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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: U.S. EPA Health Effects Test Guidelines. OPPTS 870.3050; Jul 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation:42 ± 1 days
- Weight at study initiation: males: xx g; females: xxx g
- Fasting period before study: no
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm^2).
- Diet: ground Kliba maintenance diet mouse/rat "GLP", ad libitum
- Water: supplied ad libitum from water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a dilution with corn oil, depending on the required dose

VEHICLE

- Amount of vehicle (if gavage): 4 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The studies were carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test article in corn oil at room temperature over a period of 4 days and at refrigerator over 7 days was proven before the start of the administration. Homogeneity analyses were performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning of the administration period.
The various analyses confirmed:
• the stability of the test-substance preparations for a period of 4 days at room temperature and 7 days at refrigerator conditions
• the homogeneous distribution of the test substance in the vehicle
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
450, 150 and 50 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14-day dose-range finding study
- Rationale for animal assignment: Random distribution according to weight among the individual test groups

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before the administration as well as 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and thereafter at weekly intervals. Parameters examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period and on day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 4 days and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Animals fasted: Yes
- How many animals: all
- Parameters examined: "Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Day 24 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.), Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: Home cage observations / Open field observations / Sensory motor tests/ reflexes / Motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
Organs were fixed followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.
Other examinations:
To further classify the increase in eosinophilic droplets in the kidneys of male animals in test group 3 (1000 mg/kg bw/d), the kidneys of animal Nos. 1 and 16 were immunohistochemically stained against alpha 2u globuline. The immunorelevant organs and tissues were evaluated according to the following
parameters:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio
Statistics:
• Body weight, body weight change: DUNNETT's test (two-sided)
• Feces, rearing, grip, strength forelimbs, grip strength, hindlimbs, footsplay, test, motor activity: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Blood parameters: For parameters with bidirectional changes: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05; For parameters with unidirectional changes: WILCOXON-test (one-sided)
• Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: WILCOXON-test (one-sided)
• Urine pH, volume, specific gravity, color and turbidity: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Weight parameters: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 450 mg/kg bw/d. Nearly all animals of test group 3 (450 mg/kg bw/d) ploughed nose-first into bedding after treatment on several days of the study. In addition, all animals in both sexes of test group 3 (450 mg/kg bw/d) showed slight to moderate salivation directly after treatment on several days of the study. This was also observed in animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. These effects were related to the test substance but assessed as being non-adverse as no treatment-related lesions in the upper digestive tract were observed during pathological examinations.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No impairment in body weight parameters were observed for male and female animals of test groups 1, 2 and 3 (50, 150 and 450 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed in test groups 1, 2 and 3 (50, 150 and 450 mg/kg bw/d) over the entire administration period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed over the entire administration period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After four weeks of administration, in rats of both sexes of test group 3 (450 mg/kg bw/d) total white blood cell (WBC) counts (in males not statistically significant), absolute and relative neutrophil counts and absolute monocyte counts were increased. Relative lymphocyte counts were decreased in these individuals. Additionally, in females of this test group relative monocyte counts were higher compared to controls. In rats of both sexes of test group 2 (150 mg/kg bw/d) absolute neutrophil counts (not statistically significant in females) and relative neutrophil counts (not statistically significant in males) were already increased and relative lymphocyte counts (not statistically significant in males) were decreased. In male rats of test group 3 (450 mg/kg bw/d) hemoglobin values were decreased and in females of the same test group mean corpuscular hemoglobin content (MCH) was lower compared to controls. The hemoglobin decrease in males was the only changed red blood cell parameter in these individuals and the change was minimal (mean -4% compared to controls). In females no measured red blood cell parameter (i.e. hemoglobin, hematocrit and red blood cell [RBC] counts) was altered and only the calculated MCH was decreased. Therefore, the changes of both parameters were regarded as incidental and not treatmentrelated.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After the four-week administration period, in females of test group 3 (450 mg/kg bw/d) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were increased and total bilirubin levels were decreased. In absence of an anemia the lower bilirubin levels were most probably due to an increased conjugation rate of bilirubin because of a liver enzyme induction followed by an accelerated excretion of bilirubin via the bile. This mechanism was regarded as adaptive rather than adverse.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. The following examinations were performed during FOB and have to be assessed individually:
- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed.
- Sensorimotor tests/reflexes: No test substance-related effects were observed.
- Quantitative parameters: Rearing, grip strength of fore- and hindlimbs and foot splay test did not reveal significant changes in test groups 1, 2 and 3 (50, 150 and 450 mg/kg bw/d) when compared to the control animals.
- Motor activity measurement: Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals. Comparing the single intervals with the control groups, significantly decreased values were measured for male animals of test group 2 (150 mg/kg bw/d) at intervals 1 and 3 and for male animals of test group 3 (450 mg/kg bw/d) at interval 12. The changes were regarded to be incidental and not related to treatment as single intervals were not changed in a dosedependent manner and the overall motor activity was not affected. No changes were observed for female animals in test groups 1, 2 and 3 (50, 150 and 450 mg/kg bw/d) when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and spleen weights increased (mid and high dose group). The absolute and relative liver weight increase in females of test group 3 (450 mg/kg bw/d) and the relative liver weight in males of test group 3 (450 mg/kg bw/d) were regarded to be
treatment-related. In addition, the increased spleen weights in males and females of test group 3 (450 mg/kg bw/d) were regarded to be treatment-related. All other significantly changed organ weights were regarded to be incidental or if treatmentrelated to be not adverse, because there was no histopathologic correlate and/or no doseresponse relationship.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The bone marrow of males and females of test group 2 and 3 (150 and 450 mg/kg bw/d) revealed an increase in cell numbers of the white blood cell line. This was regarded to be treatment-related.
In the submucosa of the ileum of 2 males and 2 females of test group 3 (450 mg/kg bw/d) (multi)focal granuloma were found. This finding was characterized by (multi)focal aggregates of mainly macrophages, fewer numbers of granulocytes and occasional multinucleated giant cells. The cytoplasm of the macrophages had a foamy appearance. This finding was regarded to be treatment-related. #
In the liver of males of all test groups and females of test group 2 and 3 (150 and 450 mg/kg bw/d) multiple small granuloma were observed. This finding was characterized by (multi)focal aggregates of mainly macrophages and fewer numbers of granulocytes. The cytoplasm of the macrophages had a foamy appearance. This finding was regarded to be treatment-related. One male of test group 3 (450 mg/kg bw/d) revealed a minimal hypertrophy of centrilobular hepatocytes. A treatment-related effect could not be excluded, but liver parameters were not significantly altered in male animals during clinical pathology examinations. Therefore, the minimal hypertrophy was regarded to be not adverse.
Throughout most of the mesenteric lymph nodes of males and females of all test groups a granulomatous inflammation was diagnosed. This term was used, whenever not only focal granuloma were present, but the granuloma were coalescent and most parts of the lymph node were affected. There were mainly macrophages, fewer numbers of granulocytes and occasional multinucleated giant cells. The cytoplasm of the macrophages had a foamy appearance. This finding was regarded to be treatment-related.
In the spleen extramedullary hematopoiesis was observed in 2 males of test group 3 (450 mg/kg bw/d) and each one female of test groups 2 and 3 (150 and 450 mg/kg bw/d). This was regarded to be treatment-related.
In the bone marrow of the sternum, males and females of test groups 2 and 3 (150 and 450 mg/kg bw/d) revealed an increase in cell numbers of the white blood cell line. This was regarded to be treatment-related.
Histopathological findings: neoplastic:
not examined
Details on results:
Clinical symptoms
With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 450 mg/kg bw/d.
Nearly all animals of test group 3 (450 mg/kg bw/d) ploughed nose-first into bedding after treatment on several days of the study. In addition, all animals in both sexes of test group 3 (450 mg/kg bw/d) showed slight to moderate salivation directly after treatment on several days of the study. This was also observed in animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. These effects were related to the test substance but assessed as being non-adverse as no treatment-related lesions in the upper digestive tract were observed during pathological examinations.

Pathology (see tables below)
Regarding clinical pathology, in rats of both sexes of test groups 2 and 3 (150 and 450 mg/kg bw/d) increased neutrophil cell counts indicated an acute inflammatory process. Additionally, in male and female rats of test group 3, total white blood cell (WBC) counts and monocyte counts were increased whereas relative lymphocyte counts were decreased. The increase of monocyte counts confirmed the inflammatory process because these cells act as macrophages.
Additionally, at least in females higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities indicated a slight affection of liver cells leading to a leakage enzyme molecules out of the cells.


Regarding pathology, bone marrow (femur and sternum), liver, ileum and mesenteric lymph nodes were target organs.

The increase in liver weight in males and females of test group 3 (450 mg/kg bw/day) was regarded to be treatment-related. Although no histopathologic correlate was seen (the granuloma observed were not regarded to have caused this weight increase), in combination with the altered liver parameters in clinical pathology this finding was regarded to be adverse for female animals of this test group. In males, there was no change in liver parameters in clinical pathology. Therefore, the weight increase was regarded to be treatment-related but not adverse.

The granuloma/granulomatous inflammation observed in several organs (mesenteric lymph nodes, ileum, liver) were regarded to be treatment-related. The most likely way of origin of these findings is the uptake of the test-substance probably mainly in the gastrointestinal tract. It is phagocytosed by macrophages that are either overloaded by the test-substance or are not able to digest it. This leads to a chronic, granulomatous inflammation with foreign body character (represented by the multinuclear giant cells) or single granuloma. This was regarded to be an adverse finding.
The hyperplasia of the bone marrow (femur and sternum) as well as the extramedullary hematopoiesis in the spleen, which leads to an increase of white blood cells, was seen as a secondary reaction to the inflammatory process. It was therefore also regarded to be treatment-related and adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Effect levels

Dose descriptor:
LOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: At 50 mg/kg bw there were granuloma in the liver of all males and granulomatous inflammation in the mesenteric lymph nodes in 3 males and four females.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
liver
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Changes in relative organ weights

 

Male animals

Female animals

Test group

(mg/kg bw/d)

1

(50)

2

(150)

3

(450)

1

(50)

2

(150)

3

(450)

Kidneys

95%

105%

112%*

 

 

 

Liver

102%

108%

121%**

97%

104%

118%**

Spleen

108%

114%

134%**

101%

122%

133%**

*: p0.05; **: p0.01

Table 2: Histopahtology findings in liver

Liver

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(50)

2

(150)

3

(450)

0

(0)

1

(50)

2

(150)

3

(450)

No. of animals

5

5

5

5

5

5

5

5

Hypertrophy, centrilobular

0

0

0

1

0

0

0

0

  • Grade 1

 

 

 

1

 

 

 

 

Granuloma (m)f

0

5

5

4

0

0

2

3

  • Grade 1

 

3

3

1

 

 

2

2

  • Grade 2

 

2

2

1

 

 

 

1

  • Grade 3

 

 

 

2

 

 

 

 

Table 3: Histopathology findings in ileum

Ileum

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(50)

2

(150)

3

(450)

0

(0)

1

(50)

2

(150)

3

(450)

No. of animals

5

5

5

5

5

5

5

5

Granuloma (m)f

0

0

0

2

0

0

0

2

  • Grade 1

 

 

 

1

 

 

 

2

  • Grade 2

 

 

 

1

 

 

 

 

In the submucosa of the ileum of 2 males and 2 females of test group 3 (450 mg/kg bw/d) (multi)focal granuloma were found. This finding was characterized by (multi)focal aggregates of mainly macrophages, fewer numbers of granulocytes and occasional multinucleated giant cells. The cytoplasm of the macrophages had a foamy appearance. This finding was regarded to be treatment-related.

Table 4: Histopathology findings in mesenteric lymph nodes

Mesenteric lymph nodes

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(50)

2

(150)

3

(450)

0

(0)

1

(50)

2

(150)

3

(450)

No. of animals

5

5

5

5

5

5

5

5

Inflammation, granulomatous

0

3

5

5

0

4

5

5

  • Grade 1

 

3

 

 

 

4

 

 

  • Grade 2

 

 

3

1

 

 

3

 

  • Grade 3

 

 

2

3

 

 

2

4

  • Grade 4

 

 

 

1

 

 

 

1

Throughout most of the mesenteric lymph nodes of males and females of all test groups a granulomatous inflammation was diagnosed. This term was used whenever not only focal granuloma were present, but the granuloma were coalescent and most parts of the lymph node were affected. There were mainly macrophages, fewer numbers of granulocytes and occasional multinucleated giant cells. The cytoplasm of the macrophages had a foamy appearance. This finding was regarded to be treatment-related.

Table 5: Histopathology findings in spleen

Spleen

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(50)

2

(150)

3

(450)

0

(0)

1

(50)

2

(150)

3

(450)

No. of animals

5

5

5

5

5

5

5

5

Hematopoiesis, extramedullar

0

0

0

2

0

0

1

1

  • Grade 2

 

 

 

2

 

 

1

 

  • Grade 3

 

 

 

 

 

 

 

1

Table 6: Total white and differential blood cell count in males

0 mg/kg bw/d 50 mg/kg bw/d 150 mg/kg bw/d 450 mg/kg bw/d
WBC Mean 5.86 k 6.14 6.97 8.26
[giga/L] S.d. 1.72 1.14 0.90 1.36
day 29 N 5 5 5 5
  Median 4.89 6.07 6.70 8.57
NEUTA Mean 0.82 v 0.76 1.78 * 2.62 **
[giga/L] S.d. 0.37 0.30 0.74 0.63
day 29 N 5 5 5 5
  Median 0.72 0.61 1.87 2.58
LYMPHA Mean 4.77 k 5.11 4.90 5.25
[giga/L] S.d. 1.50 1.05 0.68 0.99
day 29 N 5 5 5 5
  Median 4.28 5.46 5.08 5.34
MONOA Mean 0.12 v 0.12 0.15 0.24 *
[giga/L] S.d. 0.06 0.06 0.05 0.07
day 29 N 5 5 5 5
  Median 0.13 0.12 0.12 0.24
EOSA Mean 0.10 k 0.10 0.09 0.08
[giga/L] S.d. 0.07 0.04 0.03 0.03
day 29 N 5 5 5 5
  Median 0.07 0.10 0.08 0.07
BASOA Mean 0.02 k 0.02 0.02 0.02
[giga/L] S.d. 0.01 0.01 0.01 0.01
day 29 N 5 5 5 5
  Median 0.02 0.02 0.02 0.02
LUCA Mean 0.03 k 0.03 0.03 0.05
[giga/L] S.d. 0.01 0.02 0.01 0.03
day 29 N 5 5 5 5
  Median 0.02 0.03 0.03 0.05
NEUT Mean 14.1 v 12.5 25.3 31.8 **
[%] S.d. 4.7 5.1 9.0 6.1
day 29 N 5 5 5 5
  Median 14.6 11.4 30.7 33.0
LYMPH Mean 81.4 v 83.1 70.7 63.5 **
[%] S.d. 5.7 6.3 9.4 6.5
day 29 N 5 5 5 5
  Median 79.0 82.5 65.3 62.3
MONO Mean 2.1 k 2.0 2.1 2.9
[%] S.d. 1.3 0.8 0.5 0.7
day 29 N 5 5 5 5
  Median 1.7 2.3 2.1 2.8
EOS Mean 1.6 k 1.7 1.2 0.9
[%] S.d. 0.7 0.9 0.3 0.3
day 29 N 5 5 5 5
  Median 1.3 1.2 1.2 0.9
BASO Mean 0.3 k 0.2 0.2 0.2
[%] S.d. 0.1 0.1 0.1 0.0
day 29 N 5 5 5 5
  Median 0.3 0.2 0.3 0.2
LUC Mean 0.4 k 0.5 0.4 0.7
[%] S.d. 0.0 0.2 0.2 0.3
day 29 N 5 5 5 5
  Median 0.4 0.4 0.4 0.7

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

k=KRUSKAL-WALLIS; v=KRUSKAL-WALLIS-WILCOX

Applicant's summary and conclusion

Conclusions:
The administration of the test item by gavage to male and female Wistar rats for 4 weeks caused test substance-related, adverse findings at a dose level of 50 mg/kg bw/d and above taking histopathological findings into account. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was below 50 mg/kg bw/d in male and female Wistar rats.