2-Butyne-1,4-diol, polymer with methyloxirane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 13 Mar 2012 to 12 Apr 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 Oct 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22.10.2009

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxyethylated 2-butyne-1,4-diol

EC Number:

608-711-3

Cas Number:

32167-31-0

Molecular formula:

C4 H6 O2 (C2 H4 O) n, where 1 < n < 4.5

IUPAC Name:

Hydroxyethylated 2-butyne-1,4-diol

Details on test material:

- Name of test material (as cited in study report): Golpanol BEO
- Physical state: liquid/ yellowish, clear
- Analytical purity: 99.4%
- Lot/batch No.: 92713124U0
- Storage condition of test material: room temperature
- Stability under test conditions: for a period of 7 days at room temperature
- Homogeneity: test substance completely miscible with water

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 

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

 

Applicant's summary and conclusion

Executive summary:

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

Regarding clinical examinations, signs of general systemic toxicity in rats of either sex were observed only in the high dose test group (lower body weight change values). No signs were observed for male and female animals in the mid and low dose groups. Slight to moderate salivation after treatment was observed in animals of both sexes of the mid and high dose groups on several days of the study. Due to bad taste of the test substance or local affection of the upper digestive tract salivation is not considered to be an adverse and toxicologically relevant effect.

With regard to clinical pathology, increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in rats of both sexes of the high dose group indicated liver cell degradation. Highrγ-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, which 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 in the high dose group could be seen by reduced globulin and coagulation factor synthesis, the latter leading to a prolonged prothrombin time.

Regarding pathology, oberservations were made in different organs such as liver, ovaries, seminal vesicle, prostate, adrenal glands, and thymus. The absolute and/or relative liver weights were significantly dose-dependently increased in males of all treatment groups as well as in females of the high dose group. Histopathologically, a minimal to moderate diffuse hepatocellular hypertrophy was observed in 1/5 male and 2/5 females of the mid dose group and in all animals of the high dose group. 4/5 males and 4/5 females of the mid dose group and all males and females of the high dose group showed enlarged nuclei (karyomegaly) in the hepatocytes. The severity of hypertrophy and karyomegaly was dose-related. From affected animals, 2 females of the mid dose group as well as 3 males and 3 females of the high dose group showed in addition very few apoptotic bodies or single cell necrosis and in 2 females of the mid dose group and in 1 female of the high dose group a minimal oval cell proliferation was noted. The increased liver weights in males of the mid dose group as well as in males and females of the high dose group 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 mild increase of the relative liver weight in males of the low dose group (+10%) was assessed to be treatment-related, but regarded as non-adverse and not biologically relevant, since there was no histopathological correlate.

Additional findings in further organs were only observed in the high dose group iin presence of significant liver toxicity. A minimal vacuolation of interstitial glands was observed in the ovaries of 4 females in the high dose group. The absolute and relative weights of prostate and seminal vesicles(prostate -39%/ -31%, seminal vesicle -42%/ -34%)were significantly decreased in males of the high dose group. A reduced size of these organs was only observed in 3 males, no histopathological correlates were identified.The decreased weights of adrenal glands and thymus in females of the high dose group were regarded to be treatment-related, but since there were no histopathological correlates the weight reduction was regarded as non-adverse.

The liver was identified as the most sensitive target organ andthe no observed adverse effect level (NOAEL) was considered to be 100 mg/kg bw/d in male and female Wistar rats.