere, these findigns were considered signs of no toxicological significance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

5 deaths (3 male and 2 female rats treated at 500 mg/kg) were recorded. These animals were sacrificed for humane reasons during weeks 7, 6, 6, 3, and 13 respectively. Among these animals bad general condition, alopecia, hunched posture, breathing problems, piloerection, chromodacryorrhoea and brown/yellow staining of the skin were noted.
At the microscopic examinations, epithelial necrosis in the trachea was noted in 3 animals, indicating misgavage as the cause of moribundity. Another animal had a lymphogranulocytic meningitis affecting the brain and spinal cord, resulting in moribundity (a second - surviving - animal at 500 mg/kg was found with similar reasons. Whilst the etiology of this condition could not be determined it was considered unrelated to treatment with the test item. For a last animal, the cause of moribundity could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of males treated at 500 mg/kg had a total of 23% deficit relative to controls. The total weight gain deficit for males at 50 mg/kg/day was 8% relative to male controls. However, at no time these deviations were significant in statistical terms.
In females at 500 mg/kg, lower body weights were recorded during the second half of the study, but the deviations were statistically significant in week 12 only. THe total weight gain deficit for females at 500 mg/kg/day was 10% relative to female controls.
The deviations observed for females at 500 mg/kg/day and for males at 50 mg/kg/day were considered to be within the range of normal biological variation. Further, the statistically significant lowered body weight gain in females at 5 mg/kg during week 3 was considered not to be toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences in food consumption before or after allowance for body weight between treated and control groups.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment related opthamological findings in week 13. The only fnding was focal corneal opacity in one control male.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower red blood cell counts (males), haemoglobin concentration (males and females), lower hematocrit values (males and females). These deviations indicate anaemia.
In high dose females the condition was associated with significantly lower mean corpuscular haemoglobin concentrations. Lower platelet numbers were also recorded in males and females at 500 mg/kg/day.
Lower mean corpuscular haemoglobin concentrations found in females at 5 mg/kg, were considered to have occurred by chance due to lack of corroborative findings at this dose level.
Partial thromboplastin time was statistically significantly lowered in females at 500 mg/kg. In general, shorter partial thromboplastin time was considered not to be a toxicologically relevant finding.
Statistically significantly higher mean corpuscular volume, mean corpuscular haemoglobin and lower red cell distribution width were noted in males treated at 50 mg/kg. These findings were considered not toxicologically relevant due to to lhe lack of similar changes at 500 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly higher cholesterol levels were noted in males at 50 and 500 mg/kg (dose-relationship present) and in females at 500 mg/kg. Moreover, in females at 500 mg/kg higher values of alanine aminotransferase were found.
Inorganic phosphate was lower in males at 500 mg/kg and females at 50 mg/kg. An elevation of the albumin concentration was found in females at 50 mg/kg and in males at 50 and 500 mg/kg.
Further, in males and females at 500 mg/kg, higher values bilirubin were found and in females at 500 mg/kg, higher values potassium were found. These observations suggest that the observed anaemia was haemolytic in nature.
Higher chloride values noted in females at 5 mg/kg were considered to be incidental in the absence of any effects at higher dose levels. Lower alkaline phosphatase was noted in males at 50 and 500 mg/kg (dose-relationship was not present). These deviations are of no toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No deficiencies in hearing ability, pupillary reflex, static righting reflex or grip strength were observed in any group.
The only finding was focal corneal opacity in one control male.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative and absolute liver weights were increased in both sexes at 500 mg/kg. The microscopic correlate was diffuse midzonal/centrilobular hypertrophy.
Absolute and relative adrenal weights were increased in males at 50 and 500 mg/kg, although a clear dose-relationship was absent. No microscopic correlate was observed.
Relative brain, heart, kidneys, spleen, testes and epididymides weights were higher in males at 500 mg/kg, and liver and kidney weights an males at 50 mg/kg, and a dose-relationship of these elevations was present.
Higher relative heart and kidney weights we found in females at 500 mg/kg. These deviations can be attributed to lower terminal body weights.
Other observations were not considered to be treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Liver observations:
- Accentuated lobular pattern in 2 males at 50 mg/kg and 3 males at 500 mg/kg
- Enlarged in 6 males and 5 females at 500 mg/kg
- Pale discoloration in 1 male and 2 females at 500 mg/kg
- Nutmeg liver in 1 female at 500 mg/kg
Further, in high dose females, there was statistically significant incidence of gross findings in the stomach (thickening of the limiting ridge) and the adrenals (discolouration), but there were no microscopic correlates to suggest treatment-related changes. Discolouration of the mandibular lymph nodes was noted in rats at 50 and/or 500 mg/kg.
Incidental findings among control and/or treated animals included distention with gas of the gastro-intestinal tract (stomach, small intestines or caecum), enlargement/disolouration of the kidneys, irregular surface/disolouration of the stomach, pelvic dilation, reduced spleen size, foci on thymus and stomach, enlargement/discolouration of the mandibular lymph nodes, nodule on preputial gland, discolouration of inguinal lymph node, cysts on ovaries, foci on clitoral gland, enlargement of pituitary gland, discolouration/cysts on adrenal gland, constriction of the slpleen, reduced thymus size, discolouration of the thymus, diaphragmatic hernia of a liver lobe, foci on liver, accessory liver, exophthalamus of the right eye or cloudy eyes. These findings are occasionally seen amongst rats used in these types of study, or incidentally observed. Further, in the absence of correlated microscopic findings they were considered changes of no toxicological significance. Watery fluid in the uterus, found in several females, is related to a stage in the oestrous cycle and is a normal finding.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effects were seen in liver, kidneys and spleen. The primary alteration in the liver was diffuse midzonal/centrilobular hypertrophy seen at slight to severe severity in nineteen animals at 500 mg/kg and at a minimal degree in one male at 50 mg/kg. This was accompanied by minimal or slight degrees of single cell necrosis in ten high dose rats and increased severity of hepatocellular vacuolation (slight or moderate) in five cases at 500 mg/kg. These findings were the histological correlates to the gross observations made at necropsy.
In the kidneys there was an increase in severity - slight or moderate, of corticomedullary tubular atrophy in males at 50 and 500 mg/kg and in females at 500 mg/kg. This finding was accompanied by an increased severity - slight or moderate, of hyaline casts in both sexes at 500 mg/kg.
Hemosiderin pigment was increased - moderate or severe, in the spleen of females at 50 and 500 mg/kg.
Most animals in all dose groups, including controls were affected with a range of inflammatory alterations including: peri-vascular/bronchial inflammatory foci, alveolar inflammation, alveolar macrophage foci and lymphoid hyperplasia. These lesions are characteristic for a pathological entity, of as yet undetermined etiology, which has become incresaingly widespread and common in colonies of laboratory rats (Elwell et al., 1997). The presence of this condition was considered not to have adversely affected the outcome of the study. The remainder of the recorded microscopic findings was within the range fo background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Elwell MR, Mahler JF and Rao GN. Inflammatory lesions in the lungs of rats. Toxicol. Pathol. 25: 529-531, (1997)

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL of the test substance to rats was found to be 5 mg/kg body weight/day. The LOAEL was considered to be 50 mg/kg body weight/day based on liver and kidney toxicity. As significant toxic effects have been observed in this 90 day repeated dose toxicity study via the oral route within the guidance value ranges of 10 mg/kg body weight/day < C <=100 mg/kg body weight/day, the substance is classified as STOT RE category 2, H373.