Registration Dossier

Administrative data

Description of key information

Oral administration of the test item to CD rats for 13-weeks produced non-specific toxicity at 1000 mg/kg/day, a reduction of dopamine and adaptive change in the liver at 150 or 1000 mg/kg/day. These changes were shown to be reversible during a subsequent four-week period without treatment. There was no treatment-related change at 25 mg/kg/day and this dosage was, therefore, considered the no-observed-effect level (NOEL) in this study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: Aqueous 1 % methylcellulose with 0.1% Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The homogeneity and stability of the test substance in aqueous formulations was assessed at nominal concentrations of 2.5 mg/ml and 100 mg/ml during two trials at ambient and refrigerated storage.
All formulations were prepared freshly each day for the first 40 days of the study and, after the availability of fiirther stability data weekly thereafter. The formulations were stored in the dark and refrigerated at approximately 4°C, due to probable substance adhesion with glass and metal.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 consecutive weeks
Frequency of treatment:
daily
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 animals
Control animals:
yes, concurrent vehicle
Details on study design:
The dosages used in this study (0, 25, 150 and 1000 mg/kg/day) were selected in conjunction with the Sponsor with reference to previous work with this compound. In that study it was concluded that oral administration of the test substance to CD rats for four weeks produced reduced weight gain and food intake in males receiving 1000 mg/kg/day, marginally reduced weight gain in males receiving 100 mg/kg/day and changes in the plasma that were attributed to adaptive change in the liver. Dosages up to 1000 mg/kg/day were considered suitable for use in this study, but because of the marginal reduction in weight gain at 100 mg/kg/day in males, the lowest dosage in this 13 week study was selected to be below 100 mg/kg/day.
The test substance was administered over a period of 13 consecutive weeks. The necropsy procedures were completed in four days, during which time treatment continued, and serial observations were recorded at appropriate intervals. The duration of treatment is reported as 13 weeks. Animals assigned to the recovery phase completed a further four weeks without treatment. The recovery necropsy was completed in two days.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to freatment

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before freatment commenced (Week -1), on the day that treatment commenced (Week0), weekly throughout the treatment and recovery periods, and before necropsy. More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.

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, the weight of food supplied to each cage, that remaining and an estimate of any spilied was recorded each week throughout the freatment and. recovery periods. From these records, the mean weekly consumption per animal (g/rat/week) was calculated for each cage

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. Precise measurements were not undertaken as no treatment-related change was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, the eyes of all Main and five Recovery study animals (with the lowest identity numbers from each sex group) were examined by means of a binocular indirect ophthalmoscope. Rejected animals were replaced with animals with no adverse ocular abnormality, selected from the spare animals for the study. During Week 12 of freatment the eyes of all Main and five Recovery study animals (with the lowest identity numbers from each sex group) of Groups 1 (Confrol) and 4 (1000 mg/kg/day) were similarly examined.
Prior to each examination, the pupils of each animal were dilated using 0.5% fropicamide ophthalmic solution (Mydriacyl, Alcon Laboratories Ltd.). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.
As no treatment-related change was observed, the examination was not performed during the recovery phase or for animals of Groups 2 or 3 (25 or 150 mg/kg/day).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All Main and five Recovery study animals per group (with the lowest identity numbers from each sex group) were subject to haematology investigation during Week 13 of freatment. Five Recovery study animals from each group (with the lowest identity numbers from each sex group) were also subject to haematology investigation during Week 4 of recovery. Blood samples were obtained before dosing in Week 13 and, on both occasions, after overnight starvation. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the refro-orbital sinus.
- Parameters checked: Blood samples (nominally 0.5 ml) were collected into EDTA as anticoagulant and examined for the following characteristics:All samples obtained during Week 13 of freatment were examined for the following characteristics:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concenfration (MCHC)
Mean cell volume (MCV)
Total leucocyte count (WBC)
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)

Samples obtained during Week 4 of recovery were examined for the following characteristics:
Mean cell haemoglobin concenfration (MCHC)
Total leucocyte coimt (WBC)
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)

During Week 13 of treatment and Week 4 of recovery, additional blood samples (nominally 0.5 ml) were taken into citrate anticoagulant and examined in respect of:
Prothrombin time (PT) -using an an ACL 1000 Analyser and IL PT-Fibrinogen reagent Activated partial thromboplastin time (APTT) -using an ACL 1000 Analyser and IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 ml) were collected into hthium heparin as anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample subsequently centrifuged at 3000 rpm for 10 minutes in order to separate the plasma. After separation, the plasma was examined in respect the parameters
listed below. All samples obtained during Week 13 (all Main and five Recovery study animals with the lowest identity numbers from each sex group) were examined for the following characteristics:
Alkaline phosphatase (ALP)
Alanine aminofransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Glue)
Total cholesterol (Choi)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G) was calculated from total protein concentration and analysed albumin concentration
Samples obtained during Week 4 of recovery were examined for the following characteristics:
Glucose (Glue)
Chloride (Cl) - males only
Albumin (Alb) and albumin/globulin ratio (A/G).

MACROSCOPIC PATHOLOGY:
All Main study animals and five Recovery study animals (with lowest identity numbers from each sex group) were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external featiires and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After venfral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

ORGAN WEIGHTS:
The following organs, taken from all Main study animals and five Recovery animals (with lowest identity numbers from each sex group) after 13 weeks of freatment or four weeks of recovery were dissected free of adjacent fat and other contiguous tissue and the weights recorded:
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus with cervix
Liver
Bilateral organs were weighed together, unless otherwise specified. Organ weights were also adjusted for terminal bodyweight, using the weight recorded immediately before necropsy.
Organ weights were not recorded for animals killed or dying prematurely.

HISTOLOGY
For those animals specified in the Pathology section, the relevant tissues were subject to histological processing.
Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method.
Those tissues subject to histological processing included the following regions:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint - longitudinal section including articular surface, epiphysial plate and bone marrow
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section.

IMMUNHISTOCHEMISTRY
The brain from all Main study animals and five Recovery study animals (with the lowest identity numbers from each sex group) were subject to immunohistochemistry. Additional sections of the cerebellum, cerebrum and cerebral cortex were stained for glial fibrillary acid protein (GFAP) and glutamic acid dehydrogenase (GAD).
Sections at four levels in the brain (2.7, 3.2, 3.7 and 4.2 mm; ref The Rat Brain in Stereotaxic Co-ordinates, Academic Press, New York, 1998) were stained for tyrosine hydroxylase (TH). In addition, the number of TH-positive neurones were counted.

BRAIN CHEMISTRY
All Satellite study animals and five Recovery animals (with the highest identity numbers from each sex group) were subject to brain processing.
The brain from each animal was excised, weighed and the left and right striatae were dissected free and snap-frozen in liquid nitrogen. Samples were stored deep frozen (approximately -70°C) pending despatch to the Principal Investigator. The combined left and right striatae for each animal was analysed for dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA).
All satellite study animals and five recovery animals (with the highest identity numbers from each sex group) were discarded with no further investigation and no other tissue was retained.
Statistics:
All statistical analyses were carried out separately for males and females.
All analyses were carried out using the individual animal as the basic experimental unit.
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's Exact test (Fisher 1973) for each treated group versus the control.
For continuous data, Bartlett's test (Bartlett 1937) was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
For body weight gains and organ weights, whenever Bartlett's test was found to be statistically significant, a Behrens-Fisher test was used to perform pair wise comparisons, otherwise a Dunnett's test was used.
The following sequence of statistical tests was used for grip strength and motor activity and clinical pathology data:
Treatment groups were compared using a Mantel test for a trend in proportions (Mantel 1963) and also pair wise Fisher's Exact tests (Fisher 1973) for each dose group against the control both for i) values =c, and for ii) values <=c versus values >c, as applicable.
If Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level, then parametric analysis was applied.
If Bartlett's test was significant at the 1% level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, then non-parametric tests were applied.
Significant differences between control and treated groups were expressed at the 5% (p<0.05), 1% (p<0.01) or 0.1% (p<0.001) level. The following statistical cyphers were used throughout the report:
a – p < 0.05; b - p<0.01 - using categorical or parametric tests
A – p < 0.05; B - p<0.01 - using non-parametric tests
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Head lowering and repeated movement of forelimbs was observed immediately after dosing from Day 36 in animals receiving 1000 mg/kg/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two incidental deaths occurred: One male of the Control group was killed for humane reasons on Day 41 of treatment due to difficulty in dosing; macroscopic investigations revealed that the animal had enlarged mandibular lymph nodes due to moderate plasmacytosis, and a thickened area of oesophagus 38mm from the stomach, which was found to be minimal inflammation of the musculature. The latter finding is the likely cause of the intubation difficulties. One male which received 25 mg/kg/day, was killed for humane reasons on Day 61 due to an open wound on the scrotum, found to be moderate inflammation, oedema and epidermal necrosis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall weight gain of females receiving 1000 mg/kg/day was lower than that of the Controls and the difference attained statistical significance (p<0.05); the depression of weight gain in those animals was approximately 16%. There was no clear effect upon the bodyweight of other treated animals. The low weight gain in females receiving 25 mg/kg/day and in males receiving 150 or 1000 mg/kg/day was not attributed to treatment since differences lacked dosage-relationship and were not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was considered unaffected by treatment. Such variation that occurred was minor or lacked dosage-relationship and was, therefore, attributed to normal biological variation.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The food conversion efficiency of females receiving 1000 mg/kg/day was slightly low when compared with that of the Controls, reflecting the slightly poor bodyweight gain of these animals. The food conversion efficiency of these females during the recovery period was similar to that of the Confrols. Amongst treated animals at other dosages the food conversion efficiency was considered to have been unaffected by treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
When compared with the results of the pre-treatment examination, there was no finding at the ophthalmoscopy examination in Week 13 considered to be associated with the administration of test material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematological assessment during Week 13 of treatment revealed, when compared with Controls, slightly increased neutrophil counts amongst treated females, although statistical significance was attained only at dosages of 150 mg/kg/day or above. These differences were, however, small and lacked dosage-relationship. The neutrophil counts of previously treated females were similar to those of the Controls at the end of the recovery period. Prothrombin time was prolonged, compared with the Controls in males receiving 1000 mg/kg/day whilst in both sexes there was a slight reduction of activated partial thromboplastin time at this dosage; platelet counts were unaffected. There was no similar trend at the end of the recovery period.
Mean cell haemoglobin concentration was reduced, when compared with the Controls, in treated animals. The difference from Controls was however small and lacked dosage relationship. In addition, most individual values were within the background range. In view of this, and the absence of any other inter-group difference in erythrocyte characteristics, this finding was not considered toxicologically significant; there was no similar trend at the end of the recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical analysis of the plasma during Week 13 of treatment revealed, when compared to Controls, reduced cholesterol concentrations in males receiving 150 mg/kg/day or above. This effect, which was dosage-related, was not seen in females. Plasma triglyceride concentrations were unaffected. Recovery of the effect on cholesterol concentration was not investigated in error. In females receiving 150 or 1000 mg/kg/day there was a slight increase of total protein concentration which, at the highest dosage, was associated with a slight reduction of the albumin to globulin ratio. At the end of the recovery period the albumin to globulin ratio remained low, but the difference from Controls was slightiy less than that at the end of the treatment period, signifying partial recovery. All other inter-group differences in Week 13 were minor, lacked dosage-relationship or showed inconsistent trends and were, therefore, not attributed to treatment. Such differences included the trends seen for alanine amino-transferase and chloride concentrations in males, and glucose concentrations in males and females.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity was unaffected by freatment. There was no clear effect of treatment upon grip strength. Hind limb grip strength of males treated at 1000 mg/kg/day was slightly lower than that of the controls. In the absence of a similar effect amongst females, and since the mean value for these animals was similar to that measured for low dosage males at the end of the recovery period, it was considered that grip strength was unaffected by treatment.
Motor activity was not affected by treatment. During Week 12 of treatment the motor activity (high and low beam breaks) in females receiving the test item was higher than that of the Control during the latter part of the one hour measurement period. This was not, however, considered treatment-related since the Control animals had atypically low values when compared with background data from ten recent studies.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight-relative liver weights were slightly higher than Controls after 13 weeks in males that received 150 or 1000 mg/kg/day. This change was not present at the end of the recovery period. All other inter-group differences in organ weights were minor and considered to reflect normal biological variation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination after 13 weeks revealed no treatment-related finding. All findings were of the types encountered in young CD rats at these laboratories.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination indicated an increased incidence of inflammatory changes in the lungs of females given 1000 mg/kg/day; These were shown to be fully reversible. All other findings were of a type and severity commonly seen in rats of this age at this laboratory.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRAIN-CHEMISTRY
In females receiving 150 mg/kg/day and in males and females receiving 1000 mg/kg/day there was a reduction of dopamine concentration in the striata, though in females the difference lacked dosage relationship. There was also a slight reduction of DOPAC concentration, a metabolite of dopamine, in both sexes at 150 or 1000 mg/kg/day. Concentration of homovanillic acid, a metabolite of DOPAC, were unaffected. The differences of dopamine and DOPAC levels in the striata at the end of the recovery period were similar to those of the Controls, indicating that recovery had occurred.

IMMUNOHISTOCHEMISTRY
Histopathological evaluation of the GAD (glutamic acid dehydrogenase) and GFAP (glial fibrillary acid protein) stained sections of brain revealed no change in the pattern or distribution of positive staining which was considered to be related to administration of the test item. There was no statistically significant difference in the number of cells showing positive staining to tyrosine hydroxylase.
Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: dopamine reduction; reversible increase in liver weight
Critical effects observed:
not specified
Conclusions:
It is concluded that oral administration of the test substance to CD rats for 13-weeks produced non-specific toxicity at 1000 mg/kg/day, a reduction of dopamine and adaptive change in the liver at 150 or 1000 mg/kg/day. These changes were shown to be reversible during a subsequent four-week period without treatment. There was no treatment-related change at 25 mg/kg/day and this dosage was, therefore, considered the no-observed-effect level (NOEL) in this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a GLP-compliant subacute oral toxicity study following OECD guideline 407, the test substance was administered by gavage for 28 days at daily doses of 0, 10, 100 and 1000 mg/kg body weight to a total of 160 albino rats, 20 males and 20 females per dose group. In each dose group, 10 animals per sex and group were sacrificed at the end of the treatment period (experimental groups I and III), and 10 animals per sex and group were kept for a 2-week recovery period before sacrifice (experimental groups II and IV). Administered quantities of the test article suspension were adjusted daily to individual body weight. No relevant clinical symptoms and no signs of systemic toxicity were observed during both treatment and recovery period. No death occurred during the study. During both treatment and recovery period the mean body weights of all treated groups were comparable to the respective controls. The mean food consumption of all treated groups was comparable to the respective controls. The mean food consumption ratios of all treated groups were comparable to the respective controls. The mean water consumption of all treated groups was considered unaffected by the treatment. Neurological observations did not reveal any treatment-related effects (see chapter 7.9.1 Neurotoxicity). A minor prolongation of prothrombin time occurring in males of the high dose group (1000 mg/kg) at the end of the treatment period was reversible within the recovery period. The treatment had no effects on blood chemistry parameters. The analyses of absolute and relative mean organ weights revealed no treatment-related effects. Macroscopical examination did not reveal any treatment-related changes. Histopathological examination showed extramedullary hematopoietic activity to be more prominent in the liver of male rats from the high dose group (1000 mg/kg) than in the other treated and control males at the end of the treatment period. This finding is considered to represent a treatment-related effect. At the end of the 2 week recovery period, this slight difference still persisted. Neuropathological examination did not reveal any treatment-related changes in the central or peripheral nervous system. Under the conditions of this test, treatment with the test substance resulted in a reversible minor effect on prothrombin time and an extramedullary hematopoietic activity in the liver which was irreversible within the 2 week recovery period. Both effects were observed in males of the high dose group (1000 mg/kg) only. It can be inferred from the observations made during this study, that a "no observable effect level" for the test substance when administered by gavage over a period of 4 weeks is 100 mg/kg body weight per day in males and 1000 mg/kg body weight per day in females.

The systemic toxicity of the test item to Crl:CD® (SD)IGS BR rats by oral administration was assessed over a period of 13 weeks according to OECD guideline 408 and following GLP requirements. Three groups of ten male and ten female rats received the test item by gavage at nominal dosages of 25, 150 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, aqueous 1% methylcellulose with 0.1% Tween 80, at the same volume-dosage. A further ten male and ten female rats were assigned to each group for a four week recovery period following the 13 week treatment period. For the purposes of brain chemistry a further ten males and ten females were allocated to each group and were assigned to the satellite study. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, organ weight, macroscopic pathology, immunohistochemistry and microscopic pathology investigations were undertaken.

The oral administration of test material to at dosages up to 1000 mg/kg/day was generally well-tolerated. Treatment-related changes, which included non-specific toxicity and findings that suggested an effect upon the liver and dopamine levels in the brain, were confined to 150 mg/kg/day or above. All changes were shown to be at least partially reversible during the four-week recovery period. During the treatment period there was an overall reduction of weight gain, with a consequential reduction of food conversion efficiency, in females receiving 1000 mg/kg/day. This is attributed to a non-specific toxic response to treatment. Clinical signs were limited to head lowering and repeated movement of the forelimbs. These are likely due to the physical nature of the test formulations as they occurred immediately after dose administration. It is unlikely that they relate to the effect of treatment upon dopamine levels, discussed below, as it would be expected that the signs would be more persistent. In addition, these signs are inconsistent with those that would be expected where dopamine concentrations are depressed.

The test item at dosages of 150 mg/kg/day or more produced a reduction in the concentration of dopamine within the striata of the brain. Dopamine is metabolised to 3,4-dihydroxyphenylacetic acid (DOPAC) and the levels of this chemical in the striata were also reduced. This slight inhibitory effect upon dopamine was not associated with any evidence for tissue damage. There was no histopathological lesion in the brain, nor was there any effect upon the levels of glutamic acid dehyrogenase (GAD) and glial fibrillary acid protein (GFAP); where nervous tissue damage occurs, the number of GAD-stained cells would be expected to reduce and the amount of GFAP to increase. Furthermore, the number of tyrosine hydrolase-positive neurones did not show the reduction that would be indicative of neuronal toxicity. After withdrawal of treatment, the changes in dopamine and DOPAC concentration returned to normal, indicating full recovery. Such rapid recovery would also indicate the absence of any tissue damage. It is likely, therefore, that the test item influenced the metabolic pathway for dopamine production, but does not cause damage to dopaminergic neurons. An effect upon the liver was indicated by increased liver weight after 13 weeks in males given 150 or 1000 mg/kg/day. There was, however, no histopathological change in the liver, suggesting that the increase in liver weight was an adaptive response to the administration of a xenobiotic.

Haematological investigations performed in week 13 indicated increased neutrophil counts amongst females receiving 150 and 1000 mg/kg/day, prolonged prothrombin time in males receiving 1000 mg/kg/day and slightly reduced partial thromboplastin time in males and females receiving 1000 mg/kg/day. None of these changes was evident at the end of the recovery period. During week 13, biochemical analysis of plasma revealed reduced cholesterol concentrations in males receiving 150, or 1000 mg/kg/day, slightly increased total protein concentration in females receiving 150 or 1000 mg/kg/day and low albumin to globulin ratio in females receiving 1000 mg/kg/day. There was partial recovery in respect of the protein changes. Recovery in respect of the cholesterol change was not ascertained. The reduction of total plasma cholesterol concentration in these animals is attributed to the liver change, though there was no evidence for any impairment of fat metabolism, such as increased hepatocyte vacuolation. Slight variations in plasma protein concentration in females are attributed to an effect on the liver. In addition, the small changes in clotting time may also be due to an alteration of liver metabolism since it is the site of the biosynthesis of several clotting factors. The toxicological significance of the increased inflammatory change in the lungs of high dosage females is unclear. These changes may be the cause of the small increases in neutrophil numbers. Histopathological evaluation of the glutamic acid dehydrogenase and glial fibrillary acid protein stained sections of brain revealed no change in the pattern or distribution of positive staining due to treatment. The number of cells showing positive staining towards tyrosine hydroxylase was also unaffected.

It is concluded that oral administration of the test substance to CD rats for 13-weeks produced non-specific toxicity at 1000 mg/kg/day, a reduction of dopamine and adaptive change in the liver at 150 or 1000 mg/kg/day. These changes were shown to be reversible during a subsequent four-week period without treatment. There was no treatment-related change at 25 mg/kg/day and this dosage was, therefore, considered the no-observed-effect level (NOEL) in this study.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.The NOAEL for subchronic exposure was established at 25 mg/kg/day based on adaptive changes in the liver and a reversible reduction of dopamine at the 150 mg/kg/day. As a result the substance is not considered to be classified for repeated dose toxicity via oral route under Regulation (EC) No 1272/2008.