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REACH

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

EC number: 309-912-6 | CAS number: 101357-15-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

28-Day Study: Wragg & Brooks (1994)

A fully compliant 28-d toxicity study is available performed according to OECD guideline 407 (1995). Toxicologically significant effects were observed at a dose level of 1000 mg/kg/day but not at the lower dose levels of 150 mg/kg/day and 15 mg/kg/day.

90-Day Study: Oroszlány (2019)

Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test material is considered to be 300 mg/kg bw/day for both sexes.

The NOAEL of 300 mg/kg bw is taken forward as the dose descriptor since it is derived from a longer-term study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 15 September 1993 - 13 October 1993
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent
- Age at study initiation: approximately five to six weeks old
- Weight at study initiation: males 136 to 162 g; females 121 - 150 g
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, U.K.) was used.
- Water (e.g. ad libitum): ad libitum. Mains water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 45 - 75 % relative
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: To: 15 September 1993 - 13 October 1993
Route of administration:: oral: gavage
Vehicle:: arachis oil
Details on oral exposure:: The test material was prepared at the appropriate concentrations as a suspension in Arachis oil B.P. The stability and homogeneity of the test material formulations were determined analytically and showed the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4 °C in the dark.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
The test material was administered at a treatment volume of 4 mL/kg in the vehicle.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The Nigrosine Base Ex concentration in the test samples was determined spectrophotometrically.

Samples
The test material formulations were dilute with acetone such that the final theoretical test material concentration was approximately 0.015 mg/mL.

Standards
Standard solutions were prepared in acetone at a nominal concentration of 0.015 mg/mL.

Procedure
The absorbance of the standard and sample solutions was measured at 554 nm in 10 mm cells using acetone as the reference medium.

Homogeneity Determinations
The test material formulations were mixed thoroughly and the samples were taken from the top, middle and bottom of the container, shaking between sampling. The sampling was performed in triplicate.

Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately 4 °C in the dark for 10 days.

Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within three days of preparation.

The results indicate that the prepared formulations were within ± 10 % of the nominal concentration.
Duration of treatment / exposure:: 28 days
Frequency of treatment:: Once daily
Dose / conc.:: 15 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:: 5 animals per sex per dose
Control animals:: yes, concurrent vehicle
Details on study design:: The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil B.P.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Observations and examinations performed and frequency:: Clinical Signs
All animals were examined daily for overt signs of toxicity, ill-health or behavioural change.

Bodyweight
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at necropsy.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 29). Blood samples were obtained from the lateral tail vein. A few repeat samples were also obtained by cardiac puncture prior to necropsy. Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Total leucocyte count (WBC)
Differential leucocyte count
Platelet count (PLT)
Erythrocyte indices: mean corpuscular haemoglobin (MCH)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin concentration (MCHC)
Reticulocyte counts (Retic)
Methaemoglobin (Meth)

Clotting time (CT) was assessed by Hepato Quick time using samples collected into sodium citrate solution (0.11 mo1/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Blood urea Inorganic phosphorus
Total protein Creatinine
Albumin Alkaline phosphatase (AP)
Albumin/globulin ratio (by calculation) Alanine aminotransferase (ALAT)
Sodium Aspartate aminotransferase (ASAT)
Potassium Glucose
Chloride Total bilirubin
Calcium
Sacrifice and pathology:: Pathology
On completion of the dosing period all animals were killed by intra-venous administration of sodium pentobarbitone solution (Sagatal, 60 mg/mL) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation:
Adrenals Brain
Gonads Heart
Kidneys Liver
Pituitary Spleen

Histopathology
Samples of the following tissues were removed from all animals and preserved in 10 % buffered formalin:

Adrenals Jejunum Salivary glands
Aorta (thoracic) Kidneys Sciatic nerve
Bone & Bone Marrow (femur) Liver Seminal vesicles
Bone & Bone Marrow (sternum) Lungs Skin (hind limb)
Brain Lymph nodes (cervical and mesenteric) Spleen
Caecum Muscle (skeletal) Stomach
Colon Oesophagus Testes
Duodenum Ovaries Thymus
Eyes Pancreas Thyroid/parathyroid
Gross lesions Pituitary Trachea
Heart Prostate Urinary bladder
Ileum Rectum Uterus

The following preserved tissues from all test and control group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm
and stained with haematoxylin and eosin.
Adrenals Testes
Spleen Mesentery
Heart Kidneys
Liver

Macroscopically observed lesions were also processed.
Initially microscopic examination was performed on control and high dose tissues only, but since there were indications of treatment-related hepatic and splenic changes, examination was subsequently extended to include sections of liver and spleen from all animals in the remaining dose groups.
Statistics:: Evaluation of Data
Data were processed to give group mean values and standard deviations where appropriate.
Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating ‘F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)
Clinical signs:: no effects observed
Description (incidence and severity):: There were no deaths during the study. Animals showed no clinically observable signs of toxicity during the study.
Mortality:: no mortality observed
Description (incidence):: There were no deaths during the study. Animals showed no clinically observable signs of toxicity during the study.
Body weight and weight changes:: no effects observed
Description (incidence and severity):: All animals showed normally expected bodyweight development. Animals treated with the test material showed similar bodyweight gains to controls during the study.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: There was no adverse effect on food consumption during the study.
Food efficiency:: no effects observed
Description (incidence and severity):: Animals treated with the test material showed similar weekly food efficiency to controls.
Water consumption and compound intake (if drinking water study):: no effects observed
Description (incidence and severity):: Daily visual inspection of water bottles revealed no intergroup differences in water consumption.
Ophthalmological findings:: not examined
Haematological findings:: effects observed, treatment-related
Description (incidence and severity):: See below.
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: See below.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: See below.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: See below.
Histopathological findings: neoplastic:: not examined
Details on results:: Clinical signs:
Dark faeces were evident at all three treatment levels from Day 3 onwards; this is a common finding following oral administration of a dark-coloured test material to rats and is not indicative of toxicity.

Haematology:
Group mean values and standard deviations for test and control group animals are given in Tables 1 and 2 (statistically significant differences are indicated). High dose animals of both sexes showed a slight but statistically significant reduction in haemoglobin concentration and erythrocyte count compared with controls, and a concomitant increase in the mean corpuscular volume (MCV). Haematocrit was also reduced in these animals, although the intergroup difference failed to achieve statistical significance for the females.This was probably due to an increase in the relative number of reticulocytes in the blood, particularly in the females, although there was no clear evidence of reticulocytosis in the males at this dose level. In addition, high dose females showed a slight but statistically significant increase in mean corpuscular haemoglobin, together with a reduction in mean corpuscular haemoglobin concentration. These findings, taken with histopathological evidence of splenic haemosiderosis and splenic haematopoiesis, are consistent with haemolysis, although the animals could not be considered clinically anaemic. The changes in haematological parameters observed were largely within, or only marginally outside, the historical control ranges. A treatment-related methaemoglobinaemia was also identified at the high dose level with animals of both sexes showing a slight but statistically significant increase in the relative amount of methaemoglobin present in the blood compared with controls.No treatment-related haematological changes were detected at the remaining dose levels. High dose males showed a slight increase in neutrophils compared with controls but none of the individual values were abnormally high for rats of the strain and age used in this study and, in the absence of any convincing increase in the total leucocyte count, this finding was considered not to be toxicologically important. Intermediate and high dose males also showed a slight but statistically significant increase in clotting time compared with controls but again none of the individual values were abnormally high for rats of the strain and age used in this study and the intergroup differences were considered to be entirely due to lower than expected control values.

Blood Chemistry:
There were no treatment-related effects on the blood chemical parameters measured.Intermediate and high dose males showed a slight but statistically significant increase in plasma creatinine concentration compared with controls. None of the individual values were abnormally high for rats of the strain and age used in this study and, in the absence of any histopathological evidence of renal changes, these findings were considered not to be toxicologically important.The remaining statistically significant intergroup differences were confined to intermediate dose animals. These findings were not dose-related and, as such, were considered not to be treatment-related.

Necropsy:
All intermediate and high dose animals had abnormally pink adipose tissue at terminal kill.No macroscopic abnormalities were evident at the low dose level.Intermediate and high dose animals of either sex had black stomach contents at necropsy and several of these animals also had a stained non-glandular gastric region. Such findings are common when a dark-coloured test material is administered to rats, by gavage, and are not attributable to test material toxicity.

Organ Weights:
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Tables 3 to 6 (statistically significant differences are indicated). High dose animals of either sex showed a statistically significant increase in spleen weight, both absolute and relative to body-weight, compared with controls. High dose females also showed a slight but statistically significant increase in relative liver weight.No treatment-related organ weight changes were detected at the remaining dose levels.The remaining statistically significant intergroup differences were confined to low dose females. These findings were not dose related and, as such, were considered not to be treatment-related.

Histopathology:
A summary incidence table of histopathological findings is given In Table 7. Treatment-related hepatic and splenic changes were observed:
Liver: Periportal hepatocyte basophilla was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day.
Spleen: Increased severities of extramedullary haemopoiesis and haemosiderin pigment deposition were reported in relation to treatment for rats of either sex receiving 1000 mg/kg/day.All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Discussion:
The test material induced a methaemaglobinaemia in animals of bothr sexes dosed at 1000 mg/kg/day. In addition, haematological determinations at this dose level showed effects, when taken together with the splenic extramedullary haemopoiesis, and the increased splenic haemosiderosis (likely responsible for the increased spleen weights at this dose), suggest a chemically induced haemolysis. The nature of the haemolysis and methaemoglobinaemias is such that they are normally reversible following cessation of treatment with the causative agent. Furthermore, there was no evidence that these haematological changes indicative of an adverse process (haemolysis) were severe enough to cause hypoxic damage to tissues in the rat or any other outward in-life signs of anaemia, which were offset by increased haematopoiesis in these animals. Both haemolysis and methaemoglobinuria are both known to be caused by components used to manufacture the test article, through redox cycling (oxidative stress). The increased relative liver weight detected for females treated with 1000 mg/kg/day was probably associated with the periportal hepatocyte basophilia identified histopathologically, even though the microscopic changes were present in animals of either sex. Despite these liver changes there was, however, no convincing evidence of liver dysfunction at this dose level. The aetiology of the remaining treatment-related change at this dose level, the abnormally pink adipose tissue seen at terminal kill, is unclear at present. The abnormal discolouration was possibly due to accumulation of haemosiderin pigment or methaemoglobin in the adipose tissue but this is considered unlikely, especially as both substances typically discolour tissues brown and histopathological examination of adipose tissue failed to identify the presence of any pigment. It is more likely therefore that such discolouration results from bioaccumulation of test material metabolite(s) in the adipose tissue which, in the absence of any morphological changes, is of little toxicological concern.
Dose descriptor:: NOEL
Effect level:: 15 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: see 'Remark'
Dose descriptor:: NOAEL
Effect level:: 150 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: see 'Remark'
Critical effects observed:: not specified
Conclusions:: Repeat-dose toxicity: a fully compliant 28-d toxicity study is available (Wragg MS and Brooks PN 1994), performed according to OECD guideline 407 (1995).

The study established a NOAEL at 150 mg/kg/day, because treatment at the limit dose of 1000 mg/kg/day caused haematological effects, changes in spleen weight (and relative liver weight in females only) and histopathological changes in the spleen and liver consistent with methaemoglobin formation and haemolysis, although the animals did not become anaemic.
Executive summary:: Repeat-dose toxicity: a fully compliant 28-d toxicity study is available (Wragg MS and Brooks PN 1994), performed according to OECD guideline 407 (1995).

The study established a NOAEL at 150 mg/kg/day, because treatment at the limit dose of 1000 mg/kg/day caused haematological effects, changes in spleen weight (and relative liver weight in females only) and histopathological changes in the spleen and liver consistent with methaemoglobin formation and haemolysis, although the animals did not become anaemic.

Methaemoglobinaemia and haemolysis are known to be caused by aniline and nitrobenzene which are used to make nigrosine. Aniline is a documented component of Nigrosine (Section 1). Aniline and nitrobenzene redox cycle and their presence in erythrocytes results in the reduction haemoglobin to methemoglobin, and also oxidative stress resulting damage to, and ultimately, lysis of the erythrocyte (hemolysis). Once exposure ceases, and the responsible chemical species are cleared from the body, full recovery is made from the methemoglobinaemia and hemolysis. It is important to note that exacerbation of methaemoglobin formation, and sequalae thereof, does not occur with increased duration of dosing. For further information see Kiese, 1996 .Muller et al 2006, Stolk and Smith 1966, ECB 2004.

The criteria for labelling for specific organ toxicity (STOT) under the CLP Regulation are not met. However humans are known to be more sensitive than rats to these mechanisms resulting in methemglobinaemia and hemolysis, and specific consideration was given to the classification and labelling for hemolysis by the EU Working Group on Haemolytic Anaemia (Muller et al, 2006). The findings of the working group were accepted by the European Commission in 2004. The haemolysis observed in Wragg and Brooks does not trigger the criteria set out in Muller et al (2006) where classification for specific organ toxicity would be reguired. In addition interspecies assessment factors would also accommodate the differences in sensitivity when deriving DNELs, and there were no effects in the endpoints measured in the developmental toxicity study up to the limit dose of 1000 mg/kg. The only change noted at 150 mg/kg/day was abnormal discolouration of adipose tissue, considered likely to represent presence of test substance metabolite(s).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2018 to 28 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test material in the solvent/vehicle: The test material was formulated at appropriate concentrations in the vehicle (corn oil) up to 4 days before use (formulations were kept closed, at room temperature until use when stored). Stability of the test material in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 10 - 250 mg/mL concentration range were proven as being stable for at least 5 days when stored at room temperature.
Analysis of test material formulations for concentration and homogeneity was performed using an HPLC-UV method. Top, middle and bottom duplicate samples were taken from test material formulations four times during the study (during first, fifth, ninth and last week of the treatment), one set to analyse and one set as a back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI Wistar

Details on species / strain selection:

The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, approx. 6 weeks old at start of treatment.
- Weight at study initiation: Males: 183 - 218 g, females: 155 - 181 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Housing: Type II and/or III polycarbonate cages. Rodents were housed 2 or 3 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 5 - 6 days.

DETAILS OF FOOD AND WATER QUALITY:
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice - breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 840 33675, expiry date: 31 January 2019 and batch number: 639 38520, expiry date: 30 April 2019). The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificate for the batch used, which is archived with the raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are retained with the raw data in the archives at Citoxlab Hungary Ltd.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 23.6 °C
- Humidity (%): 25 - 73 %
- Air changes (per hr): 15 - 20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): 12 hours light daily, from 6.00 a.m. to 6.00 p.m.
- The temperature and relative humidity were recorded twice daily during the study and acclimation period.

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered daily starting from Day 0 for 91 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 4 mL/kg bw/day was administered to all animals. The actual volumes to be administered were calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test material was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation).

- VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected for vehicle of the study by the Sponsor based on the formulation and analytical trials.
- Concentration in vehicle: The formulation samples were prepared in the 10 - 250 mg/mL concentration range.
- Amount of vehicle: A constant dose volume of 4 mL/kg bw/day was administered.
- Lot/batch no.: A0395699/A0386839

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material content and homogeneity of the dosing formulation samples were determined on four analytical occasions during the test.
Analytical samples were taken from each test concentration to determine concentration and homogeneity and from the control for concentration measurement.
The samples were properly diluted with eluent into the calibrated range then analysed by an HPLC with UV detection method.

- Instrumentation:
HPLC: Knauer Azura HPLC-UV system
Column: ACE 5 C18, 150×4.6 mm, 5 µm
Column temperature: 25 °C
Mobile Phase: Acetonitrile/buffer = 6/4
Buffer: 0.05 M KH2PO4 pH 2.6
Flow: 1 mL/min
Detector (UV): 290 nm
Injection volume: 20 µL

Method Validation Results
Selectivity: No interfering component was observed
Reinjection repeatability (7 injections): RSD% ≤ 1.4%
Linear range: 2.5 - 100 µg/mL
Limit of Quantification (LOQ): 2.5 µg/mL
Recovery of the test material from corn oil (10 and 250 mg/mL): 95 and 98 %
Precision in corn oil: 1.0 and 0.9 %
Stability of test material in corn oil (5 days at room temperature and 2-8 °C): 103 and 103 %
Stability of the samples in the autosampler: At least 116 hours
Stock solution stability at 5 ± 3 °C: At least 5 days

Measured Data
Calibration:
Data of regression lines
August 2018: Intercept: -0.0926, Slope: 3.18, Correlation Coefficient: 1.0000
September 2018: Intercept: 0.493, Slope: 2.98, Correlation Coefficient: 0.9997
October 2018: Intercept: 0.061, Slope: 2.99, Correlation Coefficient: 1.0000
21 November 2018: Intercept: -0.628, Slope: 2.80, Correlation Coefficient: 1.0000

Results of the Analysis
All the formulations proved to be homogeneous. Acceptance criteria for homogeneity is that the relative standard deviation (RSD) of the replicates must be less than 10 %

Duration of treatment / exposure:

91 days

Frequency of treatment:

The dose formulations were administered daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 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:

10 animals/group/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data provided by the Sponsor. Previously, a 28-day sub-acute oral (gavage) toxicity study was conducted with the test material dissolved in arachis oil with Sprague-Dawley CD strain rats. In this study, the NOEL was set to be 15 mg/kg bw/day. Change in the colour of the adipose tissue was present in the 150 mg/kg bw/day group, and was considered not to be an adverse change, therefore the NOAEL in this study was set at 150 mg/kg bw/day. At 1 000 mg/kg bw/day, in both sexes, reduction in haemoglobin concentration, erythrocyte count, and haematocrit was present when compared to control, as well as an increase in the mean corpuscular volume. In female animals at this dose, an increase in the relative number of reticulocytes and a slight reduction in the calculated mean corpuscular haemoglobin concentration was also present, as well as a slight increase in mean corpuscular haemoglobin. Related to these haematology changes, statistically significant increase in the spleen weight (both sexes), and statistically significant increase of the relative liver weight (females) was also present, and the correlating histopathological changes were periportal hepatocyte basophilia together with an increased severity of both splenic extramedullary haemopoiesis and haemosidering pigment deposition in the spleen.
Based on the results from the above study, doses of 100, 300 and 1 000 mg/kg bw/day were selected for the main study. The aim was to use a maximum of 1 000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
The oral route was selected as it is one of the possible routes of human exposure.
- Rationale for animal assignment: During the acclimation period, the animals were assigned to their respective dose groups by randomisation based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made at least once a day at approximately the same time with minor variations as practical.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on Day 0 male/female) and weekly thereafter, in the morning hours (am) and once before necropsy.
- Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc.), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 90, and prior to necropsy, fasted on Day 91.

FOOD CONSUMPTION:
- The determination of food consumption was performed for all groups once a week.
- The food was measured on Day 0 then the remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Daily food consumption was calculated for reporting purposes.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Ophthalmoscopic examination was conducted in all animals before treatment (Day -1 or -2), and in the Control group and High dose group animals, during Week 12 (Day 85/86).
- Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride) into the conjunctival sac. The evaluation was performed using an Omega 500 ophthalmoscope. As in Week 12 no treatment related alterations were found in the Control group and High dose group animals, the remaining animals were not examined at termination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations were conducted in all animals.
- Anaesthetic used for blood collection: Yes, pentobarbital anaesthesia.
- Animals fasted: Yes. Overnight period of food deprivation of animals.
- How many animals: All animals. Three blood samples were collected by heart puncture in tubes with K3-EDTA as anticoagulant, (1.6 mg/mL blood) and for blood clotting times (in tubes with sodium citrate as anticoagulant).
- Parameters examined: Red blood cell (erythrocyte) count, (10^12/L) M/µL and white blood cell (leukocyte) count, (10^9/L) K/µL using automatic laser cell count; haemoglobin concentration, (g/dL) by determination of cyan-methemoglobin absorbance; haematocrit (relative volume of erythrocytes) (%) (computed by equipment); mean corpuscular (erythrocyte) volume (fL) by laser cell volume determination; mean corpuscular (erythrocyte) haemoglobin, (pg) (computed by equipment); mean corpuscular (erythrocyte) haemoglobin concentration, (g/dL) (computed by equipment); red cell (erythrocyte) volume (%) by distribution width laser detection; platelet (thrombocyte) count, (10^9/L) K/µL by automatic laser cell count; mean platelet thrombocyte volume (fL) cell volume determination by laser; % reticulocyte count (%) (comparative value based on laser light detection); neutrophil (%), lymphocyte (%), monocyte (%), basophil (%), eosinophil (%) and large unstained cells (%) were determined by cell differentiation based on myeloperoxidase activity; activated partial thromboplastin time (sec) and prothrombin time (sec) (Quick method (Biggs, R. and R.G. MacFarlane).
Blood smears were prepared for all animals but not examined, as the standard haematology evaluation was considered to be adequate.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations were conducted in all animals.
- Animals fasted: Yes. Overnight period of food deprivation of animals.
- How many animals: All animals.
- Parameters examined: Glucose blood sugar concentration (mmol/L) by colorimetric test (540 nm); total bilirubin concentration (μmol/L) by end-point colorimetric (dual-wavelength) test (400 & 460 nm); urea concentration (mmol/L) by colorimetric test (670 nm); cholesterol concentration (mmol/L) by colorimetric test (540 nm); creatinine concentration (μmol/L) by two-point rate test (670 nm); phosphorus concentration (mmol/L) by colorimetric test (680 nm); sodium concentration (mmol/L) and potassium concentration (mmol/L) by potentiometric test; calcium concentration (mmol/L) by colorimetric test (680 nm); chloride concentration (mmol/L) by potentiometric test; total protein concentration (g/L) by colorimetric test (540 nm); albumin concentration (g/L) by colorimetric test (630 nm); alb/glob ratio (calculated value); aspartate aminotransferase activity (U/L) and alanine aminotransferase activity (U/L) by multiple-point rate test (340 nm); gamma-glutamyl transferase activity (U/L) by γ-glutamyl-p-nitroanilide + glycylglycine. Increase in p-nitroaniline-monitored at 400 nm; alkaline phosphatase activity (U/L) by multiple-point rate test (400 nm) and bile acids (µmol/L) by colorimetric test (546 nm).

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations were conducted in all animals.
- Metabolism cages used for collection of urine: Yes. Urine collection was conducted over approximately 16 hours in metabolic cages.
- Animals fasted: Yes. Food was withdrawn during the overnight urine collection.
- Parameters examined: The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable. The following parameters were evaluated in all animals: Leukocyte, nitrite, pH, protein, glucose, urobilinogen, bilirubin, ketones, blood/erythrocytes and specific gravity using Medi-Test Stick 10; sediment by microscopic examination, volume by volumetric method and colour/appearance by observation.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the treatment period, during Week 11/12.
- Dose groups that were examined: All animals were examined in the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.
- Battery of functions tested: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were painted for any possible additional measurements.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test material. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.
- After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. In addition, bone marrow smears from the femur of each animal were prepared at necropsy. The smears were fixed, then stained but not analysed.
- Organ weight measurement: The following organs were trimmed of fat and weighed in all animals:
With precision of 0.01g: Brain, epididymides, heart, kidneys, liver, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, and uterus including cervix.
With precision of 0.001g: Adrenal glands, ovaries, thyroid with parathyroid glands and pituitary.
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.

HISTOPATHOLOGY: Yes. On completion of the macroscopic examination the following tissues and organs were retained from all animals: Adrenals, animal identification (1), aorta (10), brain (2), epididymis, eye with the optic nerve (7), oesophagus, femur with marrow, heart (3), kidney, large intestine (4), extraorbital lachrymal gland, harderian gland, liver (12), lungs with bronchi (5), lymph node (6), ovary, oviduct, pancreas, pituitary, prostate, salivary gland (including mandibular, sublingual, and parotid glands), sciatic nerve, seminal vesicle with coagulating gland, skin, subcutis with mammary gland (inguinal), skeletal muscle (quadriceps), small intestine (8), spinal cord (11), spleen, sternum with marrow, stomach, testis, thymus, thyroid with parathyroid gland (7), tongue, trachea, urinary bladder, uterus (9), and vagina.
(1) Fixation and preservation only.
(2) 7 section according to the STP recommendations.
(3) Section including both ventricles and atria, septum with papillary muscle.
(4) Caecum, colon and rectum.
(5) Lungs of euthanized animals were infused with formalin; 3 lobes, left, right cranial, right caudal.
(6) Mandibular and mesenteric.
(7) If applicable, parathyroid glands and optic nerves were examined histologically only if present in routine sections.
(8) Duodenum, ileum and jejunum with Peyer’s patches.
(9) Horns, body and cervix.
(10) Aorta thoracic and abdominal
(11) Transverse sections, 3 levels - cervical, thoracic and lumbar.
(12) Liver, 3 lobes, left lateral, right medial, caudate.
- The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
- The retained tissues and organs were embedded in paraffin wax, sections were cut at 4 - 6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose). In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination from all groups.

Other examinations:

Examination of Vaginal Smears
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Statistics:

Data were collected using the software PROVANTIS v.9. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brownish black faeces were observed in all animals for all test material related groups from Day 2 (Mid and High dose animals), and from Day 3 (Low dose group). There was no evidence of other abnormalities in the faeces, hence it is considered that the presence of test material is probably the reason for the observation; it is not considered as an adverse effect.
In one Mid dose female animal (animal number 3507) a nodule was observed on Day 91 on the left inguinal region of the animal. Considering the isolated occurrence this finding is regarded as an incidental finding.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The bodyweight values of the test material treated groups did not show any test material related effect.
Significantly higher body weight gain values were observed for some occasions and for the whole dosing period in the Low dose male and High dose female groups. These observations were considered to be incidental, and not considered to be an adverse effect. The bodyweight gain values of the Mid dose group did not show any test material related effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase of food consumption for certain measurement windows, reaching up to 13.5 %, but on average, just below 10 % was observed in all test material treated male groups, and in the Low and High dose female groups. A statistically significant decrease of the food consumption was observed on one occasion for the Mid dose female group (-6.1 %, in the Day 63-70 period). These changes were considered as differences unrelated to treatment hence not a test material related adverse effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test material related changes compared to pre-treatment were noted at ophthalmoscopy examination.
In one Control dose female (animal number 1501), moderate haemorrhage in the corpus vitreum of the left eye was observed on Day 85. This finding was considered to be incidental.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decreases, reaching statistical significance, of red blood count in the Mid and High dose group males and High dose females, and haemoglobin concentration in both sexes at the High dose (not reaching statistical significance in the males) were recorded. However, the differences were all < 6 % difference and considered to be of little biological significance. Reticulocytes were increased with biologically significant differences, by about 40 % and 25 % in High dose males and females, and higher without statistical significance by about 9 % in Mid dose males and females. The haematological changes are compatible with an almost fully compensated anaemia, correlated with the organ weight and histopathological findings. This was considered to be a test material related adverse effect in the High dose in both sexes; a similar trend was seen in the Mid dose but the small differences indicate any differences were not adverse.

No other test material-related changes were observed in the haematology parameters. Statistically significant difference of the platelet count in the Mid dose males was considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an adverse effect of the test material.

The coagulation parameters were considered normal in all study groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A few clinical chemistry parameters were significantly different in the Mid and High dose groups. Generally, there was no dose response or consistency between the sexes. Higher Bilirubin or Bile acids may be related to treatment at the High dose, but the differences relative to the historical control range indicates that these are not adverse changes.

In the Low dose groups there were no statistical differences compared to the Control.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test material related effects were noted on the urinalysis parameters evaluated.

The urinalysis parameters were comparable to the concurrent or historical controls in all test treated groups. The pH in males only showed statistical increases, but the control values were lower than other recent 90 day rat studies, the High dose values are considered to be normal. Other variations occurred and consisted of minor differences to controls, these were unrelated to treatment and considered to be normal.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A dose-dependent increase in the spleen weight was observed in both sexes. The increase reached statistical significance in the High dose males (relative to body weight), and in the High dose females (absolute, relative to body weight, and relative to brain weight). The effect was confirmed at histopathology, and considered to be test material related.
Apart from the spleen, all other statistically significant weight differences were considered to be incidental and not test material-related or ascribed to differences in body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test material-related pink discolouration of the adipose tissue was observed in all High and Mid dose animals and 5 out of 10 Low dose males and females. The digestive content in the stomach was black in some of the dosed animals, correlated with the colour of the test material. The mesenteric lymph node was grey in 3 out of 10 High dose males as well.
These findings were considered to be related to the colour of the test material (or possibly its metabolites) with no histopathological evidence of change, hence with no toxicological consequences.

Other individual findings, such as focal dark red discolouration of the eye, kidney and pulmonary lymph nodes, and dilated uterine horn and body were considered as incidental or background. In one Mid dose female, an incidental firm mass in the subcutis was observed.

Neuropathological findings:

no effects observed

Description (incidence and severity):

The detailed neurological evaluations made near the end of the study, as per OECD guidelines, showed the following neurological effects:
At the functional observation battery (FOB) performed at the end of exposure (Week 11/12), there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.
No test material related effect was observed in locomotor activity measured at the end of the study.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test material-related microscopic findings were seen in the spleen, stomach and jejunum of the High dose groups.

Minimal to mild splenic extramedullary haematopoiesis, correlated with organ weight changes and haematology data was observed in 9 out of 10 males and 4 out of 10 females in the High dose. The same finding with lower severity and incidence was present in 3 out of 10 Control males. The extramedullary haematopoiesis was considered to be a test material related effect.

Black foreign material (test material) was seen in the lumen of the stomach and jejunum (adhered to the mucosal surface). These findings, correlating with the macroscopic findings, were considered to be due to the presence of the coloured test material.

Individual alterations such as tubular basophilia, chronic progressive nephropathy (CPN), hyaline casts and mineralisation in the kidney, haemorrhage in the eye, harderian metaplasia in the lachrymal gland, vacuolation of the hepatocytes, inflammation of the prostate, tubular dilatation in the testes, congestion/haemorrhage in the lung-associated lymph nodes, and oestrus in the uterus, based on low incidence, the occurrence and/or distribution in Control and dosed groups, were considered as incidental or background.

Similarly, an adenocarcinoma in the subcutis (inguinal area) in a single Mid dose female (observed as a nodule in clinical signs and as a firm mass at necropsy) was considered to be an incidental finding unrelated to treatment.

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

Dose Formulation Analysis

All test material formulations were shown to be homogeneous. The measured concentrations of the test material evaluated for each test material-dose group varied between 95 % and 109 %. No test material was detected in the control samples. These results were within the acceptable ranges (85 % - 115 %) and were considered suitable for the study purposes.

Based on the study sample analysis, and the validation date, the concentration, homogeneity and stability of the dose formulations were considered to be fully satisfactory.

Grip Strength Means: Males

Grip strength (males)	Group /Concentration (mg/kg bw/day)
Grip strength (males)	Control (0)	Low (100)	Mid (300)	High (1000)
Forelimbs (g)	1389	1477	1394	1342
	Differences from control	6.3%	0.4%	-3.4%
	Historical control data	1397 – 2438
Hind limbs (g)	594	596	556	576
	Differences from control	0.4%	-6.4%	-3.1%
	Historical control data	286 – 989

Grip Strength Means: Females

Grip strength (females)	Group /Concentration (mg/kg bw/day)
Grip strength (females)	Control (0)	Low (100)	Mid (300)	High (1000)
Forelimbs (g)	1145	1062	1142	1120
	Differences from control	-7.3%	-0.3%	-2.2%
	Historical control data	1043 – 1765
Hind limbs (g)	430	463	413	467
	Differences from control	7.5%	-4.1%	8.6%
	Historical control data	189 – 824

Splay Test Means: Males

Splay test (males)	Group /Concentration (mg/kg bw/day)
Splay test (males)	Control (0)	Low (100)	Mid (300)	High (1000)
Distance between hindpaws (mm)	75	80	84	88
	Differences from control	6%	11.5%	16.2%
	Historical control data	33 – 133

Splay Test Means: Females

Splay test (females)	Group /Concentration (mg/kg bw/day)
Splay test (females)	Control (0)	Low (100)	Mid (300)	High (1000)
Distance between hindpaws (mm)	75	76	71	81
	Differences from control	1.7%	-5.1%	8%
	Historical control data	27 – 138

Haematology Parameters in Males

Haematology (males)	Group /Concentration (mg/kg bw/day)
Haematology (males)	Control (0)	Low (100)	Mid (300)	High (1000)
Red blood count (M/µL)	9.057	8.845	8.695*	8.690*
	Differences from control	-2.3%	-4.0%	-4.1%
	Historical control data	8.07 – 9.61
Haemoglobin concentration (g/dL)	14.72	14.71	14.13	14.13
	Differences from control	-0.1%	-4.0%	-4.0%
	Historical control data	13.6 – 16.8
Reticulocytes (%)	2.08	2.08	2.27	2.91^**
	Differences from control	0%	9.1%	39.9%
	Historical control data	1.4 – 3.2
= p<0.05, *= p<0.01; Dunnett two sided test, ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

Haematology Parameters in Females

Haematology (females)	Group /Concentration (mg/kg bw/day)
Haematology (females)	Control (0)	Low (100)	Mid (300)	High (1000)
Red blood count (M/µL)	7.996	7.828	7.880	7.523⁺
	Differences from control	-2.1%	-1.5%	-5.9%
	Historical control data	7.41 – 9.16
Haemoglobin concentration (g/dL)	14.08	13.74	13.65	13.28⁺⁺
	Differences from control	-2.4%	-3.1%	-5.7%
	Historical control data		13.4 – 16.3
Reticulocytes (%)	2.32	2.20	2.54	2.90*
	Differences from control	-5.2%	9.5%	25%
	Historical control data	1.4 – 5.8
= p<0.05, *= p<0.01; Dunnett two sided test, ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

Clinical Chemistry Parameters in Males

Clinical chemistry (males)	Group /Concentration (mg/kg bw/day)
Clinical chemistry (males)	Control (0)	Low (100)	Mid (300)	High (1000)
Potassium (mmol/L)	5.71	5.96	6.44*	6.36 *
	Differences from control	4.4%	12.8%	11.4%
	Historical control data	4.7 – 7.5
Total Bilirubin (µmol/L)	1.78	2.30	2.03	2.02
	Differences from control	29.2%	14.0%	13.5%
	Historical control data	2.8 – 7.4
Bile acid (µmol/L)	7.650	8.136	9.022	10.020 **
	Differences from control	6.4%	17.9%	31%
	Historical control data	5.53 – 35.56
= p<0.05, *= p<0.01; Dunnett two sided test, ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

Clinical Chemistry Parameters in Females

Clinical chemistry (females)	Group /Concentration (mg/kg bw/day)
Clinical chemistry (females)	Control (0)	Low (100)	Mid (300)	High (1000)
Total Bilirubin (µmol/L)	2.06	2.68	2.58	3.61 **
	Differences from control	30.1%	25.2%	75.2%
	Historical control data	2.6 – 20.0
Bile acid (µmol/L)	9.520	10.118	11.694	12.137
	Differences from control	6.3%	22.8%	27.5%
	Historical control data	8.14 – 35.55
= p<0.05, *= p<0.01; Dunnett two sided test, ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

Urinalysis Parameters in Males

Urinalysis (males)	Group /Concentration (mg/kg bw/day)
Urinalysis (males)	Control (0)	Low (100)	Mid (300)	High (1000)
pH	6.90	6.80	7.90⁺⁺	8.00⁺⁺
	Differences from control	-1.4%	14.5%	15.9%
	Historical control data	6.0 – 8.0
Specific gravity	1.0115	1.0130	1.0060	1.0050⁺
	Differences from control	0.1%	-0.5%	-0.6%
	Historical control data	1.003 – 1.020
Urine bacteria	1.5	2.0	2.9⁺⁺	2.4
	Differences from control	33.3%	93.3%	60.0%
	Historical control data	--
= p<0.05, *= p<0.01; Dunnett two sided test, ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

Spleen Organ Weights of the Male Animals

Organ weights (males)	Groups/Concentration (mg/kg bw/day)
Organ weights (males)	Control (0)	Low (100)	Mid (300)	High (1000)
Terminal bodyweight (g)	533.1	594.2	577.8	523.6
	Differences from control	11.5%	8.4%	-1.8%
	Historical control data	455 – 657
Spleen (g)	0.969	1.094	1.171	1.126
	Differences from control	12.9%	20.8%	16.2%
	Historical control data	0.69 – 1.52
Spleen / Bodyweight (%)	0.180	0.184	0.202	0.216 **
	Differences from control	2.0%	12.1%	19.6%
	Historical control data	0.128 – 0.257
Spleen / Brain weight (%)	43.84	48.64	50.99	51.31
	Differences from control	10.9%	16.3%	17.0%
	Historical control data	31.222 – 64.623
= p<0.05; *= p<0.01; Dunnett two sided test. ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test Values with significant differences are indicated with bold font.

Spleen Organ Weights of the Female Animals

Organ weights (females)	Groups/Concentration (mg/kg bw/day)
Organ weights (females)	Control (0)	Low (100)	Mid (300)	High (1000)
Terminal bodyweight (g)	272.2	289.9	282.4	295.5
	Differences from control	6.5%	3.7%	8.6%
	Historical control data	254 – 352
Spleen (g)	0.597	0.655	0.672	0.773**
	Differences from control	9.7%	12.6%	29.5%
	Historical control data	0.46 – 0.86
Spleen / Bodyweight (%)	0.220	0.225	0.237	0.261**
	Differences from control	2.3%	7.8%	18.6%
	Historical control data	0.171 – 0.307
Spleen / Brain weight (%)	28.85	31.83	33.34	38.30**
	Differences from control	10.3%	15.5%	32.7%
	Historical control data	22.439 – 43.216
= p<0.05; *= p<0.01; Dunnett two sided test. ⁺= p<0.05,⁺⁺= p<0.01; Dunn two sided test Values with significant differences are indicated with bold font.

Summary of Bodyweight (g)

Sex: Male		Day(s) Relative to Start Date
Sex: Male		0	7	14	21	28	35	42
0 mg/kg bw/day	Mean	202.7R¹	266.1I²	321.0I²	364.4I²	400.3I,a³	431.9I,a³	457.1I,a³
	SD	10.0	16.4	25.1	34.1	40.9	44.2	48.8
	Max	212	283	353	409	456	493	518
	Min	183	238	280	314	341	366	382
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	204.6	272.4	338.6	386.8	424.7	463.9	493.2
	SD	10.0	13.0	17.9	26.0	31.4	36.1	44.3
	Max	218	290	360	423	468	515	554
	Min	190	253	309	343	371	402	429
	N	10	10	10	10	10	10	10
	%Diff	0.9	2.4	5.5	6.1	6.1	7.4	7.9
300 mg/kg bw/day	Mean	204.2	272.0	335.5	385.7	425.8	462.3	493.3
	SD	8.5	13.2	18.3	23.2	25.7	32.3	35.8
	Max	216	287	357	416	461	504	542
	Min	189	250	307	350	388	412	439
	N	10	10	10	10	10	10	10
	%Diff	0.7	2.2	4.5	5.8	6.4	7.0	7.9
1000 mg/kg bw/day	Mean	203.6	269.1	321.7	361.3	391.4	423.0	448.1
	SD	9.0	13.8	17.7	22.2	26.6	31.0	32.2
	Max	214	294	357	402	436	471	494
	Min	187	245	292	322	345	370	390
	N	10	10	10	10	10	10	10
	%Diff	0.4	1.1	0.2	-0.9	-2.2	-2.1	-2.0

1 [R - AutomaticTransformation: Rank]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance

Sex: Male		Day(s) Relative to Start Date
Sex: Male		49	56	63	70	77	84	90
0 mg/kg bw/day	Mean	479.7I,a¹	499.2I,a¹	514.5I,a¹	527.0R²	540.5R²	544.9I³	553.1I,a¹
	SD	51.2	53.3	54.3	55.7	57.3	57.5	59.1
	Max	547	570	586	594	607	612	628
	Min	402	420	431	442	452	457	464
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	517.6	540.4	563.9	581.1	597.2	603.8	617.3
	SD	51.4	55.2	58.8	62.8	69.7	74.8	75.0
	Max	587	618	650	663	695	710	728
	Min	442	459	477	490	503	503	521
	N	10	10	10	10	10	10	10
	%Diff	7.9	8.3	9.6	10.3	10.5	10.8	11.6
300 mg/kg bw/day	Mean	515.1	537.5	557.6	572.1	582.9	590.0	602.7
	SD	41.6	43.4	48.9	54.5	59.5	59.2	61.3
	Max	578	604	628	644	668	675	689
	Min	453	476	487	496	496	504	506
	N	10	10	10	10	10	10	10
	%Diff	7.4	7.7	8.4	8.6	7.8	8.3	9.0
1000 mg/kg bw/day	Mean	466.4	485.0	504.9	521.3	531.6	540.3	546.6
	SD	36.1	37.0	41.0	44.8	45.4	43.6	45.3
	Max	523	545	567	592	607	615	631
	Min	403	421	436	444	459	468	476
	N	10	10	10	10	10	10	10
	%Diff	-2.8	-2.8	-1.9	-1.1	-1.6	-0.8	-1.2

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance

2 [R - Automatic Transformation: Rank]

3 [I - Automatic Transformation: Identity (NoTransformation)]

Sex: Female		Day(s) Relative to Start Date
Sex: Female		0	7	14	21	28	35	42
0 mg/kg bw/day	Mean	171.1R¹	197.8I²	215.7I²	231.0I²	238.3I²	246.9I²	256.2I²
	SD	8.6	7.9	12.8	15.6	19.4	14.5	18.7
	Max	181	207	234	252	265	267	285
	Min	157	185	199	211	207	224	234
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	167.3	200.6	218.8	231.7	241.7	255.5	264.5
	SD	8.7	12.0	14.4	16.0	11.9	16.2	16.8
	Max	178	219	244	262	264	289	294
	Min	155	181	194	204	220	229	234
	N	10	10	10	10	10	10	10
	%Diff	-2.2	1.4	1.4	0.3	1.4	3.5	3.2
300 mg/kg bw/day	Mean	166.8	197.1	215.4	230.5	241.4	255.0	260.1
	SD	6.3	11.6	13.8	15.5	19.8	18.7	21.4
	Max	175	219	239	257	275	288	305
	Min	155	181	199	211	213	233	238
	N	10	10	10	10	10	10	10
	%Diff	-2.5	-0.4	-0.1	-0.2	1.3	3.3	1.5
1000 mg/kg bw/day	Mean	167.3	202.5	218.8	235.7	245.0	262.4	274.6
	SD	6.7	8.7	13.0	16.2	12.6	13.4	17.4
	Max	174	212	232	253	263	277	304
	Min	157	185	193	205	220	235	245
	N	10	10	10	10	10	10	10
	%Diff	-2.2	2.4	1.4	2.0	2.8	6.3	7.2

1 [R - AutomaticTransformation: Rank]

2 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Female		Day(s) Relative to Start Date
Sex: Female		49	56	63	70	77	84	90
0 mg/kg bw/day	Mean	264.2I¹	269.8I¹	275.7I¹	275.0I¹	284.7I¹	283.7I¹	286.6R²
	SD	18.3	17.3	16.5	16.5	20.2	20.8	16.6
	Max	293	299	306	307	319	328	318
	Min	241	246	255	257	252	259	263
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	271.1	277.5	288.0	289.5	299.1	304.1	303.2
	SD	20.2	18.0	21.9	25.5	22.2	25.3	33.2
	Max	300	299	317	324	327	346	369
	Min	233	244	251	248	260	263	267
	N	10	10	10	10	10	10	10
	%Diff	2.6	2.9	4.5	5.3	5.1	7.2	5.8
300 mg/kg bw/day	Mean	270.0	276.8	281.6	282.4	290.3	295.2	294.7
	SD	20.8	22.6	25.1	23.8	23.8	25.0	24.8
	Max	315	324	332	331	338	341	344
	Min	248	255	251	253	265	265	259
	N	10	10	10	10	10	10	10
	%Diff	2.2	2.6	2.1	2.7	2.0	4.1	2.8
1000 mg/kg bw/day	Mean	279.2	287.9	299.0	296.6	305.3	307.1	309.8
	SD	15.3	13.6	12.8	15.3	16.2	14.4	12.8
	Max	294	311	313	316	329	324	325
	Min	251	262	271	273	277	280	285
	N	10	10	10	10	10	10	10
	%Diff	5.7	6.7	8.5	7.9	7.2	8.2	8.1

[I - Automatic Transformation: Identity(No Transformation)]

2 [R - Automatic Transformation: Rank]

Summary of Bodyweight Gain: Absolute Weight Gain (g)

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
0 → 7	Mean	63.4R¹	67.8	67.8	65.5
	SD	9.0	3.6	5.9	6.4
	Max	74	73	74	80
	Min	50	62	56	58
	N	10	10	10	10
	%Diff	.	6.9	6.9	3.3
7 → 14	Mean	54.9²	66.2d⁺	63.5	52.6
	SD	10.6	8.3	7.7	7.3
	Max	70	79	73	64
	Min	38	53	48	44
	N	10	10	10	10
	%Diff	.	20.6	15.7	-4.2
14 → 21	Mean	43.4I,a³	48.2	50.2	39.6
	SD	10.2	9.3	7.9	5.5
	Max	56	63	64	47
	Min	30	34	35	30
	N	10	10	10	10
	%Diff	.	11.1	15.7	-8.8
21 → 28	Mean	35.9R,k⁺	37.9	40.1	30.1
	SD	8.6	7.1	3.1	6.6
	Max	47	48	45	41
	Min	25	27	36	23
	N	10	10	10	10
	%Diff	.	5.6	11.7	-16.2
28 → 35	Mean	31.6I,a³	39.2d⁺	36.5	31.6
	SD	5.2	5.7	7.4	5.2
	Max	41	47	47	42
	Min	24	28	24	25
	N	10	10	10	10
	%Diff	.	24.1	15.5	0.0
35 → 42	Mean	25.2R¹	29.3	31.0	25.1
	SD	5.9	9.9	4.9	4.1
	Max	34	41	41	30
	Min	16	15	24	19
	N	10	10	10	10
	%Diff	.	16.3	23.0	-0.4

1 [R - Automatic Transformation: Rank]

2 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p <0.05]

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
42 → 49	Mean	22.6I¹	24.4	21.8	18.3
	SD	3.7	8.2	6.6	5.8
	Max	29	33	37	29
	Min	18	10	14	11
	N	10	10	10	10
	%Diff	.	8.0	-3.5	-19.0
49 → 56	Mean	19.5I¹	22.8	22.4	18.6
	SD	5.7	6.3	5.4	5.2
	Max	28	32	32	25
	Min	8	17	16	8
	N	10	10	10	10
	%Diff	.	16.9	14.9	-4.6
56 → 63	Mean	15.3I,a²	23.5dd³	20.1	19.9
	SD	2.6	7.4	6.3	5.8
	Max	20	33	29	29
	Min	11	13	10	8
	N	10	10	10	10
	%Diff	.	53.6	31.4	30.1
63 → 70	Mean	12.5I¹	17.2	14.5	16.4
	SD	4.7	7.6	7.3	5.2
	Max	18	26	27	25
	Min	3	4	5	8
	N	10	10	10	10
	%Diff	.	37.6	16.0	31.2
70 → 77	Mean	13.5I¹	16.1	10.8	10.3
	SD	4.2	8.9	8.1	5.2
	Max	23	32	24	16
	Min	9	-1	0	-2
	N	10	10	10	10
	%Diff	.	19.3	-20.0	-23.7
77 → 84	Mean	4.4I¹	6.6	7.1	8.7
	SD	6.2	6.0	2.8	4.8
	Max	16	15	11	18
	Min	-6	-2	3	3
	N	10	10	10	10
	%Diff	.	50.0	61.4	97.7

1 [I - Automatic Transformation: Identity (NoTransformation)]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [dd - Test: Dunnett 2 Sided p <0.01]

Sex: Male Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
84 → 90	Mean	8.2I,a¹	13.5	12.7	6.3
	SD	4.0	4.8	7.5	4.7
	Max	16	20	26	16
	Min	4	6	2	1
	N	10	10	10	10
	%Diff	.	64.6	54.9	-23.2
0 → 90	Mean	350.4I,a¹	412.7d²	398.5	343.0
	SD	53.6	69.6	56.8	41.6
	Max	417	513	476	430
	Min	272	327	301	289
	N	10	10	10	10
	%Diff	.	17.8	13.7	-2.1

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [d - Test: Dunnett 2 Sided p < 0.05

Sex: Female Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
0 → 7	Mean	26.7I,a¹	33.3	30.3	35.2d³
	SD	3.8	6.7	8.2	7.3
	Max	35	44	46	45
	Min	22	24	23	21
	N	10	10	10	10
	%Diff	.	24.7	13.5	31.8
7 → 14	Mean	17.9I²	18.2	18.3	16.3
	SD	6.7	4.2	4.0	5.8
	Max	27	25	27	27
	Min	4	12	13	8
	N	10	10	10	10
	%Diff	.	1.7	2.2	-8.9
14 → 21	Mean	15.3I²	12.9	15.1	16.9
	SD	5.7	3.7	3.4	6.2
	Max	24	20	22	27
	Min	4	9	11	11
	N	10	10	10	10
	%Diff	.	-15.7	-1.3	10.5
21 → 28	Mean	7.3I²	10.0	10.9	9.3
	SD	6.5	7.6	7.6	8.8
	Max	15	22	26	19
	Min	-8	2	1	-5
	N	10	10	10	10
	%Diff	.	37.0	49.3	27.4
28 → 35	Mean	8.6I,a¹	13.8	13.6	17.4dd⁺
	SD	6.9	6.4	4.4	6.4
	Max	17	25	20	29
	Min	-4	4	6	7
	N	10	10	10	10
	%Diff	.	60.5	58.1	102.3
35 → 42	Mean	9.3I²	9.0	5.1	12.2
	SD	9.9	5.4	7.8	6.5
	Max	24	20	17	27
	Min	-6	2	-5	7
	N	10	10	10	10
	%Diff	.	-3.2	-45.2	31.2

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [I - Automatic Transformation: Identity (NoTransformation)]

3 [d - Test: Dunnett 2 Sided p < 0.05]

Sex: Female Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
42 → 49	Mean	8.0I¹	6.6	9.9	4.6
	SD	6.5	6.1	6.8	7.1
	Max	19	16	21	15
	Min	-1	-4	0	-13
	N	10	10	10	10
	%Diff	.	-17.5	23.8	-42.5
49 → 56	Mean	5.6I¹	6.4	6.8	8.7
	SD	4.5	6.5	6.6	7.8
	Max	11	17	18	21
	Min	-1	-3	-2	-3
	N	10	10	10	10
	%Diff	.	14.3	21.4	55.4
56 → 63	Mean	5.9I¹	10.5	4.8	11.1
	SD	4.5	7.6	6.6	5.9
	Max	13	19	16	22
	Min	0	-5	-10	0
	N	10	10	10	10
	%Diff	.	78.0	-18.6	88.1
63 → 70	Mean	-0.7I¹	1.5	0.8	-2.4
	SD	4.4	7.1	4.6	6.7
	Max	7	13	8	7
	Min	-8	-7	-7	-11
	N	10	10	10	10
	%Diff	.	-314.3	-214.3	242.9
70 → 77	Mean	9.7I¹	9.6	7.9	8.7
	SD	8.3	5.3	3.9	8.4
	Max	24	18	12	24
	Min	-5	1	0	-3
	N	10	10	10	10
	%Diff	.	-1.0	-18.6	-10.3
77 → 84	Mean	-1.0I¹	5.0	4.9	1.8
	SD	6.1	8.1	3.6	7.9
	Max	9	21	12	10
	Min	-11	-7	0	-10
	N	10	10	10	10
	%Diff	.	-600.0	-590.0	-280.0

1 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Female Day(s) Relative to Start Date		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
84 → 90	Mean	2.9I¹	-0.9	-0.5	2.7
	SD	7.9	12.3	6.1	6.6
	Max	15	23	9	14
	Min	-10	-17	-11	-6
	N	10	10	10	10
	%Diff	.	-131.0	-117.2	-6.9
0 → 90	Mean	115.5R,k²	135.9	127.9	142.5uu³
	SD	11.0	30.9	21.8	13.8
	Max	138	200	176	159
	Min	99	95	104	119
	N	10	10	10	10
	%Diff	.	17.7	10.7	23.4

1 [I - Automatic Transformation: Identity (NoTransformation)]

2 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

3 [uu - Test: Dunn 2 Sided p <0.01]

Summary of Food Consumption

Sex: Male		Day(s) Relative to Start Date
Sex: Male		0 → 7	7 → 14	14 → 21	21 → 28	28 → 35	35 → 42	42 → 49
0 mg/kg bw/day	Mean	23.89R,k¹	26.64R,k¹	26.56R,k¹	27.01R,k¹	25.81R,k¹	25.94²	25.27R³
	SD	1.59	2.31	2.72	2.44	2.32	2.74	2.36
	Max	25.8	28.9	29.1	29.9	28.0	28.8	27.7
	Min	22.0	23.8	23.2	24.4	23.0	22.4	22.3
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	25.49	28.99	29.19	29.34	28.06u⁴	28.76u⁴	27.64
	SD	0.91	1.45	1.45	1.41	0.93	1.47	1.98
	Max	26.4	30.9	30.6	31.0	29.4	30.7	30.4
	Min	24.5	27.2	27.6	27.7	27.0	27.2	25.3
	N	10	10	10	10	10	10	10
	%Diff	6.7	8.8	9.9	8.6	8.7	10.8	9.4
300 mg/kg bw/day	Mean	25.81u⁴	29.04	29.39u⁴	29.70	28.11u⁴	28.60u⁴	27.87
	SD	0.83	1.53	1.43	1.32	0.97	0.90	1.44
	Max	27.0	31.6	31.6	31.9	29.3	29.9	29.7
	Min	24.8	27.5	27.8	28.2	26.8	27.8	26.2
	N	10	10	10	10	10	10	10
	%Diff	8.1	9.0	10.7	9.9	8.9	10.2	10.3
1000 mg/kg bw/day	Mean	25.39	27.97	27.54	28.10	27.56	27.51	27.21
	SD	1.35	1.60	1.35	0.82	1.10	0.84	1.09
	Max	27.6	30.7	29.6	28.9	28.9	28.9	28.5
	Min	24.2	26.6	25.6	26.9	26.2	26.4	25.6
	N	10	10	10	10	10	10	10
	%Diff	6.3	5.0	3.7	4.0	6.8	6.1	7.7

1 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p< 0.05]

2 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]

3 [R - AutomaticTransformation: Rank]

4 [u - Test: Dunn 2 Sided p < 0.05]

Sex: Male		Day(s) Relative to Start Date
Sex: Male		49 → 56	56 → 63	63 → 70	70 → 77	77 → 84	84 → 90
0 mg/kg bw/day	Mean	25.40R¹	24.17²	23.86R,k³	23.84²	22.87⁴	22.37⁴
	SD	2.11	1.70	2.04	2.35	2.10	2.32
	Max	27.9	26.0	25.9	25.8	25.0	24.6
	Min	23.0	22.1	21.2	20.6	20.2	19.4
	N	10	10	10	10	10	10
100 mg/kg bw/day	Mean	27.56	26.71dd⁵	26.63	26.74dd⁵	25.43u⁶	25.25
	SD	1.97	2.10	2.51	2.15	1.29	1.78
	Max	30.9	30.2	31.1	30.6	27.8	28.6
	Min	25.6	24.9	24.7	24.8	24.4	24.1
	N	10	10	10	10	10	10
	%Diff	8.5	10.5	11.6	12.2	11.2	12.9
300 mg/kg bw/day	Mean	27.90	26.56dd⁵	25.79	25.09	24.31	25.00u⁶
	SD	1.28	1.78	1.45	1.61	1.17	1.51
	Max	29.9	28.9	28.3	26.5	25.2	26.6
	Min	26.4	24.2	24.3	22.9	22.7	22.9
	N	10	10	10	10	10	10
	%Diff	9.8	9.9	8.1	5.2	6.3	11.8
1000 mg/kg bw/day	Mean	27.59	26.83dd⁵	26.50u⁶	26.39d⁺	25.96uu⁺	25.32uu⁺
	SD	1.14	0.85	0.77	1.33	1.13	0.89
	Max	29.1	28.1	27.2	27.7	27.2	26.4
	Min	26.5	26.0	25.6	24.9	24.5	24.3
	N	10	10	10	10	10	10
	%Diff	8.6	11.0	11.1	10.7	13.5	13.2

1 [R - AutomaticTransformation: Rank]

2 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of V

3 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p< 0.05]

4 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p <0.01]

Sex: Female		Day(s) Relative to Start Date
Sex: Female		0 → 7	7 → 14	14 → 21	21 → 28	28 → 35	35 → 42	42 → 49
0 mg/kg bw/day	Mean	16.97¹	17.41²	17.09²	17.49²	17.00²	16.86²	17.00²
	SD	0.86	0.87	0.43	0.91	0.61	0.64	0.64
	Max	17.9	18.3	17.5	18.4	17.9	17.5	18.1
	Min	15.8	16.3	16.4	16.5	16.5	16.0	16.4
	N	10	10	10	10	10	10	10
100 mg/kg bw/day	Mean	17.91d³	18.04	17.66	18.34	17.94	17.69	17.83u⁴
	SD	1.04	0.70	0.64	0.54	0.63	0.68	0.31
	Max	19.0	18.7	18.4	19.3	18.6	18.2	18.2
	Min	16.1	16.8	17.0	17.7	17.1	16.4	17.4
	N	10	10	10	10	10	10	10
	%Diff	5.6	3.6	3.3	4.9	5.5	4.9	4.9
300 mg/kg bw/day	Mean	16.11	16.61	16.00	16.91	16.49	15.91	16.66
	SD	0.52	0.73	0.83	0.85	0.39	0.71	0.57
	Max	16.8	17.2	16.7	17.7	17.1	16.5	17.1
	Min	15.6	15.6	14.9	15.8	16.1	14.9	15.9
	N	10	10	10	10	10	10	10
	%Diff	-5.1	-4.6	-6.4	-3.3	-3.0	-5.6	-2.0
1000 mg/kg bw/day	Mean	18.46dd⁵	18.29	18.43uu⁶	18.93u⁴	18.74uu⁶	18.93uu⁶	18.56uu⁶
	SD	0.86	0.63	0.52	0.87	0.34	0.46	1.15
	Max	19.7	18.9	18.9	20.3	19.2	19.3	20.2
	Min	17.7	17.7	17.6	18.0	18.2	18.1	17.1
	N	10	10	10	10	10	10	10
	%Diff	8.8	5.0	7.9	8.3	10.3	12.3	9.2

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of V

2 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]

3 [d - Test: Dunnett 2 Sided p< 0.05]

4 [u - Test: Dunn 2 Sided p < 0.05]

5 [dd - Test: Dunnett 2 Sided p< 0.01]

6 [uu - Test: Dunn 2 Sided p < 0.01]

Sex: Female		Day(s) Relative to Start Date
Sex: Female		49 → 56	56 → 63	63 → 70	70 → 77	77 → 84	84 → 90
0 mg/kg bw/day	Mean	16.80¹	16.86¹	15.66²	17.36¹	16.01¹	15.98¹
	SD	0.58	0.55	0.41	0.62	0.68	0.84
	Max	17.4	17.4	16.4	18.1	17.1	17.3
	Min	16.0	16.1	15.3	16.4	15.3	15.0
	N	10	10	10	10	10	10
100 mg/kg bw/day	Mean	18.43uu³	18.54u⁴	16.79dd⁵	19.09uu³	18.56uu³	17.05u⁴
	SD	0.64	1.25	0.84	0.73	0.85	0.48
	Max	19.5	20.0	17.6	19.9	20.1	17.6
	Min	17.6	16.7	15.4	18.4	17.9	16.6
	N	10	10	10	10	10	10
	%Diff	9.7	10.0	7.2	10.0	15.9	6.7
300 mg/kg bw/day	Mean	16.39	15.63	14.70d⁶	16.57	16.06	14.83
	SD	1.12	1.19	0.96	0.91	0.75	0.64
	Max	17.4	16.4	15.5	17.5	16.7	16.0
	Min	14.8	13.9	13.3	15.3	15.0	14.3
	N	10	10	10	10	10	10
	%Diff	-2.5	-7.3	-6.1	-4.5	0.3	-7.2
1000 mg/kg bw/day	Mean	19.44uu³	18.93uu³	16.84dd⁵	18.77	18.63uu³	17.02
	SD	0.97	0.90	0.82	1.33	1.09	0.45
	Max	20.9	20.1	18.2	20.6	20.1	17.5
	Min	18.3	17.5	16.0	17.1	17.6	16.5
	N	10	10	10	10	10	10
	%Diff	15.7	12.3	7.6	8.1	16.3	6.5

1 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p< 0.01]

2 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of V

3 [uu - Test: Dunn 2 Sided p< 0.01]

4 [u - Test: Dunn 2 Sided p < 0.05]

5 [dd - Test: Dunnett 2 Sided p< 0.01]

6 [d - Test: Dunnett 2 Sided p < 0.05]

Summary of Haematology Data: Day 91 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Red Blood Count. (M/uL)	Mean	9.057I,a¹	8.845	8.695 d²	8.690 d²
	SD	0.290	0.320	0.431	0.188
	Max	9.48	9.33	9.29	8.91
	Min	8.77	8.29	8.12	8.34
	N	10	10	10	10
	%Diff	-	-2.3	-4.0	-4.1
WBC. (K/uL)	Mean	5.762I³	6.112	6.505	5.414
	SD	2.281	2.956	2.243	2.012
	Max	9.53	10.44	10.37	8.96
	Min	2.13	2.25	2.14	2.40
	N	10	10	10	10
	%Diff	-	6.1	12.9	-6.0
Haemoglobin Conc. (g/dL)	Mean	14.72I³	14.71	14.13	14.13
	SD	0.55	0.69	0.76	0.53
	Max	15.7	15.6	15.0	15.0
	Min	14.1	13.8	12.7	13.0
	N	10	10	10	10
	%Diff	-	-0.1	-4.0	-4.0
Haematocrit. (%)	Mean	46.45I³	46.13	44.43	45.57
	SD	1.58	1.74	1.98	1.50
	Max	49.4	48.9	46.9	47.8
	Min	44.4	43.7	40.7	42.8
	N	10	10	10	10
	%Diff	.	-0.7	-4.3	-1.9
Mean Cell Volume. (fL)	Mean	51.27I³	52.16	51.13	52.47
	SD	0.71	1.36	1.75	1.39
	Max	52.2	55.3	53.4	54.7
	Min	50.3	50.8	48.9	50.6
	N	10	10	10	10
	%Diff	-	1.7	-0.3	2.3
Mean Cell Haemoglobin. (pg)	Mean	16.26I³	16.64	16.23	16.26
	SD	0.29	0.50	0.61	0.44
	Max	16.7	17.8	17.0	16.9
	Min	15.8	16.1	15.2	15.5
	N	10	10	10	10
	%Diff	-	2.3	-0.2	0.0

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
MCHC. (g/dL)	Mean	31.70R,k¹	31.91	31.75	30.99
	SD	0.37	0.51	0.78	0.87
	Max	32.1	33.1	33.7	32.5
	Min	31.0	31.4	31.1	30.1
	N	10	10	10	10
	%Diff	-	0.7	0.2	-2.2
Red Cell D. Width. (%)	Mean	13.04 I²	12.92	13.31	13.38
	SD	0.32	0.66	0.58	0.46
	Max	13.5	13.9	14.3	14.2
	Min	12.6	11.9	12.5	12.6
	N	10	10	10	10
	%Diff	-	-0.9	2.1	2.6
Platelet Count. (K/uL)	Mean	990.3I,a³	986.4	806.1d⁴	941.6
	SD	168.9	124.1	115.9	175.5
	Max	1180	1201	963	1300
	Min	578	767	572	737
	N	10	10	10	10
	%Diff	-	-0.4	-18.6	-4.9
Mean Plat. Volume. (fL)	Mean	7.87L⁵	7.71	8.79	8.44
	SD	1.12	0.68	1.22	0.95
	Max	10.5	9.0	11.2	9.7
	Min	6.9	6.7	7.3	6.7
	N	10	10	10	10
	%Diff	-	-2.0	11.7	7.2
Reticulocyt. Rel. (%)	Mean	2.08⁺	2.08	2.27	2.91dd⁺
	SD	0.26	0.27	0.33	0.31
	Max	2.3	2.6	2.8	3.4
	Min	1.5	1.7	1.7	2.4
	N	10	10	10	10
	%Diff	-	0.0	9.1	39.9
NEU. Rel. (%)	Mean	25.41R⁺	27.06	26.37	32.69
	SD	6.42	6.70	9.40	7.35
	Max	40.8	40.1	50.0	46.5
	Min	18.3	20.6	20.2	21.9
	N	10	10	10	10
	%Diff	-	6.5	3.8	28.7

1 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

2 [I - Automatic Transformation: Identity (NoTransformation)]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

4 [d - Test: Dunnett 2 Sided p <0.05]

5 [L - Automatic Transformation:Log]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Lymphocytes Rel. (%)	Mean	69.99R¹	67.50	68.79	62.30
	SD	6.24	7.11	9.34	7.45
	Max	76.6	73.5	76.3	72.1
	Min	55.4	53.3	45.4	48.0
	N	10	10	10	10
	%Diff	-	-3.6	-1.7	-11.0
Monocytes Rel. (%)	Mean	2.11I²	2.81	2.43	2.42
	SD	0.38	0.76	0.76	0.66
	Max	2.7	4.1	3.9	3.7
	Min	1.5	1.7	1.6	1.5
	N	10	10	10	10
	%Diff	-	33.2	15.2	14.7
Basophils Rel. (%)	Mean	0.14R¹	0.10	0.11	0.10
	SD	0.13	0.12	0.03	0.05
	Max	0.5	0.4	0.2	0.2
	Min	0.0	0.0	0.1	0.0
	N	10	10	10	10
	%Diff	-	-28.6	-21.4	-28.6
Eosinophils. Rel. (%)	Mean	1.64I²	1.86	1.56	1.92
	SD	0.38	0.37	0.38	0.40
	Max	2.2	2.4	2.2	2.5
	Min	0.9	1.4	1.0	1.4
	N	10	10	10	10
	%Diff	-	13.4	-4.9	17.1
LUC. Rel. (%)	Mean	0.71L³	0.65	0.74	0.60
	SD	0.40	0.28	0.51	0.19
	Max	1.4	1.1	2.1	1.1
	Min	0.2	0.2	0.3	0.4
	N	10	10	10	10
	%Diff	-	-8.5	4.2	-15.5
APTT. (Seconds)	Mean	11.16I²	11.46	10.67	11.12
	SD	0.71	0.77	0.84	1.14
	Max	12.4	12.5	12.4	12.4
	Min	10.3	10.4	9.7	8.7
	N	10	10	10	10
	%Diff	.	2.7	-4.4	-0.4

1 [R - Automatic Transformation:Rank]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [L - Automatic Transformation:Log]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
PTT. (Seconds)	Mean	9.90R¹	9.85	9.70	9.76
	SD	0.08	0.21	0.27	0.21
	Max	10.0	10.2	10.1	10.1
	Min	9.7	9.5	9.3	9.5
	N	10	10	10	10
	%Diff	.	-0.5	-2.0	-1.4

1 [R - Automatic Transformation: Rank

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Red Blood Count. (M/uL)	Mean	7.996R,k¹	7.828	7.880	7.523u²
	SD	0.302	0.907	0.209	0.344
	Max	8.44	8.50	8.19	7.89
	Min	7.48	5.39	7.63	7.01
	N	10	10	10	10
	%Diff	-	-2.1	-1.5	-5.9
WBC. (K/uL)	Mean	1.542L³	1.382	1.403	2.475
	SD	1.176	0.687	0.573	1.513
	Max	3.82	2.53	2.88	5.75
	Min	0.37	0.58	0.85	1.23
	N	10	10	10	10
	%Diff	-	-10.4	-9.0	60.5
Haemoglobin Conc. (g/dL)	Mean	14.08⁴	13.74	13.65	13.28uu⁵
	SD	0.23	1.63	0.40	0.64
	Max	14.4	15.1	14.4	14.1
	Min	13.6	9.4	13.0	12.1
	N	10	10	10	10
	%Diff	-	-2.4	-3.1	-5.7
Haematocrit. (%)	Mean	43.93R,k¹	43.37	43.42	42.27
	SD	1.08	5.18	1.35	1.19
	Max	45.0	47.9	45.9	43.6
	Min	41.9	29.7	41.6	39.6
	N	10	10	10	10
	%Diff	.	-1.3	-1.2	-3.8
Mean Cell Volume. (fL)	Mean	54.96I⁺	55.40	55.13	56.24
	SD	1.76	1.20	2.07	1.67
	Max	58.8	58.5	57.6	59.2
	Min	52.9	54.2	51.8	53.9
	N	10	10	10	10
	%Diff	-	0.8	0.3	2.3
Mean Cell Haemoglobin. (pg)	Mean	17.63I⁺	17.57	17.32	17.67
	SD	0.56	0.43	0.56	0.58
	Max	18.8	18.5	18.3	18.7
	Min	16.9	17.0	16.4	16.6
	N	10	10	10	10
	%Diff	-	-0.3	-1.8	0.2

1 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

2 [u - Test: Dunn 2 Sided p < 0.05]

3 [L - Automatic Transformation:Log]

4 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]

5 [uu - Test: Dunn 2 Sided p <0.01]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
MCHC. (g/dL)	Mean	32.06R¹	31.71	31.41	31.40
	SD	0.55	0.39	0.54	0.79
	Max	33.2	32.3	32.2	32.6
	Min	31.5	31.0	30.6	30.5
	N	10	10	10	10
	%Diff	-	-1.1	-2.0	-2.1
Red Cell D. Width. (%)	Mean	11.33I²	11.41	11.43	11.65
	SD	0.33	0.33	0.37	0.35
	Max	11.9	12.0	11.9	12.4
	Min	10.7	10.9	10.6	11.2
	N	10	10	10	10
	%Diff	-	0.7	0.9	2.8
Platelet Count. (K/uL)	Mean	899.5I²	820.2	870.9	863.4
	SD	116.9	87.5	133.0	176.5
	Max	1043	936	1073	1154
	Min	680	667	655	596
	N	10	10	10	10
	%Diff	-	-8.8	-3.2	-4.0
Mean Plat. Volume. (fL)	Mean	7.21R¹	7.65	7.56	7.82
	SD	0.32	0.44	0.72	1.06
	Max	7.8	8.4	9.0	10.4
	Min	6.7	6.9	6.3	6.7
	N	10	10	10	10
	%Diff	-	6.1	4.9	8.5
Reticulocyt. Rel. (%)	Mean	2.32I,a³	2.20	2.54	2.90d⁴
	SD	0.49	0.41	0.61	0.35
	Max	3.2	3.0	3.3	3.6
	Min	1.7	1.7	1.3	2.5
	N	10	10	10	10
	%Diff	-	-5.2	9.5	25.0
NEU. Rel. (%)	Mean	30.29I²	31.83	30.58	23.90
	SD	9.16	7.58	6.94	5.77
	Max	47.7	48.9	40.8	34.1
	Min	18.0	21.7	21.8	17.0
	N	10	10	10	10
	%Diff	-	5.1	1.0	-21.1

1 [R - Automatic Transformation:Rank]

2 [I - Automatic Transformation: Identity (NoTransformation)]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

4 [d - Test: Dunnett 2 Sided p <0.05]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Lymphocytes Rel. (%)	Mean	63.82I¹	62.59	63.59	70.95
	SD	9.01	7.81	6.79	5.54
	Max	77.0	72.6	73.6	77.3
	Min	47.4	44.4	54.4	60.6
	N	10	10	10	10
	%Diff	-	-1.9	-0.4	11.2
Monocytes Rel. (%)	Mean	2.33I¹	2.16	2.19	1.94
	SD	0.81	0.45	0.60	0.43
	Max	4.1	2.9	3.2	2.5
	Min	0.9	1.5	1.4	1.3
	N	10	10	10	10
	%Diff	-	-7.3	-6.0	-16.7
Basophils Rel. (%)	Mean	0.34R²	0.26	0.38	0.27
	SD	0.27	0.19	0.25	0.24
	Max	0.9	0.6	0.9	0.9
	Min	0.1	0.0	0.1	0.1
	N	10	10	10	10
	%Diff	-	-23.5	11.8	-20.6
Eosinophils. Rel. (%)	Mean	2.44I¹	2.56	2.68	2.46
	SD	1.01	1.38	1.02	0.85
	Max	3.6	4.8	4.1	3.6
	Min	0.9	0.6	1.2	1.1
	N	10	10	10	10
	%Diff	-	4.9	9.8	0.8
LUC. Rel. (%)	Mean	0.81I¹	0.62	0.58	0.49
	SD	0.43	0.44	0.40	0.24
	Max	1.5	1.5	1.4	1.0
	Min	0.3	0.0	0.0	0.2
	N	10	10	10	10
	%Diff	-	-23.5	-28.4	-39.5
APTT. (Seconds)	Mean	11.63I¹	11.45	11.76	11.98
	SD	1.09	1.00	0.71	1.23
	Max	13.4	13.1	12.6	14.2
	Min	10.3	10.1	10.2	10.8
	N	10	10	10	10
	%Diff	.	-1.5	1.1	3.0

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
PTT. (Seconds)	Mean	9.58I¹	9.51	9.40	9.56
	SD	0.23	0.20	0.17	0.15
	Max	9.9	9.8	9.8	9.9
	Min	9.1	9.2	9.2	9.4
	N	10	10	10	10
	%Diff	.	-0.7	-1.9	-0.2

1 [I - Automatic Transformation: Identity (No Transformation)]

Summary of Clinical Chemistry Data: Day 91 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Glucose. (mmol/L)	Mean	9.60L¹	10.86	10.76	10.27
	SD	0.93	2.25	1.79	1.08
	Max	10.9	14.8	13.8	12.2
	Min	7.8	8.0	9.0	8.4
	N	10	10	10	10
	%Diff	.	13.1	12.1	6.9
Total Bilirubin. (umol/L)	Mean	1.78R²	2.30	2.03	2.02
	SD	0.18	0.93	0.58	0.48
	Max	2.2	4.2	3.3	3.3
	Min	1.7	1.7	1.7	1.7
	N	10	10	10	10
	%Diff	.	29.2	14.0	13.5
Urea. (mmol/L)	Mean	5.43I³	5.64	5.28	5.57
	SD	0.54	0.53	0.53	0.60
	Max	6.2	6.7	5.9	6.6
	Min	4.7	4.9	4.3	4.6
	N	10	10	10	10
	%Diff	-	3.7	-2.9	2.5
Cholesterol. (mmol/L)	Mean	1.637L¹	1.827	1.638	1.583
	SD	0.454	0.599	0.433	0.386
	Max	2.56	3.24	2.18	2.16
	Min	1.16	1.22	1.16	1.16
	N	10	10	10	10
	%Diff	.	11.6	0.1	-3.3
Creatinine. (umol/L)	Mean	55.25I³	54.42	53.52	51.96
	SD	5.61	3.67	7.13	5.39
	Max	62.8	61.3	66.3	63.4
	Min	47.9	49.8	43.3	43.7
	N	10	10	10	10
	%Diff	-	-1.5	-3.1	-6.0
Phosphorus. (mmol/L)	Mean	2.425I³	2.531	2.539	2.574
	SD	0.334	0.254	0.399	0.225
	Max	3.00	3.01	3.07	2.99
	Min	1.95	2.15	2.03	2.29
	N	10	10	10	10
	%Diff	.	4.4	4.7	6.1

1 [L - Automatic Transformation: Log]

2 [R - Automatic Transformation:Rank]

3 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Sodium. (mmol/L)	Mean	141.7I¹	140.8	142.0	142.4
	SD	1.4	1.5	1.3	1.4
	Max	145	143	144	145
	Min	140	138	140	140
	N	10	10	10	10
	%Diff	-	-0.6	0.3	0.5
Potassium. (mmol/L)	Mean	5.71I,a²	5.96	6.44d³	6.36d³
	SD	0.44	0.49	0.66	0.55
	Max	6.3	6.9	7.4	7.1
	Min	5.1	5.3	5.6	5.3
	N	10	10	10	10
	%Diff	-	4.4	12.8	11.4
Calcium. (mmol/L)	Mean	2.544I¹	2.587	2.600	2.626
	SD	0.067	0.086	0.071	0.094
	Max	2.65	2.74	2.72	2.76
	Min	2.43	2.48	2.52	2.50
	N	10	10	10	10
	%Diff	.	1.7	2.2	3.2
Chloride. (mmol/L)	Mean	100.45 I¹	99.81	100.97	100.26
	SD	1.53	1.78	2.09	1.12
	Max	104.4	102.8	105.4	102.3
	Min	98.8	97.1	98.8	98.8
	N	10	10	10	10
	%Diff	-	-0.6	0.5	-0.2
Total Protein. (g/L)	Mean	54.52I¹	55.37	55.18	56.69
	SD	2.71	1.57	2.30	1.39
	Max	61.0	57.8	58.6	59.4
	Min	52.0	53.0	51.5	54.4
	N	10	10	10	10
	%Diff	-	1.6	1.2	4.0
Albumin. (g/L)	Mean	30.56I¹	30.50	30.78	31.82
	SD	1.68	1.14	1.80	1.15
	Max	34.7	32.5	33.0	33.3
	Min	29.1	28.6	27.5	30.0
	N	10	10	10	10
	%Diff	-	-0.2	0.7	4.1

1 [I - Automatic Transformation: Identity (NoTransformation)]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [d - Test: Dunnett 2 Sided p <0.05]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
A/G Ratio.	Mean	1.28R¹	1.23	1.27	1.28
	SD	0.06	0.05	0.08	0.06
	Max	1.4	1.3	1.4	1.4
	Min	1.2	1.2	1.1	1.2
	N	10	10	10	10
	%Diff	-	-3.9	-0.8	0.0
AST/GOT. (U/L)	Mean	105.5R¹	116.6	124.1	132.8
	SD	8.4	26.7	46.5	54.9
	Max	116	175	252	276
	Min	92	76	92	85
	N	10	10	10	10
	%Diff	.	10.5	17.6	25.9
ALT/GPT. (U/L)	Mean	43.1R¹	43.7	44.7	48.9
	SD	8.0	13.8	24.3	14.5
	Max	63	80	113	85
	Min	33	32	32	35
	N	10	10	10	10
	%Diff	.	1.4	3.7	13.5
ALKP. (U/L)	Mean	81.5L²	69.9	69.5	74.7
	SD	18.1	8.9	17.8	10.3
	Max	129	86	97	95
	Min	64	58	48	65
	N	10	10	10	10
	%Diff	.	-14.2	-14.7	-8.3
GGT. (U/L)	Mean	5.0 R¹	5.0	5.0	5.0
	SD	0.0	0.0	0.0	0.0
	Max	5	5	5	5
	Min	5	5	5	5
	N	10	10	10	10
	%Diff	.	0.0	0.0	0.0
Bile Acid. (umol/L)	Mean	7.650³	8.136	9.022	10.020dd⁴
	SD	1.277	1.082	1.831	1.873
	Max	9.50	9.63	12.34	12.64
	Min	5.32	6.75	7.08	6.31
	N	10	10	10	10
	%Diff	.	6.4	17.9	31.0

1 [R - Automatic Transformation: Rank]

2 [L - Automatic Transformation: Log]

3 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

4 [dd - Test: Dunnett 2 Sided p < 0.01]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Glucose. (mmol/L)	Mean	9.01I¹	10.37	8.77	9.63
	SD	1.90	2.12	1.35	1.66
	Max	12.5	14.8	10.8	11.3
	Min	6.8	6.9	6.5	6.4
	N	10	10	10	10
	%Diff	.	15.1	-2.7	6.9
Total Bilirubin. (umol/L)	Mean	2.06L,a²	2.68	2.58	3.61dd³
	SD	0.42	0.81	1.09	1.46
	Max	2.8	3.8	4.8	6.3
	Min	1.7	1.7	1.7	1.8
	N	10	10	10	10
	%Diff	.	30.1	25.2	75.2
Urea. (mmol/L)	Mean	6.84I¹	6.85	7.40	7.89
	SD	1.30	1.33	1.13	1.02
	Max	8.9	8.9	8.9	9.5
	Min	4.6	4.9	5.6	6.5
	N	10	10	10	10
	%Diff	-	0.1	8.2	15.3
Cholesterol. (mmol/L)	Mean	1.497L⁴	1.339	1.664	1.616
	SD	0.462	0.250	0.287	0.313
	Max	2.67	1.87	2.19	2.06
	Min	1.16	1.16	1.16	1.16
	N	10	10	10	10
	%Diff	.	-10.6	11.2	7.9
Creatinine. (umol/L)	Mean	54.85I¹	49.53	55.51	52.49
	SD	6.00	7.39	4.24	8.27
	Max	64.2	60.7	62.6	68.8
	Min	46.3	35.7	47.7	42.6
	N	10	10	10	10
	%Diff	-	-9.7	1.2	-4.3
Phosphorus. (mmol/L)	Mean	2.097L⁴	1.985	1.895	2.070
	SD	0.402	0.250	0.424	0.340
	Max	2.84	2.31	2.65	2.60
	Min	1.58	1.55	1.51	1.59
	N	10	10	10	10
	%Diff	.	-5.3	-9.6	-1.3

1 [I - Automatic Transformation: Identity (NoTransformation)]

2 [L,a - Automatic Transformation: Log, (All Groups) Test: Analysis of Variance p < 0.05]

3 [dd - Test: Dunnett 2 Sided p <0.01]

4 [L - Automatic Transformation: Log]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Sodium. (mmol/L)	Mean	143.1I¹	142.7	143.1	142.8
	SD	1.1	1.6	1.2	1.2
	Max	145	146	145	145
	Min	142	140	142	141
	N	10	10	10	10
	%Diff	-	-0.3	0.0	-0.2
Potassium. (mmol/L)	Mean	5.58R²	5.71	5.25	5.51
	SD	1.09	0.94	0.18	0.56
	Max	8.2	8.0	5.4	6.4
	Min	4.8	4.6	4.9	4.7
	N	10	10	10	9
	%Diff	-	2.3	-5.9	-1.2
Calcium. (mmol/L)	Mean	2.566R²	2.616	2.594	2.634
	SD	0.148	0.045	0.098	0.072
	Max	2.85	2.69	2.78	2.75
	Min	2.41	2.54	2.45	2.49
	N	10	10	10	10
	%Diff	.	1.9	1.1	2.7
Chloride. (mmol/L)	Mean	102.58I¹	101.84	101.91	102.30
	SD	1.92	1.68	2.48	1.41
	Max	106.1	104.9	105.6	104.1
	Min	99.3	99.1	97.7	100.8
	N	10	10	10	10
	%Diff	-	-0.7	-0.7	-0.3
Total Protein. (g/L)	Mean	57.54R²	57.03	57.09	57.14
	SD	4.49	1.83	2.43	3.05
	Max	65.3	59.7	62.1	62.4
	Min	50.9	54.9	54.3	52.3
	N	10	10	10	10
	%Diff	-	-0.9	-0.8	-0.7
Albumin. (g/L)	Mean	36.44R²	35.75	35.78	35.60
	SD	4.26	1.55	2.13	2.93
	Max	43.3	38.3	40.0	40.6
	Min	30.5	33.8	33.3	31.4
	N	10	10	10	10
	%Diff	-	-1.9	-1.8	-2.3

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
A/G Ratio.	Mean	1.73R¹	1.69	1.68	1.67
	SD	0.21	0.09	0.10	0.14
	Max	2.1	1.8	1.8	1.9
	Min	1.5	1.6	1.5	1.5
	N	10	10	10	10
	%Diff	-	-2.3	-2.9	-3.5
AST/GOT. (U/L)	Mean	190.0R¹	218.6	139.9	180.5
	SD	112.1	174.3	41.6	65.5
	Max	490	708	234	303
	Min	112	125	87	88
	N	10	10	10	10
	%Diff	.	15.1	-26.4	-5.0
ALT/GPT. (U/L)	Mean	66.4R¹	57.3	45.6	68.9
	SD	52.7	24.4	10.9	35.6
	Max	214	122	73	155
	Min	38	40	34	37
	N	10	10	10	10
	%Diff	.	-13.7	-31.3	3.8
ALKP. (U/L)	Mean	42.5L²	45.3	52.5	41.6
	SD	6.7	9.2	13.1	6.3
	Max	50	56	85	50
	Min	30	25	37	34
	N	10	10	10	10
	%Diff	.	6.6	23.5	-2.1
GGT. (U/L)	Mean	5.0R¹	5.0	5.0	5.0
	SD	0.0	0.0	0.0	0.0
	Max	5	5	5	5
	Min	5	5	5	5
	N	10	10	10	10
	%Diff	.	0.0	0.0	0.0
Bile Acid. (umol/L)	Mean	9.520I³	10.118	11.694	12.137
	SD	2.145	2.594	2.380	3.054
	Max	14.39	13.84	14.88	15.38
	Min	6.85	6.88	7.85	7.09
	N	10	10	10	10
	%Diff	.	6.3	22.8	27.5

1 [R - Automatic Transformation: Rank]

2 [L - Automatic Transformation: Log]

3 [I - Automatic Transformation: Identity (No Transformation)]

Summary of Organ Weight Data: Day 91 Relative to Start Date

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
TermBW. (g)	Mean	533.1I,a¹	594.2	577.8	523.6
	SD	57.3	73.3	59.3	43.8
	Max	599	700	662	604
	Min	449	502	489	457
	N	10	10	10	10
	%Diff	.	11.5	8.4	-1.8
Brain. (g)	Mean	2.201R²	2.248	2.293	2.201
	SD	0.175	0.074	0.049	0.130
	Max	2.57	2.36	2.40	2.39
	Min	1.95	2.08	2.23	1.99
	N	10	10	10	10
	%Diff	-	2.1	4.2	0.0
Brain /BW. (%)	Mean	0.415I³	0.383	0.400	0.422
	SD	0.034	0.041	0.037	0.028
	Max	0.47	0.43	0.46	0.46
	Min	0.36	0.32	0.35	0.38
	N	10	10	10	10
	%Diff	-	-7.8	-3.6	1.6
Adrenal Glands. (g)	Mean	0.0627R²	0.0662	0.0728	0.0587
	SD	0.0090	0.0076	0.0332	0.0101
	Max	0.077	0.075	0.157	0.075
	Min	0.049	0.052	0.048	0.038
	N	10	10	10	10
	%Diff	-	5.6	16.1	-6.4
Adrenals /BW. (%)	Mean	0.0119R²	0.0113	0.0125	0.0112
	SD	0.0019	0.0019	0.0050	0.0016
	Max	0.014	0.014	0.025	0.013
	Min	0.009	0.007	0.008	0.008
	N	10	10	10	10
	%Diff	-	-4.7	5.2	-5.6

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [R - Automatic Transformation: Rank]

3 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Adrenals /Brain. (%)	Mean	2.857R¹	2.943	3.171	2.670
	SD	0.416	0.302	1.433	0.460
	Max	3.33	3.33	6.83	3.28
	Min	2.29	2.34	2.09	1.83
	N	10	10	10	10
	%Diff	-	3.0	11.0	-6.6
Heart. (g)	Mean	1.459L²	1.469	1.491	1.447
	SD	0.232	0.120	0.097	0.123
	Max	1.89	1.64	1.58	1.68
	Min	1.17	1.30	1.29	1.30
	N	10	10	10	10
	%Diff	-	0.7	2.2	-0.8
Heart /BW. (%)	Mean	0.273³	0.249dd⁴	0.259	0.276
	SD	0.023	0.015	0.019	0.009
	Max	0.32	0.27	0.28	0.29
	Min	0.24	0.23	0.22	0.26
	N	10	10	10	10
	%Diff	-	-8.9	-5.0	1.3
Heart /Brain. (%)	Mean	66.17I⁵	65.33	65.00	65.85
	SD	8.17	4.70	3.63	5.48
	Max	81.8	72.2	69.5	73.4
	Min	56.0	58.8	57.8	57.4
	N	10	10	10	10
	%Diff	-	-1.3	-1.8	-0.5
Kidneys. (g)	Mean	3.274I⁵	3.330	3.395	3.279
	SD	0.434	0.454	0.469	0.228
	Max	3.82	4.14	4.14	3.54
	Min	2.54	2.85	2.72	2.91
	N	10	10	10	10
	%Diff	-	1.7	3.7	0.2

1 [R - Automatic Transformation: Rank]

2 [L - Automatic Transformation: Log]

3 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

4 [dd - Test: Dunnett 2 Sided p < 0.01]

5 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Kidneys /BW. (%)	Mean	0.614¹	0.560d²	0.587	0.629
	SD	0.040	0.031	0.042	0.057
	Max	0.67	0.63	0.64	0.72
	Min	0.54	0.52	0.50	0.54
	N	10	10	10	10
	%Diff	-	-8.7	-4.4	2.5
Kidneys /Brain. (%)	Mean	148.56I³	147.99	147.85	149.71
	SD	14.22	18.41	18.34	16.62
	Max	167.4	179.2	172.5	177.9
	Min	121.5	128.4	122.0	123.8
	N	10	10	10	10
	%Diff	-	-0.4	-0.5	0.8
Liver. (g)	Mean	15.272R⁴	16.906	16.414	15.052
	SD	3.341	3.378	2.395	1.373
	Max	19.99	23.02	21.18	16.89
	Min	11.60	12.82	13.84	12.94
	N	10	10	10	10
	%Diff	-	10.7	7.5	-1.4
Liver /BW. (%)	Mean	2.839R⁴	2.826	2.835	2.876
	SD	0.360	0.275	0.206	0.157
	Max	3.34	3.29	3.28	3.19
	Min	2.43	2.55	2.43	2.62
	N	10	10	10	10
	%Diff	-	-0.5	-0.1	1.3
Liver /Brain. (%)	Mean	692.11L⁵	750.86	715.63	685.57
	SD	133.96	143.33	102.03	69.40
	Max	929.8	1036.9	933.0	779.4
	Min	555.0	600.9	591.5	576.2
	N	10	10	10	10
	%Diff	-	8.5	3.4	-0.9

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [I - Automatic Transformation: Identity (No Transformation)]

4 [R - Automatic Transformation: Rank]

5 [L - Automatic Transformation: Log]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Spleen. (g)	Mean	0.969I¹	1.094	1.171	1.126
	SD	0.230	0.184	0.209	0.127
	Max	1.31	1.38	1.55	1.31
	Min	0.66	0.85	0.92	0.96
	N	10	10	10	10
	%Diff	-	12.9	20.8	16.2
Spleen /BW. (%)	Mean	0.180²	0.184	0.202	0.216dd³
	SD	0.029	0.018	0.023	0.023
	Max	0.23	0.21	0.24	0.26
	Min	0.13	0.16	0.15	0.18
	N	10	10	10	10
	%Diff	-	2.0	12.1	19.6
Spleen /Brain. (%)	Mean	43.84I¹	48.64	50.99	51.31
	SD	8.82	7.87	8.57	6.45
	Max	55.8	60.8	67.4	62.7
	Min	31.3	37.8	40.7	44.3
	N	10	10	10	10
	%Diff	-	10.9	16.3	17.0
Thymus. (g)	Mean	0.421I¹	0.501	0.509	0.433
	SD	0.075	0.123	0.129	0.082
	Max	0.53	0.69	0.73	0.56
	Min	0.25	0.29	0.36	0.33
	N	10	10	10	10
	%Diff	-	19.0	20.9	2.9
Thymus /BW. (%)	Mean	0.079I¹	0.084	0.088	0.083
	SD	0.012	0.015	0.018	0.019
	Max	0.09	0.11	0.13	0.12
	Min	0.06	0.06	0.07	0.06
	N	10	10	10	10
	%Diff	-	6.0	11.2	5.8

1 [I - Automatic Transformation: Identity (NoTransformation)]

2 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

3 [dd - Test: Dunnett 2 Sided p <0.01]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Thymus/Brain (%)	Mean	19.19I¹	22.19	22.15	19.78
	SD	3.62	5.08	5.40	4.19
	Max	24.7	30.5	31.7	26.4
	Min	12.0	13.9	15.9	14.5
	N	10	10	10	10
	%Diff	-	15.7	15.4	3.1
Thyroid/Parathyroid. (g)	Mean	0.0249I¹	0.0299	0.0287	0.0270
	SD	0.0058	0.0087	0.0078	0.0057
	Max	0.034	0.047	0.041	0.036
	Min	0.015	0.019	0.015	0.019
	N	10	10	10	10
	%Diff	-	20.1	15.3	8.4
Thyroid/Parathyr./BW (%)	Mean	0.00464I¹	0.00500	0.00495	0.00515
	SD	0.00082	0.00107	0.00111	0.00097
	Max	0.0059	0.0067	0.0065	0.0065
	Min	0.0032	0.0029	0.0026	0.0040
	N	10	10	10	10
	%Diff	-	7.8	6.6	11.1
Thyroid/Parathrd./Brain. (%)	Mean	1.121I¹	1.328	1.250	1.226
	SD	0.195	0.377	0.328	0.246
	Max	1.36	2.12	1.78	1.68
	Min	0.77	0.82	0.64	0.91
	N	10	10	10	10
	%Diff	-	18.5	11.5	9.4
Epididymides. (g)	Mean	1.717I¹	1.725	1.689	1.640
	SD	0.193	0.133	0.156	0.236
	Max	1.97	1.91	1.89	2.18
	Min	1.47	1.46	1.42	1.31
	N	10	10	10	10
	%Diff	-	0.5	-1.6	-4.5

1 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Epididymids. /BW. (%)	Mean	0.324I¹	0.296	0.295	0.313
	SD	0.036	0.053	0.038	0.037
	Max	0.37	0.37	0.36	0.38
	Min	0.26	0.21	0.23	0.26
	N	10	10	10	10
	%Diff	-	-8.6	-9.0	-3.2
Epididymids. /Brain. (%)	Mean	78.02I¹	76.81	73.67	74.44
	SD	6.47	6.44	6.78	8.93
	Max	86.8	87.0	81.8	95.2
	Min	70.8	65.8	63.7	62.7
	N	10	10	10	10
	%Diff	-	-1.6	-5.6	-4.6
Prostate Gland. (g)	Mean	1.253L²	1.332	1.083	1.295
	SD	0.225	0.313	0.417	0.252
	Max	1.62	1.88	2.11	1.67
	Min	0.95	0.90	0.73	0.95
	N	10	10	10	10
	%Diff	-	6.3	-13.6	3.4
Prostate Gland/BW. (%)	Mean	0.2352L,a³	0.2253	0.1874d⁴	0.2499
	SD	0.0343	0.0477	0.0701	0.0580
	Max	0.284	0.297	0.371	0.344
	Min	0.177	0.136	0.126	0.167
	N	10	10	10	10
	%Diff	-	-4.2	-20.3	6.2
Prostate Gland/Brain. (%)	Mean	57.08L²	59.17	47.06	59.07
	SD	10.37	13.14	17.41	12.08
	Max	75.3	83.2	90.2	74.2
	Min	45.5	39.0	31.6	42.3
	N	10	10	10	10
	%Diff	-	3.7	-17.6	3.5

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [L - Automatic Transformation:Log]

3 [L,a - Automatic Transformation: Log, (All Groups) Test: Analysis of Variance p < 0.05]

4 [d - Test: Dunnett 2 Sided p <0.05]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Testis. (g)	Mean	4.211I¹	4.216	4.134	4.209
	SD	0.339	0.312	0.348	0.449
	Max	4.79	4.83	4.71	4.80
	Min	3.75	3.90	3.54	3.30
	N	10	10	10	10
	%Diff	-	0.1	-1.8	0.0
Testis /BW. (%)	Mean	0.795I¹	0.723	0.719	0.806
	SD	0.072	0.127	0.067	0.083
	Max	0.91	0.93	0.83	0.93
	Min	0.68	0.56	0.64	0.62
	N	10	10	10	10
	%Diff	-	-9.1	-9.5	1.4
Testis /Brain. (%)	Mean	191.66I¹	187.79	180.24	191.50
	SD	12.07	16.23	14.05	19.47
	Max	211.0	216.6	203.9	210.0
	Min	167.3	171.8	158.7	144.7
	N	10	10	10	10
	%Diff	-	-2.0	-6.0	-0.1
Seminal Vesicle. (g)	Mean	2.773I¹	2.769	2.609	2.515
	SD	0.436	0.339	0.393	0.490
	Max	3.60	3.28	3.22	3.22
	Min	2.05	2.11	2.24	1.48
	N	10	10	10	10
	%Diff	-	-0.1	-5.9	-9.3
Seminal Vesicle/BW. (%)	Mean	0.524I¹	0.476	0.458	0.479
	SD	0.081	0.103	0.094	0.082
	Max	0.65	0.64	0.59	0.61
	Min	0.34	0.36	0.35	0.30
	N	10	10	10	10
	%Diff	-	-9.1	-12.6	-8.4

1 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Male		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Seminal Vscl/Brain. (%)	Mean	126.1I¹	123.4	114.0	114.3
	SD	17.7	16.6	18.2	21.0
	Max	163	148	139	141
	Min	95	92	96	66
	N	10	10	10	10
	%Diff	-	-2.1	-9.6	-9.3
Pituitary Gland. (g)	Mean	0.0121R²	0.0136	0.0161	0.0129
	SD	0.0021	0.0033	0.0071	0.0020
	Max	0.016	0.019	0.032	0.016
	Min	0.009	0.009	0.010	0.011
	N	10	10	10	10
	%Diff	-	12.4	33.1	6.6
PituitaryGl /BW. (%)	Mean	0.0023R²	0.0023	0.0028	0.0025
	SD	0.0003	0.0004	0.0012	0.0004
	Max	0.003	0.003	0.006	0.003
	Min	0.002	0.002	0.002	0.002
	N	10	10	10	10
	%Diff	-	0.3	21.8	9.2
PituitaryGl /Brain. (%)	Mean	0.547R²	0.605	0.699	0.588
	SD	0.064	0.145	0.298	0.102
	Max	0.65	0.83	1.37	0.77
	Min	0.46	0.40	0.43	0.46
	N	10	10	10	10
	%Diff	-	10.5	27.7	7.6

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
TermBW. (g)	Mean	272.2I¹	289.9	282.4	295.5
	SD	18.3	28.4	24.6	13.8
	Max	312	345	331	311
	Min	251	254	253	272
	N	10	10	10	10
	%Diff	.	6.5	3.7	8.6
Brain. (g)	Mean	2.069R²	2.060	2.016	2.022
	SD	0.064	0.100	0.074	0.056
	Max	2.18	2.16	2.11	2.11
	Min	1.98	1.81	1.92	1.95
	N	10	10	10	10
	%Diff	-	-0.4	-2.6	-2.3
Brain /BW. (%)	Mean	0.763I,a³	0.716	0.718	0.686d⁴
	SD	0.060	0.067	0.062	0.042
	Max	0.87	0.81	0.81	0.76
	Min	0.67	0.62	0.60	0.64
	N	10	10	10	10
	%Diff	-	-6.2	-5.9	-10.2
Adrenal Glands. (g)	Mean	0.0789I¹	0.0821	0.0771	0.0866
	SD	0.0114	0.0158	0.0090	0.0151
	Max	0.093	0.102	0.094	0.114
	Min	0.057	0.043	0.067	0.069
	N	10	10	10	10
	%Diff	-	4.1	-2.3	9.8
Adrenals /BW. (%)	Mean	0.0290R²	0.0288	0.0273	0.0293
	SD	0.0042	0.0066	0.0022	0.0051
	Max	0.034	0.034	0.032	0.037
	Min	0.021	0.012	0.025	0.024
	N	10	10	10	10
	%Diff	-	-0.9	-6.0	1.1

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

4 [d - Test: Dunnett 2 Sided p < 0.05]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Adrenals /Brain. (%)	Mean	3.810I¹	4.009	3.825	4.277
	SD	0.520	0.866	0.429	0.700
	Max	4.70	5.14	4.75	5.59
	Min	2.82	2.02	3.32	3.54
	N	10	10	10	10
	%Diff	-	5.2	0.4	12.3
Heart. (g)	Mean	0.906R²	0.926	0.917	0.945
	SD	0.035	0.093	0.092	0.105
	Max	0.95	1.11	1.04	1.16
	Min	0.85	0.81	0.80	0.77
	N	10	10	10	10
	%Diff	-	2.2	1.2	4.3
Heart /BW. (%)	Mean	0.334I¹	0.320	0.325	0.320
	SD	0.021	0.024	0.025	0.030
	Max	0.36	0.36	0.38	0.38
	Min	0.30	0.29	0.30	0.28
	N	10	10	10	10
	%Diff	-	-4.1	-2.6	-4.3
Heart /Brain. (%)	Mean	43.83I¹	44.99	45.51	46.77
	SD	2.11	4.27	4.49	5.51
	Max	46.5	52.1	52.5	59.2
	Min	39.0	39.7	39.3	39.5
	N	10	10	10	10
	%Diff	-	2.6	3.8	6.7
Kidneys. (g)	Mean	1.692I¹	1.839	1.743	1.816
	SD	0.155	0.210	0.168	0.151
	Max	1.96	2.30	2.00	1.96
	Min	1.51	1.55	1.53	1.54
	N	10	10	10	10
	%Diff	-	8.7	3.0	7.3

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Kidneys /BW. (%)	Mean	0.622I¹	0.634	0.618	0.614
	SD	0.037	0.028	0.034	0.038
	Max	0.67	0.67	0.68	0.67
	Min	0.57	0.60	0.57	0.55
	N	10	10	10	10
	%Diff	-	2.0	-0.6	-1.2
Kidneys /Brain. (%)	Mean	81.86I¹	89.32	86.49	89.89
	SD	8.01	9.54	8.13	8.04
	Max	94.9	108.0	101.0	98.5
	Min	70.6	75.2	76.9	78.3
	N	10	10	10	10
	%Diff	-	9.1	5.7	9.8
Liver. (g)	Mean	8.077I¹	8.667	8.400	8.398
	SD	0.770	1.367	1.000	0.840
	Max	8.85	11.75	10.24	10.21
	Min	6.67	6.59	6.85	7.45
	N	10	10	10	10
	%Diff	-	7.3	4.0	4.0
Liver /BW. (%)	Mean	2.969I¹	2.981	2.980	2.842
	SD	0.242	0.260	0.312	0.248
	Max	3.42	3.41	3.43	3.36
	Min	2.62	2.59	2.51	2.56
	N	10	10	10	10
	%Diff	-	0.4	0.4	-4.3
Liver /Brain. (%)	Mean	391.25I¹	420.94	416.82	416.19
	SD	44.26	63.49	48.50	49.26
	Max	446.5	551.6	504.4	520.9
	Min	306.0	319.9	346.4	353.1
	N	10	10	10	10
	%Diff	-	7.6	6.5	6.4

1 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Spleen. (g)	Mean	0.597¹	0.655	0.672	0.773dd²
	SD	0.054	0.107	0.128	0.115
	Max	0.68	0.81	0.89	0.90
	Min	0.50	0.52	0.54	0.51
	N	10	10	10	10
	%Diff	-	9.7	12.6	29.5
Spleen /BW. (%)	Mean	0.220I,a³	0.225	0.237	0.261dd²
	SD	0.023	0.023	0.033	0.032
	Max	0.26	0.27	0.28	0.30
	Min	0.18	0.20	0.19	0.18
	N	10	10	10	10
	%Diff	-	2.3	7.8	18.6
Spleen /Brain. (%)	Mean	28.85¹	31.83	33.34	38.30dd²
	SD	2.42	5.15	6.30	6.03
	Max	31.9	38.9	44.9	45.2
	Min	24.5	25.5	26.1	24.4
	N	10	10	10	10
	%Diff	-	10.3	15.5	32.7
Thymus. (g)	Mean	0.319I⁴	0.353	0.309	0.371
	SD	0.065	0.084	0.063	0.079
	Max	0.40	0.53	0.38	0.50
	Min	0.23	0.25	0.20	0.24
	N	10	10	10	10
	%Diff	-	10.7	-3.1	16.3
Thymus /BW. (%)	Mean	0.117I⁴	0.122	0.109	0.126
	SD	0.021	0.028	0.021	0.026
	Max	0.14	0.17	0.14	0.17
	Min	0.09	0.09	0.08	0.08
	N	10	10	10	10
	%Diff	-	4.6	-6.4	7.5

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [dd - Test: Dunnett 2 Sided p < 0.01]

3 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

4 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Thymus /Brain. (%)	Mean	15.46I¹	17.12	15.37	18.32
	SD	3.32	3.80	3.28	3.67
	Max	19.0	24.5	19.2	24.5
	Min	10.6	12.0	9.5	12.1
	N	10	10	10	10
	%Diff	-	10.7	-0.6	18.5
Thyroid/ Parathyroid. (g)	Mean	0.0202I¹	0.0187	0.0183	0.0199
	SD	0.0045	0.0035	0.0034	0.0049
	Max	0.026	0.025	0.023	0.024
	Min	0.015	0.013	0.014	0.007
	N	10	10	10	10
	%Diff	-	-7.4	-9.4	-1.5
Thyroid/ Parathyr./BW (%)	Mean	0.00742I¹	0.00649	0.00649	0.00671
	SD	0.00157	0.00132	0.00110	0.00155
	Max	0.0095	0.0087	0.0087	0.0079
	Min	0.0053	0.0049	0.0045	0.0025
	N	10	10	10	10
	%Diff	-	-12.5	-12.5	-9.6
Thyroid/Para thrd./Brain. (%)	Mean	0.979 I¹	0.915	0.909	0.988
	SD	0.230	0.214	0.172	0.246
	Max	1.31	1.38	1.16	1.21
	Min	0.69	0.63	0.69	0.33
	N	10	10	10	10
	%Diff	-	-6.5	-7.2	0.9
Ovaries. (g)	Mean	0.1422 I¹	0.1609	0.1384	0.1432
	SD	0.0269	0.0403	0.0245	0.0310
	Max	0.192	0.227	0.181	0.195
	Min	0.110	0.095	0.089	0.109
	N	10	10	10	10
	%Diff	-	13.2	-2.7	0.7

1 [I - Automatic Transformation: Identity (No Transformation)]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Ovaries /BW. (%)	Mean	0.0525I¹	0.0565	0.0494	0.0484
	SD	0.0107	0.0169	0.0106	0.0097
	Max	0.068	0.082	0.069	0.067
	Min	0.035	0.031	0.034	0.036
	N	10	10	10	10
	%Diff	-	7.5	-5.9	-8.0
Ovaries /Brain. (%)	Mean	6.89I¹	7.83	6.85	7.07
	SD	1.38	2.00	1.11	1.47
	Max	9.6	11.4	8.6	9.4
	Min	5.2	4.5	4.6	5.5
	N	10	10	10	10
	%Diff	-	13.7	-0.5	2.7
Uterus incl. Cervix. (g)	Mean	0.800L²	0.732	0.708	0.673
	SD	0.265	0.125	0.268	0.120
	Max	1.23	0.92	1.41	0.93
	Min	0.46	0.59	0.46	0.56
	N	10	10	10	10
	%Diff	-	-8.5	-11.5	-15.9
Uterus incl. Cervix/BW. (%)	Mean	0.295L²	0.254	0.255	0.227
	SD	0.098	0.046	0.108	0.037
	Max	0.46	0.34	0.53	0.31
	Min	0.15	0.20	0.15	0.19
	N	10	10	10	10
	%Diff	-	-14.0	-13.8	-22.9
Uterus incl. Crvix/Brain. (%)	Mean	38.75L²	35.49	35.30	33.34
	SD	12.98	5.52	13.91	6.24
	Max	60.9	43.2	71.2	46.7
	Min	21.9	28.4	22.7	26.5
	N	10	10	10	10
	%Diff	-	-8.4	-8.9	-14.0

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [L - Automatic Transformation: Log]

Sex: Female		0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Pituitary Gland. (g)	Mean	0.0141R¹	0.0145	0.0150	0.0166
	SD	0.0054	0.0033	0.0029	0.0041
	Max	0.022	0.019	0.021	0.026
	Min	0.002	0.007	0.012	0.012
	N	10	10	10	10
	%Diff	-	2.8	6.4	17.7
PituitaryGl /BW. (%)	Mean	0.0052R¹	0.0051	0.0053	0.0056
	SD	0.0020	0.0014	0.0006	0.0015
	Max	0.008	0.007	0.006	0.009
	Min	0.001	0.002	0.005	0.004
	N	10	10	10	10
	%Diff	-	-2.2	1.2	8.2
PituitaryGl /Brain. (%)	Mean	0.681R¹	0.709	0.744	0.819
	SD	0.260	0.180	0.142	0.191
	Max	1.03	0.99	1.06	1.23
	Min	0.10	0.33	0.63	0.61
	N	10	10	10	10
	%Diff	-	4.1	9.3	20.4

1 [R - Automatic Transformation: Rank]

Summary of Necropsy Data

Removal Reason: ALL	Male				Female
Removal Reason: ALL	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Number of Animals	10	10	10	10	10	10	10	10
Completed Animals:	10	10	9	10	10	10	10	10
AORTA ABDOMINAL
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
ADIPOSE TISSUE
Submitted	0	5	10	10	0	5	10	10
Discoloration; pink, diffuse	.	5	10	10	.	5	10	10
ADRENAL GLAND
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
AORTA THORACIC
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
BRAIN
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
CECUM
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
COLON
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
COAGULATING GLAND
Submitted	10	10	10	10	.	.	.	.
Normal	10	10	10	10	.	.	.	.
DIGESTIVE CONTENTS
Submitted	0	1	1	3	0	0	1	2
Material; black, stomach	.	3	6	9	.	.	7	6
DUODENUM
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
EPIDIDYMIS
Submitted	10	10	10	10	.	.	.	.
Normal	10	10	10	10	.	.	.	.
ESOPHAGUS
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
EYE
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	9	10	10	10
Discoloration; dark red, focal, left	0	0	0	0	1	0	0	0
FEMUR & MARROW
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
HARDERIAN GLAND
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
HEART
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
ILEUM
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
JEJUNUM
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
KIDNEY
Submitted	10	10	10	10	10	10	10	10
Normal	9	10	10	10	10	10	10	10
Discoloration; dark red, focal, right	1	0	0	0	0	0	0	0
LACRIMAL GLAND
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
LIVER
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
LUNGS
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
LYMPH NODE, LUNG-ASSOCIATED
Submitted	0	0	1	1	0	0	0	0
Enlargement	.	.	1	1	.	.	.	.
Discoloration; dark red, diffuse	.	.	1	1	.	.	.	.
LYMPH NODE, MANDIBULAR
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
LYMPH NODE, MESENTERIC
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	7	10	10	10	10
Discoloration; grey, diffuse	0	0	0	3 C¹	0	0	0	0
MAMMARY GLAND AREA, INGUINAL
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
OPTIC NERVE
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
OVARY
Submitted	.	.	.	.	10	10	10	10
Normal	.	.	.	.	10	10	10	10
OVIDUCT
Submitted	.	.	.	.	10	10	10	10
Normal	.	.	.	.	10	10	10	10
PANCREAS
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
PEYERS PATCH
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
PITUITARY
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
PROSTATE
Submitted	10	10	10	10	.	.	.	.
Normal	10	10	10	10	.	.	.	.
RECTUM
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SALIVARY GLAND, MANDIBULAR
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SALIVARY GLAND, PAROTID
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SALIVARY GLAND, SUBLINGUAL
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SCIATIC NERVE
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SEMINAL VESICLES
Submitted	10	10	10	10	.	.	.	.
Normal	10	10	10	10	.	.	.	.
SKELETAL MUSCLE
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SKIN
Submitted	0	0	0	0	0	0	1	0
Mass; firm, left, inguinal; subcutaneous	.	.	.	.	.	.	1	.
SKIN & SUBCUTIS, INGUINAL
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SPINAL CORD
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
SPLEEN
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
STERNUM & MARROW
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
STOMACH
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
TESTIS
Submitted	10	10	10	10	.	.	.	.
Normal	10	10	10	10	.	.	.	.
THYMUS
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
TONGUE
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
THYROID GLAND + PARATHYROID GLAND
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
TRACHEA
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
URINARY BLADDER
Submitted	10	10	10	10	10	10	10	10
Normal	10	10	10	10	10	10	10	10
UTERINE CERVIX+BODY+HORN
Submitted	.	.	.	.	10	10	10	10
Normal	.	.	.	.	9	9	9	10
Dilatation; body; horn	.	.	.	.	1	1	1	0
VAGINA
Submitted	.	.	.	.	10	10	10	10
Normal	.	.	.	.	10	10	10	10

Summary of Ophthalmology Data

Day numbers relative to start date.

				-	-	8	8
Group	Sex	Clinical Sign	Severity	2	1	5	6
1CD	M	Animals normal	-	-	10	-	10
2LD	M	Animals normal	-	-	10	-	-
3MD	M	Animals normal	-	-	10	-	-
4HD	M	Animals normal	-	-	10	-	10
1CD	F	Animals normal	-	10	-	10	-
		Haemorrhage, corpus vitreum	Moderate	-	-	1	-
		Haemorrhage, corpus vitreum	TOTAL	-	-	1	-
2LD	F	Animals normal	-	10	-	-	-
3MD	F	Animals normal	-	10	-	-	-
4HD	F	Animals normal	-	10	-	10	-

Group 1CD - 0 mg/kg bw/day

Group 2LD - 100 mg/kg bw/day

Group 3MD - 300 mg/kg bw/day

Group 4HD - 1000 mg/kg bw/day

Organ Weight Data

Terminal body weights of the high dose animals were not changed compared to the controls.

Males: The relative (to body) organ weight of the spleen increased in the high dose group (19.6 %) and was considered test material-related.

The relative (to body) organ weight of the heart and the kidneys decreased in the low dose and the relative (to body) organ weight of the prostate decreased in the mid dose without microscopic findings.

Females: The absolute and relative (to body and brain) organ weight of the spleen increased in the high dose group statistically significant and was considered as test material-related.

The relative (to body) organ weight of the brain decreased in the high dose without microscopic findings.

Macroscopic Findings

Test material-related pink discoloration of the adipose tissue was observed in all high and mid dose animals and 5/10 low dose males and females. The digestive content in the stomach was black in some of the dosed animals correlated with the colour of the test material. The mesenteric lymph node was gray in 3/10 high dose males as well.

The other changes like focal dark red discoloration of the eye, the kidney and the lung associated lymph nodes, the firm mass in the subcutis and dilated uterine horn and body were considered as incidental or background.

Microscopic Findings

Test material related increased extramedullary hematopoiesis was present in the high dose males and females, correlated with organ weight changes and haematology data. Black foreign material (test material) was seen in the lumen of the stomach and jejunum (adhered to the mucosal surface).

Incidence of Test Material Related Microscopic Changes in the Spleen

	Males				Females
Dose (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Number of animals	10	0	0	10	10	0	0	10
Extramedullary hematopoiesis	3	-	-	9	0	-	-	4
Minimal	3	-	-	4	0	-	-	4
Mild	0	-	-	5	0	-	-	0
Total number of tissues	10	0	0	10	10	0	0	10

The alterations such as tubular basophilia, chronic progressive nephropathy (CPN), hyaline casts and mineralisation in the kidney, haemorrhage in the eye, harderian metaplasia in the lacrimal gland, vacuolation of the hepatocytes, inflammation of the prostate, tubular dilation in the testes, congestion/haemorrhage in the lung-associated lymph nodes, adenocarcinoma in the subcutis (inguinal area) and estrus in the uterus, based on low incidence, the occurrence and/or distribution in control and dosed groups were considered as incidental or background.

The oral administration of the test material to wistar rats dosed at 100, 300 and 1000 mg/kg bw/day for 90 days was associated with test material-related increased extramedullary hematopoiesis in incidence and severity in the high dose males and females. This finding was correlated with organ weight changes and with haematology data as well.

Vaginal Smear Data: Day 91 Relative to Start Date

Group	Sex	Animal	Stage of Oestrus
1CD	f	1501	M
		1502	E
		1503	D
		1504	D
		1505	E
		1506	MD
		1507	E
		1508	M
		1509	E
		1510	M
		Mean S.D. N	. . .
2LD	f	2501	EM
		2502	E
		2503	D
		2504	E
		2505	M
		2506	EM
		2507	M
		2508	D
		2509	M
		2510	D
		Mean S.D. N	. . .
3MD	f	3501	P
		3502	M
		3503	E
		3504	MD
		3505	MD
		3506	E
		3507	D
		3508	D
		3509	D
		3510	MD
		Mean S.D. N	. . .
4HD	f	4501	MD
		4502	M
		4503	M
		4504	MD
		4505	MD
		4506	E
		4507	M
		4508	E
		4509	M
		4510	E
		Mean S.D. N	. . .

P: Proestrus

E: Estrus

M: Metestrus

D: Diestrus

Summary of Total Incidence of Clinical Signs

Observation Type: All	Male			Female
Types From Day -1 to 91	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Total Number of Animals:	10	10	10	10	10	10
% of NAD Animals:	0.0	0.0	0.0	0.0	0.0	0.0
% of Animals with Symptoms:	100.0	100.0	100.0	100.0	100.0	100.0
Faeces coloured
Number of Animals Affected	10	10	10	10	10	10
% of Affected Animals	100	100	100	100	100	100
Number of Times Recorded	1010	1020	1020	1010	1020	1020
Nodule
Number of Animals Affected	0	0	0	0	1	0
% of Affected Animals	0	0	0	0	10	0
Number of Times Recorded	0	0	0	0	1	0

Conclusions:

Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test material is considered to be 300 mg/kg bw/day for both sexes.

Executive summary:

The repeated dose oral toxicity of the test material was assessed in accordance with standardized test method OECD 408 and in compliance with GLP.

This 90-day study was performed to obtain information on the toxicity of the test material when administered by oral gavage to Wistar rats at 3 dose levels daily for 91 days.

Forty male and forty female Wistar rats (10 / sex / dose) were treated with 0, 100, 300 or 1 000 mg/kg bw/day at a concentration of 0, 25, 75 and 250 mg/mL respectively using a dose volume of 4 mL/kg bw.

Analysis of test material formulations for concentration and homogeneity demonstrated that no test material was detected in the control samples, all formulations were homogeneous and all formulations were within ± 10 % of the nominal concentrations.

The examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment (including landing foot splay, grip strength and motor activity assessment), clinical pathology, gross pathology, organ weights and histopathology. Full histopathology was performed in Groups 1 (Control) and 4 (High dose).

Daily administration of the test material to Wistar rats during the treatment period under the conditions of this study did not result in test material related mortality or adverse clinical signs.

Brownish black faeces were observed in all animals for all test material related groups from Day 2 or Day 3 up to the end of the treatment.

The bodyweight, body weight gain, and food consumption of the test material treated groups did not show any test material related effect.

At the functional observation battery (FOB) there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.

No test material related changes were noted at ophthalmoscopy examination.

At clinical pathology, slight changes in red blood cell parameters and clinical chemistry values plus a relatively large increase of reticulocytes, reaching statistical significance in the High dose groups, correlated with increased spleen weight and extramedullary haematopoiesis at histopathology.

No other clear test material-related findings were seen in the clinical pathology parameters.

Test material-related pink discoloration of the adipose tissue was observed in all High and Mid dose animals and 5 out of 10 Low dose males and females. The digestive content in the stomach was black in some of the dosed animals, correlated with the colour of the test material. The mesenteric lymph node was grey in 3 out of 10 High dose males as well. These findings were considered to be related to the colour of the test material (or possibly its metabolites) with no toxicological consequences and hence not adverse.

In the High dose group, the splenic extramedullary haematopoiesis associated with clinical pathology and spleen weight increase are correlated with the haematology findings and organ weight changes of the spleen, were consistent with a compensated anaemia. These changes were considered to be a test material related High dose adverse effect. Slight differences in the Mid-dose indicate the same trend but were small enough to be considered as non-adverse.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test material is considered to be 300 mg/kg bw/day for both sexes.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 300 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

28-Day Study: Wragg & Brooks (1994)

The 28-d study established a NOAEL at 150 mg/kg/day, because treatment at the limit dose of 1 000 mg/kg/day caused haematological effects, changes in spleen weight (and relative liver weight in females only) and histopathological changes in the spleen and liver consistent with methaemoglobin formation and haemolysis, although the animals did not become anaemic. Methaemoglobinaemia and haemolysis are known to be caused by aniline and nitrobenzene which are used to make nigrosine. Aniline and nitrobenzene redox cycle and their presence in erythrocytes results in the reduction haemoglobin to methemoglobin, and also oxidative stress resulting damage to, and ultimately, lysis of the erythrocyte (hemolysis). Once exposure ceases, and the responsible chemical species are cleared from the body, full recovery is made from the methemoglobinaemia and hemolysis. It is important to note that exacerbation of methaemoglobin formation, and sequalae thereof, does not occur with increased duration of dosing. For further information see Kiese, 1996, Muller et al 2006, Stolk and Smith 1966, ECB 2004. The criteria for labelling for specific organ toxicity (STOT) under the CLP Regulation are not met. However humans are known to be more sensitive than rats to these mechanisms resulting in methemglobinaemia and hemolysis, and specific consideration was given to the classification and labelling for hemolysis by the EU Working Group on Haemolytic Anaemia (Muller et al, 2006). The findings of the working group were accepted by the European Commission in 2004. The haemolysis observed in Wragg and Brooks does not trigger the criteria set out in Muller et al (2006) where classification for specific target organ toxicity would be reguired. In addition interspecies assessment factors would also accommodate the differences in sensitivity when deriving DNELs, and there were no effects in the endpoints measured in the developmental toxicity study up to the limit dose of 1000 mg/kg. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

90-Day Study: Oroszlány (2019)

The repeated dose oral toxicity of the test material was assessed in accordance with standardized test method OECD 408 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This 90-day study was performed to obtain information on the toxicity of the test material when administered by oral gavage to Wistar rats at 3 dose levels daily for 91 days.

Analysis of test material formulations for concentration and homogeneity demonstrated that no test item was detected in the control samples, all formulations were homogeneous and all formulations were within ± 10 % of the nominal concentrations.

Daily administration of the test material to Wistar rats during the treatment period under the conditions of this study did not result in test material related mortality or adverse clinical signs.

Brownish black faeces were observed in all animals for all test material related groups from Day 2 or Day 3 up to the end of the treatment.

The bodyweight, body weight gain, and food consumption of the test material treated groups did not show any test material related effect.

No test material related changes were noted at ophthalmoscopy examination.

No other clear test material-related findings were seen in the clinical pathology parameters.

Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test material is considered to be 300 mg/kg bw/day for both sexes.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

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Diss Factsheets

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Additional information

Justification for classification or non-classification

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