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Administrative data

Description of key information

oral
Repeated Dose Toxicity Study: NOAEL for systemic toxicity (males/females): 100 mg/kg bw /day based on the clinical, the clinical pathological and histopathological findings (Val 1, GLP, OECD 407 (rat, gavage), BASF 30C0587/11S098, 2012)
dermal
no data
inhalation
no data

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 13 Mar 2012 to 12 Apr 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 03 Oct 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
22.10.2009
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
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
- Mean body weight at study initiation: males: 153.2 ± 10.3 to 154.4 ± 9.5 g; females: 126.8 ± 6.0 to 131.4 ± 4.5 g
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: from water bottles, ad libitum.
- 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: 06 Mar 2012 To: 12 Apr 2012
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was applied as a solution. The test substance preparations were produced at least once a week.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg of body weight
- Concentration in vehicle: average concentration in drinking water as determined analytically: 1.04 g/100 mL (100 mg/kg bw/d); 2.98 g/100 mL (300 mg/kg bw/d); 9.51 g/100 mL (1000 mg/kg bw/d).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean values of the test item in water were found to be in the range of 90 % – 110 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. All rats were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. Thereby, the following parameters were examined:
abnormal behaviour during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmos, faeces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Morning blood was taken from the retro-bulbar venous plexus
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked:
• in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Parameter
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Haemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RET)
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

• Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Parameter
- Prothrombin time (Hepato Quick’s test = HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Morning blood was taken from the retro-bulbar venous plexus
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
References (ALT, AST, ALP): Recommendations of the German Society for Clinical Chemistry: "Standardization of methods for determining enzyme activities in biological liquids". J. Clin. Chem. Clin. Biochem. 8, 658-660 (1970); J. Clin. Chem. Clin. Biochem. 9, 464-465 (1971); J. Clin. Chem. Clin. Biochem. 10, 182-192 (1972); Roche working instructions

• Enzyme (systematic name and system number)
- Alanine aminotransferase (ALT); EC 2.6.1.2.
- Aspartate aminotransferase (AST); EC 2.6.1.1.
- Alkaline phosphatase (ALP); EC 3.1.3.1.
- γ-glutamyltransferase (GGT); EC 2.3.2.2.

• Blood Chemistry Parameter
- 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: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Parameter
- pH
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood
- Specific gravity
- Sediment
- Color, turbidity
- Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period, on study day 28 starting at about 10.00 h. Before the start of the FOB the animals were transferred singly to polycarbonate cages. Drinking water was provided ad libitum whereas no food was offered during the measurements.
- Dose groups that were examined: all animals per sex and group
- Battery of functions tested: passive observations without disturbing the animals (home cage observations), followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random.
Motor activity was measured on the same day as FOB.
In detail, the following parameters were examined:
- Home cage observations: posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
- Open field observations: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (number of fecal pellets/appearance/consistency within two minutes, urine (appearance/quantity) within two minutes, number of rearings within two minutes
- Sensorimotor Tests/Reflexes: approach response, touch response, vision (“visual placing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during “handling”, vocalization, pain perception (“tail pinch”), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

- Motor activity measurement (MA): Measurement was performed in the TSE Labmaster System (supplied by TSE Systems GmbH, Bad Homburg, Germany) with 18 beams per cage. The number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. The period of assessment for each animal started when the first beam was interrupted and finished exactly 60 minutes thereafter. Animals received no food and water during the measurements.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Withdrawal of food about 16 to 20 hours before necropsy.
- Weight parameters (determined in all animals sacrificed on schedule): anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix

- Organ / Tissue fixation
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
all gross lesions, brain, spinal cord (cervical, thoracic and lumbar cord), sciatic nerve, pituitary gland, salivary glands (mandibular and sublingual glands), thyroid glands / parathyroid glands, adrenal glands, prostate, seminal vesicles, coagulation glands, uterus, oviducts, vagina, mammary gland (male and female), thymus, lymph nodes (axillar and mesenteric), spleen, trachea, lungs, heart, aorta, larynx, liver, pancreas, kidneys, esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum (with Peyer’s patches), ileum, cecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), eyes with optic nerve (modified Davidson’s solution), femur with knee joint, skin, skeletal muscle, testes (modified Davidson’s solution), epididymides, cervix, extraorbital lacrimal glands, Harderian glands, nose (nasal cavity), ovaries and pharynx.

From the liver, each one slice of the Lobus dexter medialis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes
- Method: Hematoxylin and eosin (H&E) stain
- Analysed animals:
• All affected animals per group, all treatment groups: all gross lesions
• All animals per test group, all treatment groups: liver, ovaries
• All animals per test group, control and high dose group: trachea, lungs, epididymides, kidneys, spleen, adrenal glands, heart, brain, spinal cord (cervical, thoracic and lumbar cords), sciatic nerve, thyroid glands, testes, uterus, vagina, prostate, seminal vesicles, coagulating glands, thymus, lymph nodes (mesenteric and axillary lymph nodes), stomach (forestomach and glandular stomach), duodenum, cecum, colon, rectum, urinary bladder, bone marrow (femur), cervix, eyes with optic nerve, ileum, jejunum, Peyer’s patches, pituitary gland, skeletal muscle and sternum with marrow.

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

Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”.
Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
A correlation between gross lesions and histopathological findings was attempted.
Statistics:
Body weight and body weight change: A comparison of each dose group with the control group using the DUNNETT's test (two-sided) for the hypothesis of equal means; DUNNETT, C.W. (1955): JASA, Vol. 50, 1096 – 1121; DUNNETT, C.W. (1964): Biometrics, Vol. 20, 482 - 491.
Urinalysis parameter, except pH, volume, color, turbidity and specific gravity: Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions (1). Urine color and turbidity are not evaluated statistically (1).
Feces, rearing, grip strength of forelimbs and hindlimbs, foot-splay test, motor activity, urine pH, volume, specific gravity, color and turbidity (1), weight parameters of pathological examination (2, 3, 4): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians (1).
(1) SIEGEL, S. (1956): Nonparametric statistics for the behavioural sciences. McGraw-Hill New York.
(2) HETTMANNSPERGER, T.P. (1984): Statistical Inference based on Ranks, John Wiley & Sons New York, 132-140.
(3) MILLER, R.G. (1981): Simultaneous Statistical Inference, Springer-Verlag New York Inc., 165-167.
(4) NIJENHUIS, A. and S.W. WILF (1978): Combinatorial Algorithms, Academic Press, New York, 32-33.
Details on results:
CLINICAL SIGNS AND MORTALITY
- No animal died.
- All male and female animals of the high dose group (1000 mg/kg bw/d) showed slight to moderate salivation within 2 hours after treatment, i.e. male animals from study day 10 and female animals from day 13 onwards. One male animal and two female animals of the mid dose group (300 mg/kg bw/d) showed salivation within 2 hours after treatment, i.e. the male animal from study day 13 and female animals from study day 9 onwards.
From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

BODY WEIGHT AND WEIGHT GAIN
- No significant changes with regard to mean body weights were observed for male and female animals of all test groups when compared to the controls. However, body weight change values were decreased in male (significantly) and female animals (not significantly altered) of the high dose group (1000 mg/kg bw/d) from study day 7 to day 28 with a maximum of -28.1% on study day 14 in males and of -7.4% on study day 28 in females (see table 4 in any other informations on results). These changes were assessed as being related to treatment. No test substance-related changes of body weight and body weight change values were observed for male and female animals of the low and mid dose group (100 and 300 mg/kg bw/d).

HAEMATOLOGY
- Regarding hemostaseology, the prothrombin time (Hepato Quick’s test = HQT) was prolonged in rats of both sexes of the high dose group (1000 mg/kg bw/d).
- Regarding the differential blood cell counts, in females of the high dose group (1000 mg/kg bw/d) absolute (0.16 Giga/L, p≤0.05) and relative monocyte (3.4%, p≤0.05) counts were increased, and females of the mid dose group (300 mg/kg bw/d) had still marginally higher absolute monocyte counts (0.11 Giga/L, p≤0.05), but the relative cell counts (2.3%) were in the historical control range (relative monocyte counts: 1.0-2.3%, absolute monocyte counts: 0.03-0.09 Giga/L). Therefore, at least the monocyte count alterations in females of the mid dose group (300 mg/kg bw/d) were regarded as treatment-related, but not adverse, because in this test group the mentioned parameter was the only changed one.
Compared to the control group (7.59 tera/L) in females of the high dose group (1000 mg/kg bw/d) red blood cell (RBC) counts (8.23 tera/L, p≤0.01) were increased, but the mean was within the historical control range (RBC: 7.15-8.41 Tera/L) and, therefore, this alteration was regarded as incidental and not treatment-related. For detailed results see also table 3 "Significant changes in haematological and clinical chemistry parameters" in any other informations on results.

CLINICAL CHEMISTRY
• 1000 mg/kg bw/d
- Increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in rats of both sexes of the high dose group (1000 mg/kg bw/d) indicated a liver cell degradation.
- Higher γ-glutamyltransferase (GGT) activities in male and female rats of the high dose group and increased alkaline phosphatase (ALP) activities in females were due to an obstruction of the bile ductuli. This was confirmed by
- higher serum total bilirubin and total bile acid levels in rats of both sexes of the high dose group.
- The reduced metabolic capacity of the liver cells in rats of both sexes could be seen by reduced globulin and coagulation factor synthesis, the latter leading to a prolonged prothrombin time.
- Total protein levels were decreased in females.
- Creatinine levels (44.2 µmol/L) in males were not significantly decreased but lower compared to controls (47.0 µmol/L) and the values were within the historical control range (43.9-52.5 μmol/L).
- Sodium levels (140.2 mmol/L, p≤0.01) in males were lower compared to controls (42.3 mmol/L), but the mean was within the historical control range (Na: 139.1-146.0 mmol/L).
Therefore, the changes in creatinine and sodium levels in male rats were regarded as incidental and not treatment-related.

• 300 mg/kg bw/d
- Females had higher total bilirubin levels, but the mean was only marginally above the historical control range and this was the only altered clinical chemistry parameter in the rats of this test group. Therefore, the bilirubin change in females was regarded as treatment-related, but not adverse.
- In males creatinine levels (44.6 μmol/L, p≤0.01) were significantly decreased compared to controls (47.0 µmol/L) but the values were within the historical control range (43.9-52.5 μmol/L).
- Albumin levels were higher compared to controls, but the values were not dose-dependently changed.

• 100 mg/kg bw/d
- total protein and albumin levels were increased in males.

For detailed results see also table 3 "Significant changes in haematological and clinical chemistry parameters" in any other informations on results.

URINALYSIS
- No treatment-related, adverse changes among urinalysis parameters were observed.
- In males of test group 3 (1000 mg/kg bw/d), urine pH value was lower compared to controls. This change without any other alteration in urine parameters, was regarded as maybe treatment-related, but not adverse.

NEUROBEHAVIOUR
- Functional observational battery: No test substance-related effects were observed concerning the home cage observations, open field observations, sensorimotor tests/reflexes and quantitative parameters.
- Motor activity measurement: No test substance-related deviations were noted for male and female rats.

ORGAN WEIGHTS
Organs affected were the liver, ovaries, seminal vesicle, prostate, adrenal glands, and thymus.

- Absolute weights: When compared to the control group (set to 100%), the mean absolute weights of the liver were dose-dependently increased in males of all treatment groups (100 mg/kg bw/d: 107%, 300 mg/kg bw/d: 107%, 1000 mg/kg bw: 121%, p ≤ 0.05). The mean absolute weight of the prostate (100 mg/kg bw/d: 93%, 300 mg/kg bw/d: 80%, 1000 mg/kg bw: 61%, p ≤ 0.01) and seminal vesicle (100 mg/kg bw/d: 107%, 300 mg/kg bw/d: 91%, 1000 mg/kg bw: 58%, p ≤ 0.01) were dose-dependently decreased in males. In female animals the absolute weight of adrenal glands (100 mg/kg bw/d: 94%, 300 mg/kg bw/d: 89%, 1000 mg/kg bw: 71%, p ≤ 0.01) and thymus (100 mg/kg bw/d: 94%, 300 mg/kg bw/d: 100%, 1000 mg/kg bw: 67%, p ≤ 0.01 were dose-dependently decreased.
All other mean absolute weight parameters did not show significant differences when compared to the control group.

- Relative organ weights (see table 1): When compared to the control group 0 (set to 100%), the mean relative weight of the liver (100 mg/kg bw: 110%, p ≤ 0.01; 300 mg/kg bw: 117%, p ≤ 0.01; 1000 mg/kg bw: 138%, p ≤ 0.01) and the brain (1000 mg/kg bw: 113%, p ≤ 0.05) in males and of the liver in females (100 mg/kg bw/d: 107%, 300 mg/kg bw/d: 112%, 1000 mg/kg bw: 129%, p ≤ 0.01) were increased.
Decreased mean relative weight of the prostate (100 mg/kg bw/d: 95%, 300 mg/kg bw/d: 87%, 1000 mg/kg bw: 69%, p ≤ 0.05) and seminal vesicles (100 mg/kg bw/d: 111%, 300 mg/kg bw/d: 98%, 1000 mg/kg bw: 66%, p ≤ 0.05) were observed in males and of the adrenal glands (100 mg/kg bw/d: 93%, 300 mg/kg bw/d: 91%, 1000 mg/kg bw: 76%, p ≤ 0.05) and thymus (100 mg/kg bw/d: 92%, 300 mg/kg bw/d: 102%, 1000 mg/kg bw: 71%, p ≤ 0.05) in females.
In males, the dose-related increase of liver weights in all test groups as well as the decreased weights of prostate and seminal vesicles in the high dose test group (1000 mg/kg bw/d) were regarded to be treatment-related.
In females, the increased relative liver weights as well as the decreased weights of adrenal glands and thymus in the high dose group (1000 mg/kg bw/d) were considered to be treatment-related.
The increased relative brain weight in males of the high dose group (1000 mg/kg bw/d) was related to the slightly but not significantly reduced terminal body weight (-13%) in these males.

GROSS PATHOLOGY
All findings occurred individually. They were considered to be incidental in nature and not related to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver
A minimal to moderate diffuse hepatocellular hypertrophy was observed in one male and 2 females of the mid dose group (300 mg/kg bw/d) as well as in all males and females of the high dose group (1000 mg/kg bw/d). The hypertrophy was characterized by enlarged hepatocytes. The cytoplasm was eosinophilic with granular weak basophilic structures. The nuclei in these hepatocytes were enlarged (karyomegaly). In the mid dose group (300 mg/kg bw/d) three further males and two further females showed a minimal karyomegaly in normal sized hepatocytes. The severity of hypertrophy and karyomegaly was dose-related (see table 2).
From animals with hepatocellular hypertrophy, 2 females of the mid dose group (300 mg/kg bw/d) as well as 3 males and 3 females of the high dose group (1000 mg/kg bw/d) showed in addition very few apoptotic bodies or single cell necrosis; in 2 females of the mid dose group (300 mg/kg bw/d) as well as in one female of the high dose group (1000 mg/kg bw/d) a minimal oval cell proliferation was noted.
The increased liver weights in males of the mid dose group (300 mg/kg bw/d) as well as in males and females of the high dose group (1000 mg/kg bw/d) were related to these findings. The occurrence of diffuse hepatocellular hypertrophy, karyomegaly, apoptosis/single cell necrosis and oval cell proliferation were considered to be treatment-related and assessed as being adverse. The increase of the relative liver weight in males of the low dose group (100 mg/kg bw/d) was assessed to be treatment-related, but regarded as non-adverse because there was no histopathological correlate.

- Ovaries
A minimal vacuolation of interstitial glands was observed in 4 females of the high dose group (1000 mg/kg bw/d). This finding was regarded to be treatment-related and assessed as being adverse.

- Prostate and seminal vesicle
The decreased weights of prostate and seminal vesicles in males of the high dose group (1000 mg/kg bw/d) correlated with a reduced size of these organs in 3 males of this test group. The severe absolute and relative organ weight decrease (prostate -39%/ -31%, seminal vesicle -42%/ -34%) was considered to be treatment-related and adverse.
The decreased weights of adrenal glands and thymus in females of high dose group (1000 mg/kg bw/d) were regarded to be treatment-related, but because there were no histopathological correlates the weight reduction was regarded as non-adverse.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased liver weight acompanied by histopathological correlate
Critical effects observed:
not specified

 

Table 1 Relative organ weight

 

 

Male animals

Female animals

Dose

(mg/kg bw/d)

100

300

1000

100

300

1000

Adrenal glands

 

 

 

93%

91%

76%*

Brain

104%

107%

113%*

 

 

 

Liver

110%**

117%**

138%**

107%

112%

129%**

Prostate

95%

87%

69%*

 

 

 

Seminal vesicle

111%

98%

66%*

 

 

 

Thymus

 

 

 

92%

102%

71%*

p ≤ 0.05; **p ≤ 0.01

 

 

Table 2 Liver hypertrophy

 

 

Male animals

Female animals

Dose

(mg/kg bw/d)

100

300

1000

100

300

1000

Animals examined

5

5

5

5

5

5

Hypertrophy, diffuse

 

1

5

 

2

5

• Grade 1

 

 

2

 

 

 

• Grade 2

 

1

 

 

2

2

• Grade 3

 

 

3

 

 

3

Karyomegaly

 

4

5

 

4

5

• Grade 1

 

3

1

 

3

2

• Grade 2

 

1

2

 

1

1

• Grade 3

 

 

2

 

 

2

 

The codes used for the grading system took into consideration either the severity or the number or the size of a microscopic finding.

 

Severity

Number

Size

Grade 1

Minimal

Very few

Very small

Grade 2

Slight

Few

Small

Grade 3

Moderate

Moderate number

Moderate size

Grade 4

Marked; severe

Many

Large

Grade 5

Massive; extreme

Extensive number

Extensive size

 


 

Table 3 Haematological and clinical chemistry parameters

 

 

Male animals (N=5)

Female animals (N=5)

Dose

(mg/kg bw/d)

0

100

300

1000

0

100

300

1000

Red blood cell parameters

 

 

 

 

 

 

 

 

RBC [tera/L]

Mean ± SD

7.97± 0.43 k

8.10± 0.60

8.07± 0.48

8.18± 0.45

7.59± 0.21 v

7.33± 0.49

7.78± 0.26

8.23± 0.45**

HQT [sec]

Mean ± SD

37.5± 3.0 k

38.9± 1.4

38.5± 2.4

45.0± 5.2

36.2± 1.8 v

36.1± 2.4

37.8± 4.8

51.0± 4.3**

White blood cell parameters

 

 

 

 

 

 

 

 

MONOA [giga/L]

Mean ± SD

0.12± 0.04 k

0.12± 0.03

0.12± 0.03

0.16± 0.05

0.06± 0.03 v

0.07± 0.02

0.11± 0.03*

0.16± 0.07*

MONO [%]

Mean ± SD

1.8± 0.2 k

2.0± 0.3

1.9± 0.4

2.5± 0.6

1.7± 0.6 v

1.8± 0.3

2.3± 0.4

3.4± 1.2*

Enzymes

 

 

 

 

 

 

 

 

ALT [µkat/L]

Mean ± SD

0.61 ± 0.23 v

0.76 ± 0.04

0.95 ± 0.55

2.25 ± 0.94**

0.54 ± 0.14 v

0.50 ± 0.13

1.09 ± 0.99

2.02 ± 0.89**

AST [µkat/L]

Mean ± SD

1.67 ± 0.33 v

1.92 ± 0.25

1.70 ± 0.41

2.54 ± 0.43*

1.89 ± 0.94 k

1.56 ± 0.21

2.36 ± 1.44

3.07 ± 0.82

ALP [µkat/L]

Mean ± SD

1.91 ± 0.27 k

2.15 ± 0.43

1.98 ± 0.22

2.14 ± 0.65

1.12 ± 0.15 v

1.15 ± 0.31

1.31 ± 0.35

2.00 ± 0.30**

GGT_C [nkat/L]

Mean ± SD

0 v

0

0

27± 39

0 v

0

0

129± 20**

Blood Chemistry Parameter

 

 

 

 

 

 

 

 

CREA [µmol/L]

Mean ± SD

47.0 ± 1.8 v

47.9 ± 2.2

44.6 ± 1.3**

44.2 ± 2.0

51.6 ± 4.0 k

52.0 ± 2.8

49.7 ± 1.1

47.7 ± 2.0

TBIL [µmol/L]

Mean ± SD

1.60 ± 0.38 v

1.86 ± 0.28

2.29 ± 0.52

3.54 ± 1.19**

1.48 ± 0.82 v

1.61 ± 0.33

3.22 ± 0.85*

5.68 ± 2.09**

TBA [µmol/L]

Mean ± SD

37.2 ± 28.1 k

35.7 ± 10.1

37.8 ± 18.7

52.5 ± 22.5

23.0 ± 10.8 v

26.0 ± 13.7

41.8 ± 12.8

65.1 ± 31.5**

TPROT [µmol/L]

Mean ± SD

59.57 ± 1.78 v

62.71 ± 1.49*

61.64 ± 1.06

58.04 ± 2.49

62.82 ± 2.32 k

62.79 ± 1.79

62.45 ± 1.92

58.29 ± 0.93**

ALB [g/L]

Mean ± SD

37.77 ± 0.60 v

39.57 ± 0.49**

39.50 ± 0.42**

37.93 ± 1.45

41.02 ± 1.46 k

41.11 ± 1.44

41.80 ± 1.17

39.13 ± 0.63

GLOB [µmol/L]

Mean ± SD

21.80 ± 1.23 v

23.14 ± 1.34

22.14 ± 0.87

20.11 ± 1.23

21.80 ± 1.10 v

21.68 ± 0.66

20.65 ± 1.04

19.16 ± 0.76**

Electrolytes and minerals

 

 

 

 

 

 

 

 

NA [mmol/L]

Mean ± SD

142.3 ± 0.4 v

142.6 ± 1.3

141.6 ± 0.7

140.2 ± 1.0**

140.8 ± 1.0 k

140.9 ± 1.4

141.5 ± 1.1

139.8 ± 1.1

Urinalyses

 

 

 

 

 

 

 

 

PH_C

Mean ± SD

6.2 ± 0.3 v

6.3 ± 0.3

6.2 ± 0.3

5.2 ± 0.4*

5.6 ± 0.5 k

5.4 ± 0.5

5.4 ± 0.5

5.3 ± 0.7

RBC = red blood cells (erythrocytes); HQT = prothrombin time (Hepato Quick's test); MONOA = monocytes (absolute); MONO = monocytes; ALT = alanine aminotransferase;

AST = aspartate aminotransferase; ALP = alkaline phosphatase; GGT_C = serum-γ-glutamyltransferase; TBIL = total bilirubin; TBA = bile acids; TPROT = total protein; ALB = albumin; GLOB = globulins;

NA = sodium; pH_C = pH value;* p ≤ 0.05, ** p ≤ 0.01; v=KRUSKAL-WALLIS-WILCOX; k=KRUSKAL-WALLIS

 

Table 4 Body weight changes

 

Male animals

Female animals

Dose

(mg/kg bw/d)

0

100

300

1000

0

100

300

1000

d 0 – 7

Mean

SD

N

Deviation vs control

43,4n

4.6

5

-

39.5

3.0

5

-8.9

37.7

5.4

5

-13.2

33.3**

4.9

5

-23.2

17.8n

6.0

5

-

15.7

4.5

5

-11.6

15.1

6.3

5

-14.9

18.2

3.8

5

2.0

d 0 – 14

Mean

SD

N

Deviation vs control

85,1n

9.3

5

-

78.5

2.8

5

-7.7

71.4

10.1

5

-16.1

61.1**

16.1

5

-28.1

30.3n

8.3

5

-

31.7

6.9

5

4.8

30.0

8.7

5

-0.9

29.9

4.7

5

-1.3

d 0 – 21

Mean

SD

N

Deviation vs control

113,9n

10.1

5

-

105.1

6.0

5

-7.7

94.2

19.1

5

-17.3

85.1*

18.7

5

-25.3

39.7n

6.5

5

-

43.9

7.1

5

10.5

31.1

4.9

5

-21.6

30.9

6.2

5

-22.1

d 0 – 28

Mean

SD

N

Deviation vs control

133,9n

12.3

5

-

123.3

4.6

5

-7.9

108.8

23.6

5

-18.7

100.3*

16.6

5

-25.1

51.5n

12.2

5

-

51.0

4.9

5

-0.9

47.8

4.0

5

-7.2

40.8

7.7

5

-20.9

n=Dunnett test (two-sided), * p<=0.05, ** p <=0.01

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

There is a reliable study available to assess the potential of the substance for repeated dose toxicity after oral dosing.

Oral

In a repeated dose toxicity study according to GLP and OECD test guideline 407 (Repeated Dose 28-day Oral Toxicity Study in Rodents), the test substance was administered orally via gavage to groups of 5 male and 5 female Wistar rats (99.4% a.i. purity) at dose levels of 100, 300 and 1000 mg/kg bw/day over a period of 4 weeks (BASF 30C0587/11S098, 2012). Drinking water served as vehicle.

Detailed clinical observations were performed in all animals at weekly intervals. Food consumption and body weights of the animals were determined once weekly. Clinical and haematological examinations as well as clinical chemistry, and urinalyses were performed in all animals per sex and group at the end of the administration period. Functional observational battery was performed towards the end of the administration period and motor activity was measured in all animals per sex and test group. All animals were sacrificed and assessed by gross pathology. Weights of selected organs were recorded and histopathological examination was performed.

Oral administration of the test item by gavage to male and female Wistar rats over a period of 4 weeks resulted in signs of systemic toxicity which were determined at a dose level of 300 mg/kg bw/d and above.

A treatment related decrease in body weight changes was apparent in the high dose group, from study days 7-28, though this did not significantly influence the mean body weights at study termination.

The liver was identified as the most sensitive target organ. Increases in liver weight were apparent at all dose levels in male animals (110, 117, 138% at 100, 300, 1000mg/kg bw, compared to control), and at 1000mg/kg bw (129%) in female animals.Increases in liver weight were accompanied by a histopathological correlate: histopathological findings in the mid and high dose group included diffuse hepatocellular hypertrophy, karyomegaly and very few apoptotic bodies or single cell necrosis. Further signs of liver toxicity were identified at 1000 mg/kg bw/d, as defined by indicators of liver cell degeneration (increased ALT, AST activities) or obstruction of the bile ductuli (higher GGT, ALP activities; higher serum bilirubin) etc. 

Further target organs were only affected in the high dose group and in presence of the described pronounced liver toxicity: minimal vacuolation of interstitial glands in ovaries and a decrease of absolute and relative organ weight of prostate and seminal vesicles. Although these observations were considered to be treatment-related and assessed as being adverse in character, their biological relevance is unclear since (a) they were only observed in the high dose group in presence of severe liver toxicity and (b) no histopathological correlate was observed in prostate and seminal vesicles.

The no observed adverse effect level (NOAEL) was considered to be 100 mg/kg bw/d in male and female Wistar rats.

CONCLUSION

The oral administration of the test item by gavage to male and female Wistar rats over a period of 4 weeks resulted in adverse signs of systemic liver toxicity which were determined at a dose level of 300 mg/kg bw/d and above.

Therefore, the no observed adverse effect level (NOAEL) was considered to be 100 mg/kg bw/d in male and female Wistar rats.


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No signs of systemic toxicity were observed at dose levels of 100 mg/kg bw after subacute exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Due to diffuse hepatocellular hypertrophy, karyomegaly and apoptotic bodies or single cell necrosis as well as oval cell proliferation the liver was identified as target organ in the 28 day repeated dose study. The no observed adverse effect level (NOAEL) was considered to be 100 mg/kg bw/d in male and female Wistar rats. According to Haber`s rule an equivalent value of ≤ 300 mg/kg bw/d was determined. As a result the substance is considered to be classified for repeated dose toxicity (Cat 2, STOT RE 2, H373) under Regulation (EC) No. 1272/2008.