Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-09-2011 to 24-10-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, (2000).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2013 ; signature: May 2013
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
EC Number:
939-400-3
Molecular formula:
C13H22O
IUPAC Name:
Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: clear colourless liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males (mean of groups) 149 - 154 g and females (mean of groups) 125 – 129 g; individuals were randomly allocated to treatment groups using a randomization procedure. All animals within ± 20% of the sex mean.
- Fasting period before study: None
- Housing: Macrolon cages with sawdust bedding and paper, changed at appropriate intervals; group housed (5 per group) by sex. During locomotor investigations were housed individually in polycarbonate cages without cage-enrichment or bedding material.
- Diet (e.g. ad libitum): pelleted rodent diet, certified (recognised supplier), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: pelleted rodent diet, certified (recognised supplier) – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 – actual: 18 – 24 °C
- Humidity (%): 55 ± 15 (or 40 to 70)
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 02-09-2013 to 15-10-2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Propylene Glycol 400. Formulations (w/w) were prepared daily within 6 hours prior to dosing. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least 6 hours. Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test item and vehicle. No correction was made for the purity of the test item.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Propylene Glycol 400 was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable in vehicle.
- Concentration in vehicle: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No significant test item was detected in the Group 1 formulations.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Other: Dose-formulations were analysed during the study and were reported as within ± 10% applied limits.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability: Samples of the test item formulations were taken on at least two occasions and analyzed for concentration of test item (method of analysis provided in full study report). The results concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The mean recoveries of the procedural recovery samples fell within the criterion of 85-115%. It demonstrated that the analytical method was adequate for the determination of the test item in the test samples.
- The analysis consisted of GC-MSD analysis with internal standards and external calibration (within a dedicated formulation analysis report attached to the full study report). Samples in Propylene Glycol were accurately fortified with known amounts of test item in an end solution of cyclohexane. These were then subjected to analysis by GC-MSD analysis using external calibration, with linear regression to calibration standard. The analytical method was validated (details available within the full study report). The test item samples were subject to a known dilution factor (details available within the full study report) to place the end samples in the calibration range within cyclohexane.
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low – Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate – Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High – Group
No. of animals per sex per dose:
5 per sex per dose (5 male / 5 female)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 5-day sighting study (Report number attached to and cited in the full study report). Dose levels in the 5-day sighting test were: Group 1: 500 mg/kg bw/day (in Propylene Glycol) Group 2: 1000 mg/kg bw/day. In the 5-day range finder (administered consecutively, for 5-days) in six females per group the following effects were determined. High dose females: indicated no mortality, and demonstrated hunched posture and salivation (all females, Day 4). Uncoordinated movements (one female, Day 2). Bodyweights and food consumption were normal. No abnormalities were observed on macroscopic examination. Liver weights slightly higher than normal. Kidney weights considered to be normal. At 500 mg/kg bw/day (in Propylene Glycol) females indicated: no mortality. Hunched posture and salivation (all females, Day 4). Bodyweights and food consumption were normal. No abnormalities were observed on macroscopic examination. Liver and kidney weights were considered to be normal. Basis: other: nominal in vehicle (Propylene glycol).
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: None.
- Section schedule rationale: Random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment, immediately after dosing and at weekly intervals, this was also performed outside the home cage in a standard arena. These clinical observations were conducted at least between 1 and 2 hours after dosing. Additional functional observations were made as ‘special evaluations’. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination.

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: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: Yes.
- Body weight gain % was determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals.
- Anaesthetic used for blood collection: Yes. isoflurane (recognised supplier)
- Animals fasted: Yes. Overnight (maximum 24 hours).
- How many animals: All animals
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Additionally: Prothrombin time (CT) was assessed and Activated partial thromboplastin time (APTT) was assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 28) for all test and control group individuals.
- Animals fasted: Yes. Overnight (maximum 20 hours).
- How many animals: All animals
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot.Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Triglycerides (Tri), Chloride (Cl-), Bile acids, Calcium (Ca++), Inorganic phosphosphate (P)

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate1, Liver, Seminal vesicles including coagulating glands1, Ovaries, Thyroid including parathyroid1.
Where marked 1: weighed when fixed for at least 24 hours.

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Identification marks: not processed, Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain, [cerebellum, mid-brain, cortex], (Pituitary gland), Caecum, (Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides *, Seminal vesicles including coagulating gland, Eyes (including optic nerve [if detectable] and harderian gland) *, Skeletal muscle (Skin), (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes *, Kidneys, Thymus (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals
- liver, spleen and sternum with bone marrow (both sexes) and kidneys and colon (males) from all animals of groups 2 and 3, based on (possible) treatment-related changes in these organs in group 4
- all gross lesions
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. (Ref. 2)
- The exact Fisher-test was applied to frequency data. (Ref. 3)

Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

1 C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
2 R.G. Miller, Simultaneous Statistical Inference, Springer Verlag, New York (1981).
3 R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
4 Kruskal W.H. and Wallis W.A., Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
At 1000 mg/kg bw and 300 mg/kg bw (and all females at 100 mg/kg bw): salivation seen after dosing and was considered to be a physiological response rather than a sign of systemic toxicity. Applicant assessment: palatability of the test item could be considered responsible for this observation.
Mortality:
no mortality observed
Description (incidence):
No mortalities occurred during the observation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treatment group individuals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was similar between treatment group individuals and controls over the study period.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food consumption during the study. Body weight gains in treatment group individuals was similar to that of controls over the study period.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined in treatment group individuals relative to and/or including that of controls over the study period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the haematological parameters examined at the end of the treatment period.

At 1000 mg/kg bw: statistically significant changes from controls included: lower red blood cell counts in males, higher reticulocyte counts in males and females, higher red cell distribution width (RDW) in males, lower haemoglobin level in males, lower haematocrit level in males and higher mean corpuscular haemoglobin concentration (MCHC) in males. All the changes remained within a change considered normal for rats of this strain and age.

Two control females (#22 and #25) showed notably lower white blood cell counts, attributable to lower lymphocyte counts. No apparent cause was found for these variations. This had no impact on study assessment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw: changes in clinical biochemistry parameters distinguished treated individuals from controls (statistically significant, unless indicated otherwise) were:
higher alanine aminotransferase activity (ALAT) in females (primarily due to female #36 and #40), higher aspartate aminotransferase activity (ASAT) for females #36 and #40 (mean not statistically significant), higher total protein level in females, higher cholesterol level in females, lower chloride level in males and females, higher inorganic phosphate level in males.

Other changes included: higher total bilirubin level in males and females at 300 and 1000 mg/kg, higher creatinine level and lower glucose level in males at 100, 300 and 1000 mg/kg.

With the exception of the higher total bilirubin level at 1000 mg/kg in both sexes, all above changes either remained within or marginally exceeded the range of values considered normal for rats of this age and strain.

At 300 mg/kg bw: statistically significant lower aspartate aminotransferase activity and bile acid level in males were considered to be of no toxicological significance as these variations occurred in the absence of a dose -related trend and remained (essentially) within the range considered normal for rats of this age and strain.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the behavioural parameters or in sensory reactivity. There was no toxicologically relevant changes in functional performance.

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the in the organ weights examined at the end of the treatment period.

At 600 mg/kg bw/day dose level: males showed a statistically significant higher liver to body weight ratio. Liver weights of females were also slightly higher, but not statistically significant (and the mean was within the range considered normal). Heart to body weight ratio was also slightly lower in these females (statistically significant, but within the range considered normal). This was considered of no toxicological significance as there was no morphological correlates and observations were slight in nature.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in absolute organ weights and relative organ weights (organ to body weight ratio) were considered to be related to treatment:
At 1000 mg/kg bw: higher kidney weights and kidney to body weight ratio in males (relative kidney weights were increased with approximately 20%) ; plus higher spleen weight and spleen to body weight ratio in males (relative spleen weights were increased with approximately 28%).

At 1000 and 300 mg/kg bw: higher liver weights and liver to body weight ratio in males and females (relative liver weights were increased with approximately 10% and 37% (males) or 24% and 57% (females) at 300 and 1000 mg/kg, respectively).

Statistically significantly lower relative prostate weight at 300 mg/kg and heart weight of females at 100 mg/kg occurred in the absence of a dose-related trend, and means remained within the range considered normal for rats of this age and strain. No toxicological relevance was therefore ascribed to these changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment related effects were observed:
- Liver:
Hypertrophy, centrilobular to diffuse, hepatocellular was present in 5/5 males (1 minimal, 3 slight, 1 moderate) and in 5/5 females treated (1 minimal, 3 slight, 1 moderate) at 1000 mg/kg. This was present in 5/5 males treated at 300 mg/kg (all minimal), but the minor degrees of diffuse/centrilobular hypertrophy in the liver in males at 300 mg/kg bw may be regarded as an adaptive physiological response to treatment. At 1000 mg/kg bw, higher severities, accompanied by other indicators of liver. damage such as necrosis and vacuolation, are considered adverse.
Coagulative necrosis was present in 1/5 males (minimal) and 2/5 females (1 minimal, 1 slight) treated at 1000 mg/kg bw only.
Hepatocellular vacuolation was present at slightly increased incidence and/or severity in 2/5 males (2 slight) and 3/5 females (1 minimal, 2 slight) treated at 1000 mg/kg bw compared to a minimal background degree in 1/5 treated male, 1/5 treated female at 300 mg/kg bw and 1/5 control.

- Kidneys:
Hyaline droplets were present at increased severity in 5/5 males: 1000 mg/kg bw treated males (4 moderate, 1 marked) and at 300 mg/kg bw (3 slight, 2 moderate), compared to background degrees in 2/5 control (1 minimal, 1 slight) and 100 mg/kg bw (2 minimal, 3 slight) treated males.
Cortical hyaline droplets are considered to represent alpha2uglobulin, which is a male rat-specific lesion. The alteration was not accompanied by an increase in indicators of tubular damage or any other renal damage. Therefore this is not considered an adverse lesion.

- Spleen:
Hemopoietic foci, primarily erythropoiesis was present at increased severity in 5/5 males (2 slight, 3 moderate) treated at 1000 mg/kg bw, compared to minimal degrees in 5/5 control, 3/5 at 100 mg/kg bw and 5/5 at 300 mg/kg bw treated males. For females the incidence and severity was within the normal background range.

- Bone marrow, sternum:
Erythroid hyperplasia was present at minimal degree in 3/5 males and 4/5 females treated at 1000 mg/kg bw.

- Colon:
Hypertrophy villous epithelium, accompanied by an inflammatory cell infiltrate and an increase in mitotic figures, was present in 4/5 males (3 slight, 1 moderate) treated at 1000 mg/kg bw.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities detected. There were no microscopic findings that were considered to be related to treatment with the test item.
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 300 mg/kg body weight per day.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7 and US EPA OPPTS 870.3050 guidelines under GLP conditions. Following a previously conducted 5-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day oral gavage study in Wistar: Crl:WI(Han) rats. Three groups, each comprising five male and five female rats, received test item at doses of 100, 300 or 1000 mg/kg bw/day/day. A control group of five males and five females was dosed with vehicle alone (Propylene Glycol 400). Chemical analyses of formulations were conducted once during the study treatment phase to assess accuracy, homogeneity and stability over 6 hours. Separately, dedicated formulation analyses confirmed that formulations of test item in propylene glycol 400 were prepared accurately and homogenously, and were stable over at least 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. At 1000 mg/kg bw/day, adverse findings consisted of centrilobular to diffuse hepatocellular hypertrophy in both sexes (up to moderate degree), with coagulative necrosis (up to slight degree), a slightly increased incidence and/or severity of hepatocellular vacuolation (up to slight degree), higher liver weights in both sexes (relative liver weights were increased with approximately 37% (males) or 57% (females)), and enlargement and/or an accentuated lobular pattern of the liver in all animals. These liver findings were considered to be related to the observed changes in clinical biochemistry parameters at 1000 mg/kg bw/day including higher alanine/aspartate aminotransferase activity and higher total protein and cholesterol level in females, higher total bilirubin level in males and females, and lower glucose level in males. The increased severity of hemopoietic foci (primarily erythropoiesis) in the spleen of males (up to moderate degree), enlargement of the spleen in most males and higher spleen weight (relative spleen weights were increased with approximately 28%), and erythroid hyperplasia of the sternal bone marrow in both sexes (at minimal degree) were indicative of extramedullary haematopoiesis, and supported the (relatively slight) haematological changes in males at 1000 mg/kg bw/day, consisting of higher reticulocyte counts, red cell distribution width and mean corpuscular haemoglobin concentration , and lower red blood cell counts, haemoglobin and haematocrit level. Females at 1000 mg/kg bw/day also showed higher reticulocyte counts, but in the absence of any supportive changes in other red blood cell parameters or concurrent histopathological changes, this was considered not toxicologically relevant in females. Histopathology additionally showed villous hypertrophy of the colon epithelium in males at 1000 mg/kg bw/day (up to moderate degree). The increase in severity of cortical hyaline droplets in all male kidneys at 1000 mg/kg bw/day (up to marked degree) is considered to represent alpha2uglobulin. This is a male rat specific lesion which is not observed in female rats and is considered to be of no relevance to humans. This finding occurred in conjunction with greenish discolouration of both kidneys in several males, higher kidney weights (relative kidney weights were increased with approximately 20%), and higher creatinine and inorganic phosphate level. Overall, it was considered that the renal findings at 1000 mg/kg bw/day should be considered adverse in nature based on the combination of histopathology findings with higher kidney weights and accompanying clinical biochemistry changes. Other changes in clinical biochemistry parameters at 1000 mg/kg bw/day that could be related to the above histopathological lesions consisted of higher total bilirubin level in males and females, lower glucose level in males, higher cholesterol level in females, and lower chloride level in males and females. All findings observed at 100 and 300 mg/kg bw/day were considered not to be adverse in nature as they were generally minor in nature (i.e. within the range considered normal for rats of this age and strain), showed no clear dose-related response and/or had no correlating adverse histopathological lesions. No toxicologically significant changes were noted in any of the other parameters investigated in this study (i.e. mortality, clinical appearance, functional observations, body weight and food consumption). Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 300 mg/kg bw/day bw/day for males/females, excluding the considered male rat-specific kidney response relating to alpha2uglobulin, which was not considered relevant to human health risk assessment.