Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2011 to March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
In addtion to the standard protocol a neurotoxicity test battery was added according to US EPA OPPTS 870.6200 (1988) and US EPA NTS publication 91-154617 (1991)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan lab. UK
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: males: 227 - 263 g, females: 162 to 191 g
- Fasting period before study: no
- Housing: goups of 5 to 6 animals,. polycarbonate cages
- Diet : ad libitum
- Water :ad libitum):
- Acclimation period:up to 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2
- Humidity (%):55 +-15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark

IN-LIFE DATES: From: 01.09.2011 To: 14.10.2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE: distilled water
- Concentration in vehicle: 0, 6, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material solution were taken and analysed for substance concentration. Stability and homogenity of the suspensions were determined in a previous experiment and it was demonstrated that the suspensions were stable and homogen for 13 days.
Duration of treatment / exposure:
28-days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
A recovery high dose group of 10 animals per sex per dose receiving 1000 mg/kg bw/day of the test item was also included.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selcected based on the results of a 7-day dose range finding study with rats of the same strain and origin.
- Animals were randomly assigned to the dose groups.
- Post-exposure recovery period in satellite groups: high dose group and control
.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
weekdays:
immediately before dosing, up to thirty minutes post dosing and one and five hours after
dosing during the working week.
weekends
immediately before and after
dosing and one hour after dosing..
Recovery animals during treatment-free period,
daily.
- Cage side observations:.
All animals were examined for overt signs of toxicity, ill-health or behavioural change
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
- Prior to the start of treatment,
- following treatment on Day 1 and weekly thereafter, all
Observations were carried out from approximately two hours after dosing on each occasion.
Observations: animals were observed for signs of functional/behavioural toxicity, together with an assessment of sensory reactivity to different stimuli.
Detailed individual clinical observations were performed for each animal using a purpose
built arena. The following parameters were observed:
Gait and co-ordination 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
BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and weekly thereafter, before necropsy

FOOD CONSUMPTION
- Food consumption for each cage group was determined weekly

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
Dayly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28 for main study animals, day 42 for recovery animals (if necessary second sampling directly before necropsy day 29 and 43)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals
- Parameters examined:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
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)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28 for main study animals, day 42 for recovery animals (if necessary second sampling directly before necropsy day 29 and 43)
- Animals fasted: No
- How many animals: all animals
- Parameters examined:
Urea Inorganic phosphorus (P), Glucose, Gamma glutamyltranspeptidase (γGT), 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+), Triglycerides (Tri), Chloride (Cl-) , Total cholesterol (Chol), Calcium (Ca++), Total bilirubin (Bili), Bile acids

URINALYSIS: Yes
- Time schedule for collection of urine:during week 4 of main study and week 6 for reciovery animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes during the overnight urine collection
- Parameters examined: Volume, Specific Gravity, pH, Ketones, , Bilirubin, Urobilinogen, Protein, Blood, Glucose,
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment, following treatment on Day 1 and weekly thereafter, all
observations were carried out from approximately two hours after dosing on each occasion. These tests were also
performed on recovery animals during the treatment-free period, on a weekly basis.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength fore and hindlimb / motor activity /

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period, or in the case of recovery group animals, at the end
of the treatment-free period, all main test animals were killed by intravenous overdose of
sodium pentobarbitone followed by exsanguination.
Animals assigned to the neuropathology groups were killed by intravenous overdose of
sodium pentobarbitone, followed by whole body perfusion with glutaraldehyde:
paraformaldehyde fixative, via the heart, following initial perfusion with heparinised saline.
All animals were subjected to a full external and internal examination, and any
macroscopic abnormalities were recorded.
The following organ weights were determined:
Adrenals
Brain
Epididymides ♦
Heart
Liver
Kidneys
Ovaries
Pituitary (post-fixation)
Prostate and Seminal Vesicles
(with coagulating glands and fluids)
Spleen
Testes
Thymus
Thyroid/Parathyroid (post fixation)
Uterus with Cervix

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in
buffered 10% formalin except where stated:
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
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Skin (hind limb)
* Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes ♦
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina
* Eyes fixed in Davidson’s fluid
♦ Preserved in Bouin’s fluid then transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

Histopathology was perfomed on the following organs from high dose and main study control animals from paraffin sections and Haematoxilin/Eosin stained preparations.
Adrenals
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and
pons)
Caecum
Colon
Duodenum
Epididymides
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Oesophagus
Ovaries
Pituitary
Prostate
Rectum
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina
Any macroscopically observed lesions were also processed together with the liver and spleen from all 30 and 300 mg/kg bw/day dose group animals. In addition, sections of testes and epididymides from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined. Kidneys, adrenal glands,liver and spleen sections from animals in the non-perfused low and intermediate treatment groups were also subsequently examined..

For the animals assigned to the neuropathology groups samples of the following tissues from control and 1000 mg/kg
bw/day were processed and examined:
Brain: at levels to include olfactory bulb,
forebrain, centre of cerebrum (including
hippocampus), midbrain, cerebellum,
pons and medulla oblongata.
Dorsal root ganglia
Dorsal and Ventral root fibres
Eyes
Optic Nerve
Sciatic Nerve
Skeletal (calf) muscle
Spinal Cord
Tibial Nerve
Statistics:
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 nonparametric 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 (nonparametric) 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)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
no unscheduled deaths, no treatment related clincal observations
Mortality:
no mortality observed
Description (incidence):
no unscheduled deaths, no treatment related clincal observations
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences between treated and control groups were observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences between treated and control groups were observed
Food efficiency:
no effects observed
Description (incidence and severity):
No differences between treated and control groups were observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No differences between treated and control groups were observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant treatment related effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant treatment related effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
ally relevant treatment related effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment related effects were observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant organ weight changes were observed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
the obsered effects were not considered of toxicological significance and not considered adverse
Histopathological findings: neoplastic:
not specified
Details on results:
MORTALITY, BODY WEIGHTS, CLINICAL SIGNS, FOOD and WATER CONSUMPTION, FOOD EFFICIENCY
No unschdeuled deaths or treatment related clinical signs were observed throughout the study in any of the treatment groups.
Body weight and body weight gain was comparable between treated and contol groups and no statisitcally significant difference was observed. Food and waterconsumption of treated grouips was comparble to controls and no significant difference in food efficiency between treatment and cotnrol groups was observed.
HAEMATOLOGY
No test substance related toxicologically relevant effects were observed.
In mid dose males a statistically significant reduction in lymphocyte counts compared to concurrent controls was observed. The significance was minimal and in the absence of a dose response this finding was considered incidental rahter than treatment related. High dose group males showed a statistically significant increase in reticulocyte counts when comopared to controls. However, in the absence of changes of other hematological parameters and due to the fact that all individual values were in the normally expected range for this parameter, while controls showed on lower than expected value, the minimal increase was considered due to the low control values rather than treatment related. Recovery high dose males showed a statisticlly significant increase in neutrophil counts compared to concurrent controls. In isolation this finding was not consdidered of toxicological significance. Equally an isolated finding of a statisitcally significant increase in eosinophil counts in female recover animals was not considered of toxicological significance.
CLINICAL CHEMISTRY
No toxicologically significant treatment related effects were observed.
Males of the high dose group showed a slight but statsitcally significant increase in blood Ca2+ levels compared to concurrent controls, females of the high dose group showed a reduction in triglyuceride values when compared to concurrent controls and recovery high dose females showed a slight reduction in A/G ratio when compared to concurrent controls. All indicvidual values in this cases were within the normally expected ranges and were therefore not considered of toxicological significance. Females in the mid and high dose groups showed a slight increase in blood chloride values compared to concurrent controls. The significance was minimal and there was no dose response. Therfore this finding was considered incidental.Alkaline phosphatase levels were statisitcally significantly reduced compared to concurrent control values in females of all dose groups. There was however no convincing dose response and the finding was not considered of toxicological significance. An increase in creatinine levels of minimal statistical significance compared to concurrent control levels was observed in females of the mid and high dose groups. Again, a dose response was not observed, the individual values were within the expected ranges and the finding was not considered of toxicological significance. A slight but statistically significant increase in blood creatinine levels was observed in recovery high dose males compared to concurrent controls. As the significance wa minimal the finding in isolation was considered incidental rather than treatment related.
URINALYSIS
No treatment related findings were observed. The only statistically significant obsevation was a slight reduction in urinary volume of females of the high dose group compared to concurrent control. This correlated with a slightly reduced water intake, but no histopathological correlates were found and the findign was not related to test item toxicity.
NEUROBEHAVIOUR
Behavioural assessments: Assessments of transfe/arousal, urination, defecation and tail elevation did not show any test substance related effects or statistical differences between treament and control groups. All observations were within normal variation for rats of this strain.
Functional peformance tests:
In the test prior to test item administration females allocated to the main test high dose group showed a significant increase in forelimb grip strength in one of 3 test runs.
In the test on day 1 forelimb grip strength of females of the mid and high dose group was statistically significantly increased compared to controls in one of 3 tests. As this was only seen at one test occasion and no dose-response reslationship was observed, this was not considered treatment related. Furthermore all individual values were within the normally expected ranges.
On day 1 of treatment the overall activity of males of the mid and high dose main study groups was statistically significantly reduced compared to controls. In females of the mid dose group the overall activity was statistically significantly increased compared to controls. The absence of a dose response and a consistent effect between sexes as well as the absence of any other clinical signs indicates that these differences are mot treatment related. In the nueropathology groups no statistically significant changes compared to controls were observed prior to treatment or on day 1. No statistically significant differences between treated and control groups were observed at the day 7 assessment of the main and recovery groups. In the nueropathology group a statsitically increased forelimb grip strength was observed in high dose males and a decrease of forelimb grip strength in high dose females compared to controls in one of 3 tests only. The inidivual values were all in the normally expeced ranges. In the absence of consistent effects between sexes and due to the fact that it was only observed in one group of the subgroup and not in the animals of the main study, the findings were considered as incidental rather than test substance realted. In the day 14 assessment females of the 300 and 1000 mg/kg/day dose groups showed a statisitcally significant increase in hindlimb grip strength in one of 3 tests only. Males of the 300 mg/gk/day dose group showed a forelimb grip strenghth that was statistically significantly increased compared to controls in one of 3 tests. However , no dose-response was seen for the effect size With the exception of one value all individual values were in the normally expected ranges. Therefore the findings are not considered test substance related. Overall activity was slightly, but statistically significantly decreased in high dose males of the neuropathology group on day 14 compared to concurrent controls. As the significance was minimal and not observed in the main study and recovery groups, this finding was considered incidental. At the day 21 assessment overall activity of males of all dose groups was statistically significantly lower than that of controls. There was however no dose response (in effect an inverse dose response), a simialr finding was not observed in females or the neuropathology groups. Therefore the effect was not considered treatment related. In the day 26 assessment hindlimb grip strength was slightly increased, forelimb grip strength decreased compared to controls in females of the high dose main study/recovery group in on e of 3 tests only. In males no statisitcally significant differences to controls were observed in any of the test groups. In the neuropathology subgroup hindlimb grip strength of females in the mid and high dose groups was slighty decreased (stat. significant) compared to controls in one of 3 tests and forelimb grip strength increase in the high and mid dose group females in one of 3 tests. All individual data were within the expected ranges. The findings were therefore not considered treatment related. A statistically significant increase in overall activity was observed in males of the neuropathology high dose group at day 26 compared to controls. As the animals of the main study/recovey group did not show a similar effect, this finding was considered incidnetal and not treatment related. Testing after the recovery period revealed no ststistically singinfanct differences between high dose and control males and only a slight but significant increase in hindlimb grip strength of high dose females in one of three tests only. This finding was minimal and considered incidental.
Overall the functional testing did not reveal any treatment related effects. Statistically significant differences did not show a consistent pattern and were mostly related to single tests and in most cases all individual values were in the expected normal range for this strain of rats.
Sensory function:
No statistically significant differences in sensory reactivity between control and main/recovery group animals were abserved. The same is true for the neuropathology subgroups.
ORGAN WEIGHTS
In the main test group all absolute and relative organ weights of treated groups were comparable to the concurrent control groups. In the neuropathology group perfused brain weights of males and females of the mid dose group were statisically significantly higher than those of concurrent controls. There were no macroscopic abnormalities detected and in the absence of a dose response and a pathological correlate this finding is considered unrelated to test item toxicity.
GROSS PATHOLOGY
No macroscopic findings suggestive of systemic toxicity of the test item were reported. One female of the mid dose group had a fluid filled sac on the right ovary and enlarged vagina with no apparent opening as well as a malformed uterus and cervix. This was considered a congenital abnormality and not treatment related. Reddened lungs were observed in in two males and one female of the high dose group. In the absence of any significant histopathological findings in the changed tissues, the findings were not considered of toxicological significance.
HISTOPATHOLOGY: NON-NEOPLASTIC
Liver: Centrilobular hepatocellular hypertrophy was recorded in 3 male and 2 female high dose animals at minimal severity. After the recovery period no such changes were observed. The changes are considered adaptive in nature and not indicative of an adverse effect.
Spleen: Two high dose males showed minot lymphoid depletion. no such changes were seen after the 14 day recovery period. In the absence of any organ weight changes and given the low incidence and severity, this minot stress induced response was not considered clearly treatment related.
Kidneys: Tubular vacuolation was observed in 4 of 5 males in the high dose group inthe corticomedullary junction tubes. No such findings were observed in females or in the recovery group animals. In the absence of any degenerative of inflammatory changes, this finding was considered not to represent an adverse effect.
Adrenals: Minimal to slight diffuse hypertrophy of the cortical zona fasciculata was observed in 3 males and 2 females of the mid dose group and 1 male and 3 females in the high dose group. No such findings were observed in the 14-day recovery group animals. In the absence of a clear dose response (neither of incidence nor of severity) and any correlating organ weight changes, these findings were considered not to represent an adverse health effect.
Neuropathology: No lesion indicative of an effect was observed in any animals of the study.
Sperm staging: No effects of the test item were observed on the completeness of stages or cell population. There was no indication of maturation arrest, reabsorption or any other degenerative effect.
All other histopathological investigations revealed no substance related alterations and were comparable to control animals.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All treatment related effects were adaptive in nature and not observed in the recovery animals. Therefore the effects observed are not considered adverse.
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight histopathological changes in the liver, spleen, kidneys and adrenal glands were observed in the 1000 and 300 mg/kg bw/day dose groups. The effects were not considered adverse, but adaptive and were not observed in the recovery group animals.
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The lowest dose level at which the slight histopathological changes were observed was 300 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of the test substance to groups of 5 male and 5 female Wistar rats with 0, 30, 300 and 1000 mg/kg bw day of test item did not result in test substance related adverse effects up to the highest dose tested. An NOAEL of 1000 mg/kg bw/day was derived from this study. Minor test substance related changes were considered of an adaptive rather than adverse nature. The study included an addtional neuorological screening battery and neurohistopathology as well as extended histopathology of the sex organs includig sperm staging. No clearly treatment related effects were observed in hematology anmd clinical chemistry. Functional testing did not reveal any treatment related effects. Statistically significant differences did not show a consistent pattern and were mostly related to single tests and in most cases all individual values were in the expected normal range for this strain of rats. Histopathological findings included minimal hypertrophy of the liver in a few animlas of the high dose group, tubular vacuolation in the corticomedullary tubules of the kidneys was observed in high dose males only. The alteration was not associated with any further injury affecting the integrity of the renal tissues. Stress-related changes were observed by reduced size of periarteriolar sheets and mantle zone in the spleen of two high dose males, and diffuse hypertrophy of the cortical zona fasciculata in some mid and high dose male and female animals (no clear dose-response). All those effects were not observed in the 14-day recovery high dose animals and can thus be considered reversible in nature.
Executive summary:

Groups of Wistar rats per sex were administered 0 (vehicle (distilled water) control), 30, 300 and 1000 mg/kg bw of the test item on 28 consecutive days by oral gavage. Addtional recovery groups of 5 animals per sex were administered the vehicle and the test item at a dose level of 1000 mg/kg bw/day for 28 days followed by a 14 -day treatment free period. In addition three dose groups receiving the same dose levels as the main group animals of 5 males and 5 females per group for neuropathological examination were included with an additional vehicle control group. The study followed OECD guideline 407 (2008) and was also performed in accordance with EU guideline 94/54/EC Method B7, Japenese METI guidelines of 2003 and US EPA OPPTS 870.30.50 (2000) and was conducted under GLP conditions. It included an additiional neurofuctional test battery in accordance with US EPA guideline OPPTS 850.370.6200 (1998) and US EPA NTIS publication 91 -154617 (1991). Clincal signs, behavioral and functional performance, body weights and body weight cahnges, food and water consumption were monitored during the study at regular interval for both the main study animals and the neuropathology groups. At the end of the treatment period all main study animals except the recovery group animals were subjected to standard haematology, clinical chemistry and urinalysis investigations. After The recovery group animals were subject to the same set of investigations at the end of the recovery period. All animals of the main and recovery groups were subjected to gross pathology. An extended set of histopathological investigations was performed in all animals of the high dose and control groups. In addition all tissues with macroscopic lesions, and liver, kidneys, spleen and adrenals of all dose gropus were investigated histopathologically. In the neuropathology subgroup all animals were subjeted to gross necropsy after the 28 -day treatment period, followed by whole body perfusion and determination of brain weights. Neuropathological investigations on all neural tissues were performed on all animals of the high dose group and controls of this subgroup. No toxicologically significant adverse effects were observed in any of the dose groups. Therefore an NOAEL of 1000 mg/kg body weight per day was derived from this study. Minor test substance related changes were considered of an adaptive rather than adverse nature. The study included an addtional neuorological screening battery and neurohistopathology as well as extended histopathology of the sex organs includig sperm staging. No clearly treatment related effects were observed in hematology anmd clinical chemistry. Functional testing did not reveal any treatment related effects. Statistically significant differences did not show a consistent pattern and were mostly related to single tests and in most cases all individual values were in the expected normal range for this strain of rats. Histopathological findings included minimal hypertrophy of the liver in a few animlas of the high dose group, tubular vacuolation in the corticomedullary tubules of the kidneys was observed in high dose males only. The alteration was not associated with any further injury affecting the integrity of the renal tissues. Stress-related changes were observed by reduced size of periarteriolar sheets and mantle zone in the spleen of two high dose males, and diffuse hypertrophy of the cortical zona fasciculata in some mid and high dose male and female animals (no clear dose-response). All those effects were not observed in the 14-day recovery high dose animals and can thus be considered reversible in nature.