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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994-02-08 to 1997-03-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted with a structural analogous read-across substance (free acid of the registered substance) in compliance to GLP and similar to current guidelines. Please refer to IUCLID section 13 for read-across justification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Breeding Laboratories, CH 4414-Füllingsdorf, Switzerland
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: body weight was within +/- 20 % of the mean value for each sex
- Housing: 4 animals of the same sex per cage
- Diet: KLIBA powdered diet no. 343 (Klingentalmuehle AG, Basel, CH-4303 Kaiseraugst, Switzerland) ad libitum, offered freshly weekly.
- Water: Municipal supply of Muttenz/Basel, ad libitum from polyethylene bottles, offered freshly each week.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 +/-2°C
- Humidity: 40 to 80 %
- Air changes: 10 - 15 per hour
- Photoperiod: 12/12 (hours dark / hours light)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
A premix was prepared weekly by mixing powdered test material with powdered diet. This premix was stored at room temperature until use. Final diets were prepared weekly by mixing the premix with additional powdered diet. The validity of the dose preparation procedure was certified by satisfactory results of dietary test substance analyses.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each batch of animal feed was analysed for chemical and microbiological contaminants before delivery to the facility. These analyses were routinely performed (including bacteriological counts). Test substance homogeneity in diet mixes was verified at beginning of the study on diet prepared for week 1 . Stability of test substance in the diet was demonstrated beforehand. During the study, diet mixes were analysed in weeks 0, 2, 3, 7, 11 ,14, 19, 26, 39, 42 and 52 for confirmation of test article content.
Duration of treatment / exposure:
104 weeks
52 weeks for satellite groups
Frequency of treatment:
daily with diet
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1500, 5000 , 10000 ppm corresponding to 22, 69, 235 and 517 mg/kg bw/day for males and 29, 93, 323 and 696 mg/kg bw/day for females, respectively
Basis:

No. of animals per sex per dose:
104 weeks exposure: 52 animals per sex per dose
52 weeks exposure (satellite grous): 20 aninmals s per sex per dose
Batch health check animals: 12 animals per sex per dose
Sentinel health check animals: 10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:


- Dose selection rationale: Based on previous experience with the test item, a broad dose range was applied (0 - 10000 ppm)
- Rationale for animal assignment: Animals assigned in a randomised manner
- Rationale for selecting satellite groups: satellite groups of 20 males and 20 females per dose group were allocated to interim sacrifice after 52 weeks of treatment.
- Post-exposure recovery period in satellite groups: none

Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
Health check
During the acclimatisation phase, all animals were examined by the study veterinarian, and blood was collected for differential white blood cell (WBC) counts, red blood cell (RBC) morphology and serology.
Macroscopic abnormalities. Lungs, liver, kidney, spleen, heart were fixed in buffered 10’% formalin and examined histologically in-house.
Sentinel animals were maintained as control animals and were sampled from blood (but were not fasten overnight before) on weeks 26, 36, 80 and 103, and serology investigation were performed on each occasion. WBC counts were also performed on week 103.

Clinical Observations and Mortality
All animals were checked for viability and signs of illness twice daily (except at weekends and bank holidays, when animals were checked only once). All animals were subjected to a detailed health check, which included a palpation, on a weekly basis. All findings were recorded with a computerised data capture system.

Ophthalmoscopy
Prior to treatment and to the scheduled sacrifices, the eyes of all rats at 0 and 10000 ppm were examined with an ophthalmoscope, following administration of mydriatic solution. In the absence of treatment-related changes, no ophthalmoscopic examinations were performed on animlas at 500, 1500, and 5000 ppm.

Bodyweight and Feed Consumption
Animals were weighed weekly, starting one week before the commencement of treatment Feed consumption was also determined weekly, by calculating the difference in feed given and feed remaining in the hopper at the end of the week. Weight determinations were made with a electronic balance in conjunction with a computerised data capture system. The efficiency of feed utilisation per week was assessed during the first 13 weeks of the study by calculating feed conversion ratios.
Sacrifice and pathology:
Clinical Pathology
During weeks 13, 25, 51, 77 and 103, blood and urine samples were collected from 10 males and 10 females of each main group for clinical pathology investigations. Rats were deprived of food and water during overnight urine collection. Urine was cooled by ice during collection. Blood samples were drawn following urine collection by venepuncture of sublingual vein under Metophane anaesthesia. Additional blood samples were collected at these same time points form 10 males and 10 females o each of the satellite groups for haematological investigations on (weeks 13, 25, 51). These satellite group rats were also deprived of food overnight (but not water) before blood sampling.

Blood smears for differential WBC counts were also prepared in weeks 50 and 102/103 form all survivors, excluding those allocated for the above mentioned week 51 and week 103 investigations. Blood smears were also prepared from animals killed in extremis during the study. Blood was drawn by superficial venesection of the tail vein without anaesthesia.

Heamatological investigations
The following investigations were performed using a Sysmex E-2500 automaed haematology analyser:
haematocrit, haemoglobin concentration, rrythrocytes count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpucuscular haemoglobin concentration, Platelets count, Leucocytes count.

Blood smears were prepared and following paramters were evaluated:
reticulocytes, banded neutrophils, segmended neutrophils, lymphocytes, monocytes, eosinophils, basophils.

Clinical Chemistry Investigations:
Total Bilirubin, total protein, albumin, total globulin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, creatine kinase, lactate dehydrogenase, triglycerde, toal cholesterol, creatinine, fastening glucose, urea, calcium, chloride, inorganic phosphate, sodium, potassium.

Rinalysis:
Volume, specific gravity, pH, protein, glucose, keones, occult blood, urobilinogen, leucocyts, nitrite, urine sediment analysis

Terminal investigations
Moribund animals which were killed for human reasons during the course of the study, and surviving animals killed at scheduled necropsies after 52 were all sacrificed by carbon dioxide asphyxiation.

Macroscopic Examination
All animals on study, whether sacrificed or found dead, were subjected to a complete post-mortem examination, which included examination of the following:
external surfaces
all orifices
the cranial cavity and brain
thoracic
abdominal and pelvic cavities with associated organs and tissues
the neck with its tissues.
All abnormalities, including all suspected tumours

Organ Weights
The following organs were weighed at scheduled necropsies by use of electronic balance:
spleen
adrenals
brain
liver
kidneys
testes
heart
ituitary
ovaries (including oviduct)

Organs were trimmed of fat and connective tissue before weighing. Organ to bodyweight and brain weight ratios were calculated

Histopathology
Samples of the following tissues were preserved in 10% buffered formalin (except for eyes, which were fixed in Davidson's solution) until histoprocessing:

Brain
Stomach
Uterus (+ Cervix)
Pituitary
Duodenum
Ovaries
Tongue
Ileum
Mammary glands
Trachea
Jejunum
Skin
Thyroids
Caecum
Lymph nodes
Parathyroids
Colon
Salivary glands
Oesophagus
Rectum
Sciatic nerve
Thymus
Kidneys
Spinal cord (3 levels)
Heart
Adrenals
Femur (+ stiffle joint)
Lungs
Testes
Skeletal muscle
Liver
Epididymis
Sternum (+ bone marrow)
Spleen
Seminal vesicles
Eyes (+ optic nerve)
Pancreas
Prostate
Skull
Aorta
Urinary bladder
Lacrymal
Glands
(Harderian and exorbital)
Vagina
Gall bladder
All abnormalities

Statistics:
All animal data (as bodyweights, feed consumption and clinical observations) were recorded on-line and processed by a VAX-computer based software package (PSA, Scientific Computer Consultants Inc., Ringwood, NJ 07456, USA). Organ weights data were recorded manually and processed by PSA. Means, standard deviations and statistical analyses of the various data were calculated, where appropriate.
The following statistical tests were performed:
- Parametric data: ANOVA followed by Dunnets test.
If ANOVA failed, the following non-parametric methodology was used:
- Count data: Chi-squar, followed by Fishers exact test.
- Non-Parametric data: Kruskall Wallis, followed by Mann-Whitney-U.

Results and discussion

Results of examinations

Details on results:
52-week interim evaluation

1000 ppm
Reduced bodyweight gain (-20% vs. control) and reduced food utilisation efficiency in both sexes. Lower glucose and triglyceride levels in males in week 13 and 25 (approximately - 35 and -50 %, respectively) and 51 (triglyceride only), whereas phosphate was slightly increased (+17 %, p< 0.01) at the same time points.

5000 ppm
Reduced bodyweight gain in male rats only (male rats; -9% vs. control, p<0.01) (female rats; -4% vs. control. p>0.05), lower triglyceride levels in males in week 13, 25 and 51 and higher phosphate levels in week 13 in male rats only.
1500 ppm
Slight bodyweight gain deficit was observed in males only (-6% vs. controls, p<0.05).

500 ppm
No effects were observed

Results after termination of the study

Analysis of diets
 The results of the diet analyses demonstrated adequate exposure of the animals to the test substance at dietary concentrations well within the limits that are considered tolerable for feeding studies (i.e. ±20% of nominal for single analytical results, ±10 % of nominal for averaged concentrations over the whole dosing period), with the exception of Dose 500 ppm week 0.

Health check
The investigations performed during the acclimatisation phase did not reveal any signs of disease and the animals were regarded to be generally healthy and suitable for the study. The serological investigations on the presence of pathogens in sentinel animals were largely negative. Antibodies againstBacillus piliformis,which can cause Tyzzer's disease in adolescent rats, were detected in 11/19 rats in week 26, but were no longer detected in week 36. An immune response toB piliformisis observed frequently in the Han Wistar strain of rat, but outbreaks of nTyzzer's disease have not been observed in recent years. Accordingly, this finding was not
considered to be of clinical relevance. Furthermore, all macroscopic and microscopic findings observed in sentinel animals were considered to be incidental and commonly diagnosed in rats of this strain and age. No lesions were noted that were considered due to an infectious disease.

Mortality
The overall group distribution of deaths during the 104 week treatment period did not distinguish
treated animals from controls. Less males in the treated groups died compared with the control
mortality. Among females, there was a slightly higher mortality among treated groups compared
with the controls although the incidences did not show any dose relationship and the survival
was within the expected range.

Clinical observations
The incidence and severity of clinical signs, which were mainly related to skin and fur, or of palpable masses, did not distinguish treated animals from controls.

Ophthalmoscopy
All changes detected were considered as incidental otr as spontaneous background lesions, and therefore not related to treatment.

Body weights
Treatment-related and statistically significant effects on body weight development were observed in males at 10000 ppm, 5000 ppm and 1500 ppm in males and in females at 10000 ppm.
The body weight gain deficit resulted in mean body weight values at termination (week 104) of 23%, 11% and 8% lower than the control value for males at 10000 ppm, 5000 ppm or 1500 ppm, respectively, and 21% lower than the control value for females a 10000 pp. However, the differences only developed in the last three month of the study and did not attain a level of statistical significance. Therefore, the biological relevance of these differences is in doubt.

Fod consumption
The overall mean food consumption values did not distinguish treated animals form controls. Significant reductions in mean food consumptions which were observed in males and females at 10000 ppm (weeks 18-19, 73-74,77 80, 87, 90 and 91-92) are considered possibly treatment-related. Other significant deviations form weekly control feed consumption means were occasional and within the range of normal biological variation, and were therefore considered to be incidental in nature.

Test material intake
Mean daily test item intakes at 500, 1500 and 5000 ppm were linearly related to dietary concentration. The reduced weight gains at 10000 ppm caused disproportionally higher test item exposure levels. As a result of body weight development with largely unchanged food intake over time. the exposure levels within treatment groups gradually decreased in the course of the treatment period.

Haematology
There were no changes in haematology parameters that distinguished treated animals from controls. A l l statistically significant deviations (e.g. males: platelets, lymphocytes, monocytes segmented neutrophils, eosinophils, banded neutrophils, WBC, MCV, MCHC' females: haematocrit, haemoglobin, WBC, monocytes, eosinophils, banded neutrophils platelets lymphocytes, segmented neutrophils) were not clearly dose-dependent. These values were within the range of observed control values. Therefore, the effects were attributed to normal biological variation and considered to be incidental in nature. Reticulocyte counts showed a slight increase at week 25 only in females treated at 10000 ppm. On all other occasions, the differences between reticulocyte values for controls and the males and females receiving 10000 ppm were not statistically different. During red blood cells morphology evaluation, a slight increase of polychromasia and anisocytosis was noticed at weeks 25 and 51 in females treated at 10000 ppm or 5000 ppm and a slight increase of anisocytosis at week 13. Minor differences between control and high dose groups for male animals were evident only at week 13 of treatment. However, it is stressed that such increases were only marginal, and not strictly dose-related. Therefore, these differences are considered to have little or no biological relevance.

Clinical chemistry
Blood chemistry investigations gave no clear indication o f a treatment-related effect in anv organ
System. However, the following statistically significant deviations from control mean values were not excluded as possible treatment-related effects:
-Lower triglyceride levels in males at 5000 and 10000 ppm in weeks 13, 25, 51 and 77 with a dose-related trend. However, there were no indications of triglyceride accumulation in liver parenchyma at 5000 and 10000 ppm and the biological relevance of the lower plasma triglyceride levels is, therefore, uncertain.
-Lower glucose levels in males at 10000 ppm in weeks 13, 25, 51 and 77.
-Higher phosphate levels in males at 5000 ppm in week 13, and in males at 10000 ppm in weeks 13 and 25.
All other statistically significant deviations from control mean values were, considered to be mincidental in nature.

Urinalysis
Throughout the study, a slight tendency was present for increased numbers of amorphous urate and/or uric acid crystals m the urinary sediment of both males and females at 10000 ppm or 5000 ppm and at week 25 only in males at 1500 ppm. These crystals are the most common form of crystals seen in normal urine of an acidic pH. Since there were no relationships to dose or time these observations are considered to be of no biological relevance. Furthermore, no indication of a renal dysfunction was obvious from the blood chemistry results or the histopathology examinations.

Organ weight
At the 52-week interim sacrifice, all statistically significant deviations from the control mean absolute and relative organ weights were attributed to reduced body weights at 5000 and 10000.
After 104 weeks of treatment, final bodyweights for males and females at 10000 ppm and for males at 5000 ppm were statistically significantly lower than control values. Absolute and relative adrenal gland and kidney weights for females at 10000 ppm were higher than control values, with the relative weights attaining a level of Statistical significance No histological correlates could be found at microscopic examination.
Other values which attained a level of Statistical significance were considered to be the consequence oft he reduced bodyweights at 10000 ppm and 5000 ppm.

Histopathology
Macroscopic Findings
A number of macroscopic findings were diagnosed in rats that died or were sacrificed in extremis during the course of the study, and in those rats that were sacrificed on schedule after 52 or 104 weeks of treatment. The type, incidence and severity of these gross lesions were not considered to distinguish treated animals from controls.

Microscopic Findings
The non-neoplastic changes that were observed microscopically mainly affected the endocrine, reproductive and large parenchymatous organs. They were generally categorized as inflammatory, degenerative or hyperplastic lesions, and their incidence, type and severity did not distinguish treated from control rats. The type, incidence and organ distribution of neoplasm were similar in both treated and control rats and the diagnosed neoplastic lesions were not considered to be related to the administration of the test substance.




Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
69 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on changes in body weight and blood chemistry parameter.
Dose descriptor:
NOAEL
Effect level:
93 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on changes in body weight and blood chemistry parameter.
Dose descriptor:
NOAEL
Remarks:
After 52-weeks interim toxicity evaluation
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on body weight and haematology parameters
Dose descriptor:
NOAEL
Remarks:
After 52-weeks interim toxicity evaluation
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on body weight and haematology parameters

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion