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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 allclose all

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

Table 1 Group Mean Haematological Values and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)

 

Hb (g/dL)

RBC (10^12/L)

Hct (%)

MCH (pg)

MCV (fL)

MCHC (g/dL)

WBC (10^9/L)

Meth (%)

Differential (10^9/L)

CT (secs)

PLT (10^9/L)

Retic (%)

Neut

Lymph

Mono

Eos

Bas

Control

Mean

SD

15.3

0.5

7.52

0.33

43.6

1.4

20.4

0.4

58.0

1.9

35.2

0.6

15.2

1.8

1.51

0.87

1.82

0.89

13.29

1.51

0.03

0.06

0.06

0.09

0.00

0.00

25

1

978

252

4

2

15

Mean

SD

15.1

0.4

7.30

0.36

42.7

1.4

20.7

0.5

58.6

1.2

35.4

0.4

12.7

0.5

0.59

0.61

2.01

0.28

10.63

0.69

0.00

0.00

0.10

0.11

0.00

0.00

26

1

1104

91

4

0

150

Mean

SD

15.0

0.6

7.36

0.38

42.8

1.8

20.4

0.5

58.1

1.1

35.0

0.5

18.1

3.4

1.77

0.51

2.30

0.99

15.71

2.47

0.07

0.10

0.03

0.08

0.00

0.00

27**

1

1150

173

4

1

1000

Mean

SD

13.8***

0.2

6.60***

0.10

39.7***

0.8

20.9

0.4

60.2*

1.0

34.7

0.3

16.9

4..5

4.07***

1.62

3.05*

1.15

13.70

3.46

0.08

0.12

0.05

0.10

0.00

0.00

28***

1

1076

74

6

2

* = statistically different from control group value p<0.05

** = statistically different from control group value p<0.01

*** = statistically different from control group value p<0.001

Table 2 Group Mean Haematological Values and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)

 

Hb (g/dL)

RBC (10^12/L)

Hct (%)

MCH (pg)

MCV (fL)

MCHC (g/dL)

WBC (10^9/L)

Meth (%)

Differential (10^9/L)

CT (secs)

PLT (10^9/L)

Retic (%)

Neut

Lymph

Mono

Eos

Bas

Control

Mean

SD

14.8

0.7

7.38

0.30

41.6

1.4

20.1

0.7

56.3

2.2

35.7

0.6

9.9

1.2

1.21

0.22

0.94

0.35

8.82

0.92

0.08

0.08

0.06

0.06

0.00

0.00

27

3

988

72

3

1

15

Mean

SD

14.9

0.7

7.30

0.27

41.8

2.1

20.4

0.5

57.3

1.6

35.6

0.2

12.4

3.6

1.36

0.70

1.23

0.76

11.10

3.06

0.00

0.00

0.11

0.07

0.00

0.00

26

2

1020

46

3

2

150

Mean

SD

14.7

0.3

7.32

0.34

41.2

0.9

20.1

0.8

56.3

1.7

35.7

0.4

8.4

1.5

1.60

0.71

1.07

0.69

7.26

4.54

0.03

0.05

0.06

0.13

0.00

0.00

27

1

1001

62

4

1

1000

Mean

SD

13.8*

0.6

6.48***

0.22

39.7

2.1

21.3**

0.3

61.2***

1.2

34.7**

0.4

12.8

2.9

2.44**

0.44

1.03

0.65

11.57

2.33

0.10

0.13

0.06

0.14

0.00

0.00

26

1

1110

217

9***

2

* = statistically different from control group value p<0.05

** = statistically different from control group value p<0.01

*** = statistically different from control group value p<0.001

Table 3 Group Mean Organ Weights and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)

 

Organ weight (g)

Adrenals

Brain

Gonads

Heart

Kidneys

Liver

Pituitary

Spleen

Control

Mean

SD

0.0446

0.0082

1.9398

0.0772

4.0364

0.1427

1.6256

0.1915

2.4498

0.1393

14.7514

1.1256

0.0096

0.0011

0.8547

0.1127

15

Mean

SD

0.0491

0.0075

1.9718

0.1064

4.2128

0.3621

1.4812

0.2590

2.4939

0.3821

14.2238

1.9802

0.0098

0.0018

0.8168

0.1077

150

Mean

SD

0.0478

0.0096

1.9883

0.0765

3.9597

0.5100

1.3715

0.2161

2.2811

0.2595

13.2898

1.9012

0.0087

0.0008

0.7041

0.1406

1000

Mean

SD

0.0462

0.0095

1.9837

0.0625

3.9796

0.2056

1.7059

0.2486

2.5502

0.1904

14.6910

0.5582

0.0101

0.0009

1.3585***

0.1677

***significantly different from control group value p<0.001

 

Table 4 Group Mean Organ Weights and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)

 

Organ weight (g)

Adrenals

Brain

Gonads

Heart

Kidneys

Liver

Pituitary

Spleen

Control

Mean

SD

0.0603

0.0165

1.8509

0.0554

0.1186

0.0149

0.9062

0.0790

1.6575

0.1615

7.8554

0.8467

0.0107

0.0010

0.5551

0.0652

15

Mean

SD

0.0743*

0.0067

1.8609

0.0647

0.1258

0.0211

0.9640

0.0413

1.7904

0.1622

9.2305**

0.7362

0.0110

0.0014

0.6583*

0.0791

150

Mean

SD

0.0623

0.0059

1.8310

0.0571

0.1133

0.0091

0.8809

0.0531

1.5607

0.1568

7.4615

0.6158

0.0103

0.0009

0.5012

0.0167

1000

Mean

SD

0.0692

0.0047

1.8148

0.0986

0.1158

0.0176

0.8832

0.0981

1.7631

0.1217

8.5693

0.5878

0.0105

0.0021

0.7275***

0.0623

**significantly different from control group value p<0.01

***significantly different from control group value p<0.001

 

Table 5 Group Mean Relative Organ Weights (% of Bodyweight) and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)

 

Relative Organ weight (%)

Adrenals

Brain

Gonads

Heart

Kidneys

Liver

Pituitary

Spleen

Control

Mean

SD

0.0119

0.0022

0.5154

0.0223

1.0727

0.0510

0.4329

0.0611

0.6504

0.0286

3.9162

0.2526

0.0025

0.0002

0.2279

0.0368

15

Mean

SD

0.0127

0.0013

0.5148

0.0355

1.0992

0.0751

0.3881

0.0793

0.6472

0.0602

3.6916

0.2531

0.0025

0.0003

0.2123

0.0194

150

Mean

SD

0.0133

0.0020

0.5589

0.0467

1.1077

0.1101

0.3836

0.0507

0.6369

0.0294

3.7037

0.2738

0.0025

0.0002

0.1957

0.0287

1000

Mean

SD

0.0122

0.0027

0.5214

0.0205

1.0476

0.0889

0.4474

0.0588

0.6692

0.0316

3.8606

0.1433

0.0026

0.0003

0.3577***

0.0499

***significantly different from control group value p<0.001

 

Table 6 Group Mean Relative Organ Weights (% of Bodyweight) and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)

 

Relative Organ weight (%)

Adrenals

Brain

Gonads

Heart

Kidneys

Liver

Pituitary

Spleen

Control

Mean

SD

0.0266

0.0071

0.8182

0.0510

0.0522

0.0052

0.3995

0.0236

0.7301

0.0445

3.4604

0.2536

0.0047

0.0004

0.2447

0.0249

15

Mean

SD

0.0300

0.0028

0.7505

0.0308

0.0506

0.0072

0.3890

0.0241

0.7199

0.0311

3.7151

0.1579

0.0045

0.0007

0.2648

0.0254

150

Mean

SD

0.0286

0.0025

0.8415

0.0533

0.0522

0.0061

0.4043

0.0235

0.7148

0.0484

3.4202

0.2023

0.0047

0.0005

0.2303

0.0132

1000

Mean

SD

0.0306

0.0024

0.7999

0.0247

0.0512

0.0086

0.3891

0.0375

0.7773

0.0435

3.7788*

0.2309

0.0046

0.0010

0.3210***

0.0277

*significantly different from control group value p<0.05

***significantly different from control group value p<0.001

 

Table 7 Histopathological Findings - Summary Incidence

 

Males

 

Females

Dose Level (mg/kg/day)

Dose Level (mg/kg/day)

Control

15

150

1000

Control

15

150

1000

No. of Animals

5

5

5

5

5

5

5

5

Heart

Focal myocarditis

No data

Absent

Minimal

Slight

 

0

2

2

1

 

5

0

0

0

 

5

0

0

0

 

0

2

3

0

 

0

2

3

-

 

5

0

0

-

 

5

0

0

-

 

0

4

1

-

Kidneys

Groups of basophilic/dilated tubules

No data

Absent

Minimal

 

 

 

0

1

4

 

 

 

5

0

0

 

 

 

5

0

0

 

 

 

0

4

1

 

 

 

0

1

4

 

 

 

5

0

0

 

 

 

5

0

0

 

 

 

0

3

2

Liver

Mononuclear cell foci

Minimal

Slight

Moderate

 

Basophilic periportal hepatocytes

Absent

Slight

Moderate

 

 

3

2

0

 

 

 

 

5

0

0

 

 

5

0

0

 

 

 

 

5

0

0

 

 

5

0

0

 

 

 

 

5

0

0

 

 

1

3

1

 

 

 

 

2

2

1

 

 

4

1

-

 

 

 

 

5

0

-

 

 

5

0

-

 

 

 

 

5

0

-

 

 

5

0

-

 

 

 

 

5

0

-

 

 

5

0

-

 

 

 

 

4

1

-

Spleen

Extramedullary haemopoiesis

Minimal

Slight

Moderate

Marked

 

Pigment deposition

Minimal

Slight

Moderate

 

 

3

2

0

0

 

 

5

0

-

 

 

4

1

0

0

 

 

5

0

-

 

 

4

1

0

0

 

 

5

0

-

 

 

0

0

3

2

 

 

0

5

-

 

 

5

0

0

-

 

 

2

3

0

 

 

5

0

0

-

 

 

1

4

0

 

 

5

0

0

-

 

 

1

4

0

 

 

2

2

1

-

 

 

0

0

5

Statistical Information

Mode of death

Terminal kill

 

5

 

5

 

5

 

5

 

5

 

5

 

5

 

5

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).

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:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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:
no

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)

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)

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)

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)

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)

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)

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)

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)

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)

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)

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)

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

0

7

14

21

28

35

42

0

mg/kg

bw/day

Mean

202.7

266.1

321.0

364.4

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

49

56

63

70

77

84

90

0

mg/kg

bw/day

Mean

479.7I,a¹

499.2I,a¹

514.5I,a¹

527.0

540.5

544.9

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

0

7

14

21

28

35

42

0

mg/kg

bw/day

Mean

171.1

197.8

215.7

231.0

238.3

246.9

256.2

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

49

56

63

70

77

84

90

0

mg/kg

bw/day

Mean

264.2

269.8

275.7

275.0

284.7

283.7

286.6

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

63.4

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

25.2

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

22.6

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

19.5

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

12.5

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

13.5

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

4.4

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

412.7

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

35.2

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

17.9

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

15.3

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

7.3

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

9.3

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

8.0

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

5.6

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

5.9

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

-0.7

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

9.7

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

-1.0

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

2.9

-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

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

25.27

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

49 → 56

56 → 63

63 → 70

70 → 77

77 → 84

84 → 90

0

mg/kg

bw/day

Mean

25.40

24.17²

23.86R,k³

23.84²

22.87

22.37

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

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

17.91

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

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

14.72

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

46.45

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

51.27

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

16.26

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 

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

69.99

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

2.11

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

0.14

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

1.64

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

0.71

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

11.16

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

9.90

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

7.523

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

1.542

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

14.08

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

32.06

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

11.33

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

899.5

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

7.21

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

30.29

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

63.82

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

2.33

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

0.34

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

2.44

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

0.81

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

11.63

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

9.58

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

9.60

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

1.78

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

5.43

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

1.637

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

55.25

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

2.425

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

141.7

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

6.44

6.36

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

2.544

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

54.52

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

30.56

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

1.28

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

105.5

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

43.1

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

81.5

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  

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

9.01

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

6.84

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

54.85

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

143.1

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

5.58

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

2.566

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

57.54

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

36.44

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

1.73

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

190.0

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

66.4

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

42.5

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

5.0

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

9.520

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

2.201

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

0.415

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

1.459

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

0.560

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

43.84

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

0.421

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

0.079

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

19.19

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

0.00464

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

1.121

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

0.324

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

78.02

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

1.253

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

57.08

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

4.211

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

0.524

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

126.1

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

0.547

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

272.2

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

2.069

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

0.0789

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

0.0290

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

0.334

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

43.83

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

81.86

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

8.077

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

15.46

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

0.00742

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

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

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

6.89

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

0.295

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

38.75

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

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

-

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.

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). This correlated with minimal to mild splenic extramedullary haematopoiesis.

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.

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 item was detected in the control samples, all formulations were homogeneous and all formulations were within ± 10 % of the nominal concentrations.