Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct - 21 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
screening study (OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Oct - 21 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Limit test:
no
Species:
rat
Strain:
other: Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 63 - 66 days (males), 84 - 86 days (females)
- Weight at study initiation: 250 - 306 g (males), 189 - 236 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Details on route of administration:
The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was formulated in sunflower oil at in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration.


VEHICLE
- Justification for use and choice of vehicle: The suitability of the vehicle for the test item was analytically verified up front. The test material was stable in sunflower oil at concentrations of 1 mg/mL and 250 mg/mL at room temperature for 1 day and in a refrigerator (at 5 ± 3 °C) for 3 days.
- Concentration in vehicle: 25, 75 and 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: 1604-4373 and 1607-4569
- Quality: Ph.Hg. VIII
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 10 mL of each formulation (25, 75 and 150 mg/mL) and five aliquots of 10 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 94% to 99% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from sunflower oil formulations was 101 and 103% at ~1 and ~250 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 97% of starting concentrations at 1 mg/mL and 96% at 250 mg/mL) and at 5 ± 3°C for 3 days (recovery was 99% of starting concentrations at 1 mg/mL and 94% at 250 mg/mL).
Duration of treatment / exposure:
49 days (males, control and test groups)
55 days (females, control and test groups)
49 days and 14 day post-exposure observation period (recovery groups)
Frequency of treatment:
once daily at similar time (± 2 h), 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Treatment period:
Control: 12/sex
100 mg/kg bw/day: 12/sex
300 mg/kg bw/day: 12/sex
600 mg/kg bw/day: 12 females and 13 males

Treatment and recovery period:
Control: 5/sex
600 mg/kg bw/day: 5 females and 4 males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the test material in rats. The high was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations on parental animals (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for for animals showing clinical signs at 600 mg/kg bw/day, prior to the first exposure and once weekly thereafter on all animals, weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.


FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

IMMUNOLOGY: No

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 54.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of deep narcosis by Isofluran and were subjected to gross necropsy (Parental male animals: after the optionally extended post-mating period on Day 49; Dams not selected for toxicology examinations: on post-partum day 13 or shortly thereafter (Day 55); Dams selected for toxicology examinations: shortly after post-partum day 13 (Day 55); Recovery animals after 14-day post-treatment observation period (Day 63). Examination of the external appearance and the appearance of the tissues and organs. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded. The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected from each group: adrenal glands, aorta, bone with bone marrow and joint (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), gross lesions, heart, kidneys, large intestines (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder.
The following organs of male adults were weighed: brain, testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole. In addition, for 5 males and females randomly selected from each group, and for recovery animals, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.


HISTOPATHOLOGY: Yes
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (600 mg/kg bw/day) including recovery groups. In addition, stomach was also processed and examined histologically in the low and mid dose groups due to necropsy findings. The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES
300 mg/kg bw/day: salivation (6/12 animals) and nuzzling up the bedding material (12/12 animals) shortly after the daily administration; scar on the skin of the neck during the last week of the treatment period in 1/12 animal
600 mg/kg bw/day: salivation (16/17 animals), nuzzling up the bedding material (17/17 animals), compulsive biting of the bedding material (17/17 animals), prone position (17/17 animals) and closed eyes (17/17 animals) with variable incidence and duration; prone position and closed eye ceased between Day 13 and 24

FEMALES
Control: piloerection and paleness at parturition (1/12 animal)
300 mg/kg bw/day: salivation and nuzzling up the bedding material during the pre-mating (8/12 and 12/12 animals, respectively), gestation (2/12 and 12/12 animals, respectively) and lactation (1/12 and 12/12 animals, respectively) periods
600 mg/kg bw/day: salivation (7/17 animals), nuzzling up the bedding material (17/17 animals), compulsive biting of the bedding material (17/17 animals), prone position (17/17 animals) and closed eyes (17/17 animals) during the pre-mating period (main groups) or during the treatment period (recovery groups); compulsive biting of the bedding material and nuzzling up the bedding material during gestation (12/12 animals) and salivation during gestation and lactation (3/12 and 2/12, respectively) for a short period of time after the treatment; alopecia of the abdomen in 1/12 dam between gestation days 19 and lactation day 8
Mortality:
mortality observed, treatment-related
Description (incidence):
600 mg/kg bw/day: 1/17 males euthanized
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistical signigicant increased mean body weight gain between Days 7 and 13 (non-treatment related)
600 mg/kg bw/day: statistical signigicant decreased mean body weight gain between Days 34 and 41 and for the whole treatment period (between Days 0 and 48); increased mean body weight gain at each occasion of the recovery period; slightly increased mean body weight during recovery period (not statistically significant)

FEMALES
100 mg/kg bw/day: statistical significant decreased mean body weight gain between Days 7 and 13 (non-treatment related)
300 mg/kg bw/day: statistical significant increased mean body weight gain between Days 0 and 7 (non-treatment related); statistical significant decreased mean body weight gain between Days 7 and 13 (non-treatment related)
600 mg/kg bw/day: statistical significant increased mean body weight gain between Days 0 and 7 (non-treatment related)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
MALES
600 mg/kg bw/day: statistical significant decreased mean daily food consumption during the entire treatment period with statistical significance during the premating period and last week of the treatment period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: slightly longer activated thromboplastin time (not significant)
600 mg/kg bw/day: statistical significant increased mean white blood cell count and mean percentage of neutrophil granulocytes; statistical significant decreased percentage of lymphocytes

FEMALES
600 mg/kg bw/day: statistical significant increased mean white blood cell count; slightly shorter prothrombin time; statistical significant increased percentage of neutrophil granulocytes and decreased percentage of lymphocytes (both not statistically significant); slightly increased mean white blood cell count at the end of the recovery period
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
600 mg/kg bw/day: statistical significant slightly increased mean concentrations of urea and bile acids at the end of the recovery period (not significant)

FEMALES
100 mg/kg bw/day: statistical significant increased mean total protein concentration
600 mg/kg bw/day: statistical significant slightly increased mean cholesterol concentration; statistical significant slightly increased concentrations of total bilirubin, cholesterol and bile acids at the end of the recovery period; statistical significant slightly decreased albumin level at the end of the recovery period
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
600 mg/kg bw/day: statistical significant decreased mean thymus weight; statistical significant slightly increased liver weight relative to body weight; statistical significant increased thymus weight (absolute and relative to body and brain weights) at the end of the recovery period

FEMALES
100 and 300 mg/kg bw/day: statistical significant slightly decreased mean thymus weights (absolute and relative to body and brain weights)
600 mg/kg bw/day: statistical significant slightly decreased mean thymus weights (absolute and relative to body and brain weights); statistical significant slightly increased weights of liver, spleen and adrenal glands (absolute and relative to body and brain weights for each organ); minor differences in thymus weights; statistical significant slightly increased mean liver weight relative to body weight and slightly decreased mean adrenal glands weight relative to body weight at the end of the recovery period
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES
300 mg/kg bw/day: thickened mucous membrane in the stomach (6/12 animals); thymic hemorrhage (1/12 animal); scar on the neck (1/12 animal; non-treatment related)
600 mg/kg bw/day: thickened mucous membrane in the stomach in the prematurely euthanized animal; thickened mucous membrane in the stomach (12/12 animals); adhesion of the stomach and liver (1/12 animal); thickened mucous membrane in the stomach at the end of the recovery period (4/4 animals)

FEMALES
Control: thymic hemorrhage (1/12 animal); moderate hydrometra (1/12 animal); slight or marked hydrometra at the end of the recovery period (1/5)
300 mg/kg bw/day: thickened mucous membrane in the stomach (2/12 animals)
600 mg/kg bw/day: thickened mucous membrane in the stomach (12/12 animals); slight hydrometra (1/12 animal); slight or marked hydrometra at the end of the recovery period (2/5)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES
Control: alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (2/5 animals; non-treatment related); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period (1/5 animal; non-treatment related)
300 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals)
600 mg/kg bw/day: serious deep ulceration and necrosis in the forestomach and purulent inflammation in the kidney (purulent nephritis) and brain (purulent leptomeningitis), as well as centrilobular vacuolation in the liver of the prematurely euthanized animal; squamous cell hyperplasia in the forestomach (12/12 animals); ulceration in the forestomach (10/12 animals); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period (1/4 animal; non-treatment related)

FEMALES
Control: alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); acute hemorrhage in the thymus (1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (1/5 animal; non-treatment related); dilatation of uterine horns (1/12 animal; non-treatment related); dilatation of uterine horns at the end of the recovery period (1/5 animal; non-treatment related); alveolar emphysema at the end of the recovery period (1/5 animal; non-treatment related)
300 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals)
600 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals); ulceration in the forestomach (10/12 animals); alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (1/5 animal; non-treatment related); dilatation of uterine horns (1/12 animal; non-treatment related); dilatation of uterine horns at the end of the recovery period (2/5 animals; non-treatment related); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period(1/5 animal; non-treatment related)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.
Details on results:
CLINICAL SIGNS
Dermal changes (scar in 1/12 male of the mid dose group and alopecia in 1/12 female of the high dose group) are common spontaneous finding in this strain of experimental rats and are seen also in untreated rats. These were considered to be individual signs and not related to the test item.

MORTALITY
The male of the high dose group was euthanized due to its moribund condition and subjected to necropsy on Day 9. Nuzzling up the bedding material, compulsive biting of the bedding material, prone position, closed eyes, piloerection, decreased muscle tone, sanguineous fur around the nose and decreased activity were noted for this animal between Days 1 and 9. The body weight of this animal decreased significantly during the first week of study and animal became moribund on Day 9. Histological examinations revealed test item related serious deep ulceration and necrosis in the forestomach accompanied by purulent inflammation in the kidney (purulent nephritis) and brain (purulent leptomeningitis) as well as centrilobular vacuolation in the liver in accordance with necropsy findings (thickened mucous membrane in the stomach).

BODY WEIGHT AND WEIGHT CHANGES
The slight changes in the body weight gain at the low dose in males and at all doses in females had no influence on the mean body weight. These changes were not considered treatment-related.

FOOD CONSUMPTION
The effect on the food consumption in the high dose males was reversible during the recovery period.

CLINICAL BIOCHEMISTRY FINDINGS
The slight, but statistically significant differences with respect to controls in total protein and cholesterol concentrations in females at 100 and 600 mg/kg bw/day, respectively, were considered to have little or no toxicological importance because all these changes were with low degree, values remained within the historical control ranges or there were no dose relevance. The differences with respect to the control in urea and bile acids in males at the highest dose as well as in total bilirubin and albumin levels in females at the highest dose were detected at the end of the recovery period but not at the termination of the treatment. Moreover, there were no histological alterations to substantiate the relevance of these changes.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus in one control female was due to circulatory disturbance developed during exsanguination. Hydrometra in one control females, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) it was judged not to be toxicologically relevant.
Scars on the skin, as observed in 1/12 male at 300 mg/kg bw/day, is a common spontaneous finding in this strain of experimental rats and was considered to be individual sign and not related to the test item.

ORGAN WEIGHT FINDINGS
The differences in thymus weights in high dose females were minor and not related to doses. In the lack of histological changes, thymus weight alterations were not considered to be related to the test item.
All changes in organ weights were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
Treatment related findings: The purulent lesions observed in prematurely euthanized male probably were in connection with bacterial infection, which was a consequence of the deep ulceration and necrosis in the forestomach. The centrilobular vacuolation in the liver of this animal could be developed due to inanition during the disease. The development of squamous cell hyperplasia and ulceration was considered to be a consequence of the local effect of the test item. Spontaneous occurrence of erosion or ulceration of the stomach is uncommon in rats. In the animals belonging to the 300 mg/kg bw/day treated groups, the intensity of squamous cell hyperplasia was milder than in the high dose group and ulceration was not detected. In the 100 mg/kg bw/day treated male and female animals the squamous cell hyperplasia and ulceration were not detectable. No histopathological changes were present in the stomach of recovery animals at 600 mg/kg bw/day (male or female) therefore these were considered to be reversible.
Non-treatment related findings: Acute changes in the lungs and thymus in control or high dose treated animals (alveolar emphysema, acute hemorrhage in the thymus) were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations. Hyperplasia of bronchus associated lymphoid tissue as observed in control animals and high dose treated females is a physiological immune-morphological phenomenon, without toxicological significance. The dilatation of uterine horns observed in control and high dose treated females - without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
haematology
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

17/17

5/5

12/12

0/12

0/17

4/4

Salivation

0/17

0/5

0/12

6/12

16/17

0/4

Compulsive biting of the bedding material

0/17

0/5

0/12

0/12

17/17

0/4

Nuzzling up the bedding material

0/17

0/5

0/12

12/12

17/17

0/4

Prone position

0/17

0/5

0/12

0/12

17/17

0/4

Closed eyes

0/17

0/5

0/12

0/12

17/17

0/4

Skin: scar

0/17

0/5

0/12

1/12

0/17

0/4

Piloerectiona

0/17

0/5

0/12

0/12

1/17

0/4

Decreased activitya

0/17

0/5

0/12

0/12

1/17

0/4

Decreased muscle tonea

0/17

0/5

0/12

0/12

1/17

0/4

Sanguineous fur around the nosea

0/17

0/5

0/12

0/12

1/17

0/4

Moribund

0/17

0/5

0/12

0/12

1/17

0/4

One animal of the high dose recovery group was re-located to main group due to early euthanazia of moribund animal.

* Including animals of recovery group

aNoted for moribund animal only

 

Table 2: Summary of clinical signs in females

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Pre-mating and mating periods

Normal

12/12

12/12

0/12

0/12

Salivation

0/12

0/12

8/12

6/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Prone position

0/12

0/12

0/12

12/12

Closed eyes

0/12

0/12

0/12

12/12

Gestation period

Normal

12/12

12/12

0/12

0/12

Salivation

0/12

0/12

2/12

3/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Alopecia

0/12

0/12

0/12

1/12

Lactation period

Normal

11/12

12/12

0/12

0/12

Salivation

0/12

0/12

1/12

2/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Pale

1/12

0/12

0/12

0/12

Piloerection

1/12

0/12

0/12

0/12

Alopecia

0/12

0/12

0/12

1/12

 

 

Table 3: Summary of clinical signs in females (recovery)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

5/5

5/5

-

-

0/5

5/5

Salivation

0/5

0/5

-

-

1/5

0/5

Nuzzling up the bedding material

0/5

0/5

-

-

5/5

0/5

Compulsive biting of the bedding material

0/5

0/5

-

-

5/5

0/5

Prone position

0/5

0/5

-

-

5/5

0/5

Closed eyes

0/5

0/5

-

-

5/5

0/5

* Including animals of recovery group

- no data

 

Table 4: Summary of body weight gain in males

Group

 

Body weight gain (g) between

Pre-mating days

Mating days

Post-mating days

Total

0 – 7

7 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 48

Control

Mean

29.2

16.9

19.6

21.1

18.5

14.5

14.2

134.2

SD

9.4

5.0

5.4

5.3

3.7

4.3

4.0

25.3

n

17

17

17

17

17

17

17

17

100 mg/kg bw/day

Mean

29.8

22.5**

22.0

19.5

18.7

16.3

12.1

140.8

SD

6.6

4.7

4.6

4.5

4.1

6.1

4.0

25.9

n

12

12

12

12

12

12

12

12

300 mg/kg bw/day

Mean

24.2

18.3

17.4

18.9

17.9

16.9

12.8

126.4

SD

6.1

4.3

8.3

6.1

4.0

7.1

4.6

21.8

n

12

12

12

12

12

12

12

12

600 mg/kg bw/day

Mean

11.0**

10.7*

17.6

19.0

18.2

8.4**

10.1

97.9**

SD

13.1

9.4

5.6

7.1

6.0

2.8

10.5

28.4

n

17

16

16

16

16

16

16

16

 

U

U

NS

NS

NS

U

NS

DN

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 5: Summary of body weight gain in males of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

7.2

0.8

8.0

SD

4.2

1.9

5.3

n

5

5

5

600 mg/kg bw/day

Mean

16.0*

9.8**

25.8**

SD

6.0

2.1

4.7

n

4

4

4

 

T

T

T

* p < 0.05, **p < 0.01

T = t-test vs. Control

 

Table 6: Summary of body weight gain in females

Group

 

Body weight gain (g) between

Pre-mating days

Total

Treatment days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 48

Control

Mean

2.6

8.4

11.0

6.8

1.8

3.2

8.2

2.0

34.8

SD

4.2

6.0

5.3

2.5

3.3

3.1

4.4

2.3

8.3

n

17

17

17

5

5

5

5

5

5

100 mg/kg bw/day

Mean

7.3

3.6*

10.9

-

-

-

-

-

-

SD

9.4

4.4

10.1

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

300 mg/kg bw/day

Mean

7.3*

3.5*

10.8

-

-

-

-

-

-

SD

4.8

4.7

4.1

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

600 mg/kg bw/day

Mean

6.8*

4.8

11.5

10.8

4.0

8.2

8.4

6.6

53.6

SD

4.9

5.4

6.9

4.5

5.4

5.5

5.9

4.2

27.2

n

17

17

17

5

5

5

5

5

5

 

U

DN

NS

NS

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 7: Summary of body weight gain in females of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

0.8

2.0

2.8

SD

4.7

4.8

2.9

n

5

5

5

600 mg/kg bw/day

Mean

2.4

0.0

2.4

SD

7.1

6.5

4.8

n

5

5

5

 

NS

NS

NS

NS = Not Significant

 

 

Table 8: Summary of food consumption in males

Group

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

20 – 27

27 – 34

34 – 41

41 – 48

Day 0 – 6

Day 6 – 13

Control

Mean

23.1

23.4

21.9

21.9

22.4

22.7

23.9

26.2

SD

1.6

1.2

2.2

2.0

1.9

2.3

1.9

2.4

n

8

8

8

8

8

8

2

2

100 mg/kg bw/day

Mean

22.6

24.1

22.3

21.9

22.4

21.5

-

-

SD

0.9

1.2

1.8

1.5

2.0

1.5

-

-

n

6

6

6

6

6

6

-

-

±%

-2

3

2

0

0

-5

-

-

300 mg/kg bw/day

Mean

21.8

22.3

21.8

21.6

22.4

21.9

-

-

SD

1.2

0.6

1.0

1.2

1.5

1.7

-

-

n

6

6

6

6

6

6

-

-

±%

-6

-5

0

-2

0

-4

-

-

600 mg/kg bw/day

Mean

18.7**

19.3**

21.4

20.7

20.6

19.7**

24.5

23.4

SD

1.6

2.1

2.4

2.5

1.6

1.5

0.5

3.9

n

8

8

8

8

8

8

2

2

±%

-19

-18

-2

-6

-8

-13

3

-11

 

 

DN

DN

NS

NS

DN

NS

NS

**p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 9: Summary of hematology

Group

 

White blood cell count [×10E9/L]

Neutrophil [%]

Lymphocyte [%]

Prothrombin time [sec]

Activated partial thromboplastin time [sec]

MALES – TREATMENT PERIOD

Control

Mean

9.39

18.56

77.68

20.30

21.30

SD

2.46

4.02

3.60

1.81

1.24

n

5

5

5

5

5

100 mg/kg bw/day

Mean

8.99

20.32

75.36

22.94

24.64*

SD

1.68

6.74

7.03

0.99

2.09

n

5

5

5

5

5

±%

-4

9

-3

13

16

300 mg/kg bw/day

Mean

9.83

21.66

74.10

21.78

23.46

SD

1.19

8.11

9.04

1.92

2.70

n

5

5

5

5

5

±%

5

17

-5

7

10

600 mg/kg bw/day

Mean

13.33*

39.50**

55.60**

22.12

22.34

SD

2.55

4.09

5.08

1.60

1.00

n

5

5

5

5

5

±%

42

113

-28

9

5

 

DN

DN

DN

NS

DN

MALES – RECOVERY PERIOD

Control

Mean

9.37

19.30

75.00

22.10

20.25

SD

1.19

3.49

3.87

1.49

1.59

n

5

5

5

4

4

600 mg/kg bw/day

Mean

11.00

20.45

73.70

23.78

22.65

SD

2.39

5.97

6.52

0.99

1.52

n

4

4

4

4

4

±%

17

6

-2

8

12

 

NS

NS

NS

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

5.00

32.14

63.82

21.98

19.26

SD

1.81

19.16

21.56

1.61

0.98

n

5

5

5

5

5

100 mg/kg bw/day

Mean

5.26

26.40

70.14

21.42

19.48

SD

0.94

12.80

12.49

1.33

1.19

n

5

5

5

5

5

±%

5

-18

10

-3

1

300 mg/kg bw/day

Mean

4.60

24.60

72.36

22.68

20.48

SD

1.01

7.24

7.22

1.59

1.39

n

5

5

5

5

5

±%

-8

-23

13

3

6

600 mg/kg bw/day

Mean

8.84**

41.82

54.72

17.66*

17.96

SD

2.68

3.72

4.45

4.91

2.95

n

5

5

5

5

5

±%

77

30

-14

-20

-7

 

DN

NS

NS

U

NS

FEMALES – RECOVERY PERIOD

Control

Mean

5.67

19.44

75.82

22.32

22.50

SD

0.54

4.27

4.48

0.50

1.47

n

5

5

5

5

5

600 mg/kg bw/day

Mean

7.32*

17.74

76.36

21.24

20.80

SD

1.29

5.28

5.51

1.35

0.83

n

5

5

5

5

5

±%

29

-9

1

-5

-8

 

*

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of necropsy findings

Organs

Observations

Frequency of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Main group

Recovery group

 

 

Main group - survivors

Main group - moribund

Recovery group

MALES

 

No macroscopic finding

12/12

5/5

12/12

4/12

0/12

0/1

0/4

Stomach

Thickened mucous membrane

0/12

0/5

0/12

6/12

12/12

1/1

4/4

Adhesion to liver

0/12

0/5

0/12

0/12

1/12

0/1

0/4

Thymus

Hemorrhages

0/12

0/5

0/12

1/12

0/12

0/1

0/4

Skin

Scar

0/12

0/5

0/12

1/12

0/12

0/1

0/4

FEMALES

 

No macroscopic finding

11/12

4/5

12/12

10/12

0/12

NA

3/5

Stomach

Thickened mucous membrane

0/12

0/5

0/12

2/12

12/12

NA

0/5

Thymus

Hemorrhages

1/12

0/5

0/12

0/12

0/12

NA

0/5

Uterus

Hydrometra

1/12

1/5

0/12

0/12

1/12

NA

2/5

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 11: Summary of organ weights of males

MALES – MAIN GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

386.1

9.96

0.43

0.63

0.077

SD

33.94

0.89

0.06

0.06

0.012

n

12

5

5

5

5

100 mg/kg bw/day

Mean

402.4

2.27

0.46

0.63

0.076

SD

37.96

0.32

0.14

0.10

0.010

n

12

5

5

5

5

±%

4

0

8

0

-2

300 mg/kg bw/day

Mean

387.5

2.41

0.43

0.70

0.077

SD

29.46

0.26

0.09

0.06

0.009

n

12

5

5

5

5

±%

0

6

0

11

0

600 mg/kg bw/day

Mean

354.0*

2.26

0.30*

0.67

0.077

SD

40.18

0.14

0.05

0.07

0.013

n

12

5

5

5

5

±%

-8

-1

-29

6

0

 

DN

NS

DN

NS

NS

MALES – RECOVERY GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

433.8

11.68

0.37

0.74

0.081

SD

28.99

1.78

0.04

0.12

0.010

n

5

5

5

5

5

600 mg/kg bw/day

Mean

400.8

10.54

0.44*

0.68

0.072

SD

23.26

0.75

0.03

0.13

0.017

n

4

4

4

4

4

±%

-8

-10

18

-8

-11

 

NS

NS

*

NS

NS

MALES – MAIN GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.519

0.109

0.161

0.020

SD

NA

0.074

0.023

0.025

0.004

n

NA

5

5

5

5

100 mg/kg bw/day

Mean

NA

2.545

0.118

0.163

0.020

SD

NA

0.148

0.032

0.027

0.002

n

NA

5

5

5

5

±%

NA

1

8

1

-1

300 mg/kg bw/day

Mean

NA

2.638

0.109

0.180

0.020

SD

NA

0.172

0.026

0.022

0.001

n

NA

5

5

5

5

±%

NA

5

0

12

0

600 mg/kg bw/day

Mean

NA

2.861**

0.083

0.187

0.021

SD

NA

0.111

0.013

0.035

0.003

n

NA

5

5

5

5

±%

NA

14

-24

16

7

 

 

DN

NS

NS

NS

MALES – RECOVERY GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.694

0.086

0.170

0.0188

SD

NA

0.379

0.011

0.024

0.0023

n

NA

5

5

5

5

600 mg/kg bw/day

Mean

NA

2.631

0.109**

0.168

0.0181

SD

NA

0.138

0.005

0.030

0.0045

n

NA

4

4

4

4

±%

NA

-2

27

-1

-4

 

 

NS

**

NS

NS

MALES – MAIN GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

18489.8

477.27

20.33

30.19

3.68

SD

1862.54

50.70

2.52

1.85

0.45

n

12

5

5

5

5

100 mg/kg bw/day

Mean

19297.8

466.17

21.75

29.95

3.57

SD

2093.34

47.69

6.88

6.16

0.34

n

12

5

5

5

5

±%

4

-2

7

-1

-3

300 mg/kg bw/day

Mean

18597.0

489.87

20.21

33.31

3.66

SD

1177.17

44.17

4.17

3.06

0.39

n

12

5

5

5

5

±%

1

3

-1

10

-1

600 mg/kg bw/day

Mean

17266.1

499.26

14.40

32.09

3.67

SD

1634.65

63.52

1.99

3.48

0.50

n

12

5

5

5

5

±%

-7

5

-29

6

0

 

NS

NS

NS

NS

NS

MALES – RECOVERY GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

19765.2

532.95

16.85

33.47

3.71

SD

1437.51

90.11

1.81

4.87

0.44

n

5

5

5

5

5

600 mg/kg bw/day

Mean

18984.2

498.58

20.63*

31.89

3.42

SD

1026.01

11.25

1.85

5.66

0.79

n

4

4

4

4

4

±%

-4

-6

22

-5

-8

 

NS

NS

*

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

NA = Not Applicable

 

Table 12: Summary of organ weights of females

FEMALES – MAIN GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

240.2

8.39

0.31

0.53

0.077

SD

15.63

0.58

0.04

0.07

0.012

n

5

5

5

5

5

100 mg/kg bw/day

Mean

244.2

9.04

0.24*

0.64

0.079

SD

12.76

0.76

0.05

0.08

0.016

n

5

5

5

5

5

±%

2

8

-22

20

2

300 mg/kg bw/day

Mean

245.0

8.72

0.21**

0.56

0.083

SD

14.98

0.93

0.04

0.07

0.022

n

5

5

5

5

5

±%

2

4

-30

5

7

600 mg/kg bw/day

Mean

240.4

9.89*

0.23*

0.70**

0.109**

SD

5.86

0.90

0.04

0.11

0.009

n

5

5

5

5

5

±%

0

18

-26

32

41

 

NS

DN

DN

DN

DN

FEMALES – RECOVERY GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

244.6

7.30

0.34

0.51

0.086

SD

18.16

0.65

0.03

0.08

0.018

n

5

5

5

5

5

600 mg/kg bw/day

Mean

260.0

8.50

0.31

0.58

0.073

SD

34.20

1.54

0.08

0.10

0.012

n

5

5

5

5

5

±%

6

16

-9

15

-15

 

NS

NS

NS

NS

NS

FEMALES – MAIN GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

3.497

0.128

0.22

0.0322

SD

NA

0.225

0.020

0.037

0.0039

n

NA

5

5

5

5

100 mg/kg bw/day

Mean

NA

3.700

0.098*

0.261

0.0323

SD

NA

0.175

0.019

0.027

0.0060

n

NA

5

5

5

5

±%

NA

6

-24

18

1

300 mg/kg bw/day

Mean

NA

3.557

0.087**

0.226

0.0336

SD

NA

0.282

0.015

0.017

0.0068

n

NA

5

5

5

5

±%

NA

2

-32

2

4

600 mg/kg bw/day

Mean

NA

4.113**

0.094*

0.290**

0.0453**

SD

NA

0.315

0.017

0.043

0.0030

n

NA

5

5

5

5

±%

NA

18

-27

31

41

 

 

DN

DN

DN

DN

FEMALES – RECOVERY GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.985

0.139

0.208

0.0351

SD

NA

0.175

0.013

0.037

0.0059

n

NA

5

5

5

5

600 mg/kg bw/day

Mean

NA

3.258*

0.122

0.224

0.0280*

SD

NA

0.190

0.040

0.024

0.0032

n

NA

5

5

5

5

±%

NA

9

-12

8

-20

 

 

*

NS

NS

*

FEMALES – MAIN GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

12217.7

426.56

15.58

27.07

3.94

SD

670.13

23.66

1.99

4.52

0.64

n

5

5

5

5

5

100 mg/kg bw/day

Mean

12460.5

461.21

43.80

32.46

4.00

SD

650.10

34.91

3.52

3.15

0.62

n

5

5

5

5

5

±%

2

8

1

20

2

300 mg/kg bw/day

Mean

12309.8

438.67

43.56

27.93

4.15

SD

568.78

48.81

6.67

3.24

0.99

n

5

5

5

5

5

±%

1

3

1

3

5

600 mg/kg bw/day

Mean

12954.2

532.84**

45.80

37.46**

5.88**

SD

607.70

48.28

3.06

4.63

0.55

n

5

5

5

5

5

±%

6

25

6

38

49

 

NS

DN

NS

DN

DN

FEMALES – RECOVERY GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

12557.0

374.60

17.49

26.03

4.41

SD

754.69

26.79

1.85

4.49

0.84

n

5

5

5

5

5

600 mg/kg bw/day

Mean

13902.4

455.15

16.63

31.15

3.90

SD

1803.32

85.56

4.30

4.94

0.62

n

5

5

5

5

5

±%

11

22

-5

20

-12

 

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

NA = Not Applicable

 

 

Table 13: Summary of histopathological findings

Organs

Observations

Incidence of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Main group

Recovery group

 

 

Main group - survivors

Main group - moribund

Recovery group

MALES

Brain

Purulent leptomeningitis

0/5

0/5

/

/

0/5

1/1

0/4

Epididymides

Storage of mature spermatozoa

12/12

5/5

/

/

12/12

1/1

4/4

Kidneys

Purulent nephritis

0/5

0/5

/

/

0/5

1/1

0/4

Liver

Centrilobular vacuolation

0/5

0/5

/

/

0/5

1/1

0/4

Lungs

Alveolar emphysema

1/5

0/5

/

/

0/5

0/1

0/4

Hyperplasia of BALT

2/5

1/5

/

/

0/5

0/1

1/4

Stomach - forestomach

Squamous cell hyperplasia

0/12

0/5

0/12

12/12

12/12

1/1

0/4

Ulceration

0/12

0/5

0/12

0/12

10/12

1/1

0/4

FEMALES

Lungs

Alveolar emphysema

1/5

1/5

/

/

1/5

NA

0/5

Hyperplasia of BALT

2/5

1/5

/

/

1/5

NA

1/5

Stomach - forestomach

Squamous cell hyperplasia

0/12

0/5

0/12

12/12

12/12

NA

0/5

Ulceration

0/12

0/5

0/12

0/12

10/12

NA

0/5

Thymus

Acute hemorrhage

1/12

0/5

/

/

0/5

NA

0/5

Uterus

Dilatation

1/12

1/5

/

/

1/12

NA

2/5

Incidence of observation =number of animals with observations / number of animals examined

/ = not examined

BALT = bronchus associated lymphoid tissue

NA = not applicable

 


 

Table 14: Summary of litter weight and litter weight gain of offspring

Group

 

Litter weight (g) on post-natal days

Litter weight gain (g) between post-natal days

0

4

13

0 - 4

4 - 13

0 - 13

Control

Mean

57.6

108.9

232.0

51.3

147.6

187.5

SD

11.6

14.7

18.6

11.7

15.9

19.8

n

11

11

11

11

11

11

100 mg/kg bw/day

Mean

65.1

114.9

231.2

50.2

146.3

184.3

SD

13.3

22.6

51.8

11.9

32.1

42.3

n

12

12

12

12

12

12

300 mg/kg bw/day

Mean

63.3

104.7

230.4

44.2

145.4

181.6

SD

11.2

16.3

10.8

5.5

9.4

9.9

n

12

12

12

12

12

12

600 mg/kg bw/day

Mean

58.0

92.7

184.9**

35.2**

115.6**

141.7**

SD

15.7

27.0

41.6

12.1

31.6

36.9

n

12

12

12

12

12

12

 

NS

NS

U

DN

U

U

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

U = Mann-Whitney U-test vs. Control

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity after oral administration via gavage of the test substance to male and female Hsd.Han: Wistar rats was 300 mg/kg bw/day in both sexes. The NOAEL for local toxicity after oral administration via gavage of the test substance to male and female Hsd.Han: Wistar rats was 100 mg/kg bw/day in both sexes.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 63 - 66 days (males), 84 - 86 days (females)
- Weight at study initiation: 250 - 306 g (males), 189 - 236 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthii annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was formulated in sunflower oil at in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the vehicle for the test item was analytically verified up front. The test material was stable in sunflower oil at concentrations of 1 mg/mL and 250 mg/mL at room temperature for 1 day and in a refrigerator (at 5 ± 3 °C) for 3 days.
- Concentration in vehicle: 25, 75 and 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no.: 1604-4373 and 1607-4569
- Quality: Ph.Hg. VIII
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred
- Proof of pregnancy:vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 10 mL of each formulation (25, 75 and 150 mg/mL) and five aliquots of 10 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 94% to 99% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from sunflower oil formulations was 101 and 103% at ~1 and ~250 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 97% of starting concentrations at 1 mg/mL and 96% at 250 mg/mL) and at 5 ± 3°C for 3 days (recovery was 99% of starting concentrations at 1 mg/mL and 94% at 250 mg/mL).
Duration of treatment / exposure:
49 days (females, control and test groups)
55 days (females, control and test groups)
49 days and 14 day post-exposure observation period (recovery groups)
Frequency of treatment:
once daily at similar time (± 2 h), 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Treatment period:
Control: 12/sex
100 mg/kg bw/day: 12/sex
300 mg/kg bw/day: 12/sex
600 mg/kg bw/day: 12 females and 13 males

Treatment and recovery period:
Control: 5/sex
600 mg/kg bw/day: 5 females and 4 males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the test material in rats. The high was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations on parental animals (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily for for animals showing clinical signs at 600 mg/kg bw/day, prior to the first exposure and once weekly thereafter on all animals, weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 54.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization; however, several animals that failed to exhibit typical 4-5 days cycles were included in the study due to the large number of animals with irregular cycle. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smears were also prepared on the day of the necropsy. Vaginal smears were stained with 1% aqueous methylene blue solution and were examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in male parental generation:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies, postnatal mortality, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the optionally extended post-mating period on Day 49.
- Maternal animals: All surviving animals not selected for toxicology examinations on post-partum day 13 or shortly thereafter (Day 55). All surviving animals selected for toxicology examinations shortly after post-partum day 13 (Day 55).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively: ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (600 mg/kg bw/day).
Postmortem examinations (offspring):
SACRIFICE
- All offspring were sacrificed on postnatal day 13 or shortly thereafter.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of examinations for gross abnormalities.

OTHER: The serum level of T4 (thyroid hormone) was determined in pups sacrificed on Day 13.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
Reproductive indices:
Copulatory index males [%] = Number of males with confirmed mating/Total number of males cohabited x 100
Copulatory index females [%] = Number of sperm positive females/Total number of females cohabited x 100
Fertility index males [%] = Number of males impregnating a female/Total number of males with confirmed mating x 100
Fertility index females [%] = Number of pregnant females/Number of sperm positive females x 100
Gestation index [%] = Number of females with live born pups/Number of pregnant females x 100
Offspring viability indices:
Post-implantation mortality (intrauterine mortality) [%] = (Number of implantations - Number of liveborns)/Number of implantations x 100
Post-natal mortality [%] = (Number of liveborns - Number of live pups on postnatal day 13)/Number of pups born x 100
Survival index [%] = Number of live pups on postnatal day 13/Number of pups born x 100
Sex ratio [%] = (Number of pups examined - Number of males (females)/Number of pups examined x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES
300 mg/kg bw/day: salivation (6/12 animals) and nuzzling up the bedding material (12/12 animals) shortly after the daily administration; scar on the skin of the neck during the last week of the treatment period in 1/12 animal
600 mg/kg bw/day: salivation (16/17 animals), nuzzling up the bedding material (17/17 animals), compulsive biting of the bedding material (17/17 animals), prone position (17/17 animals) and closed eyes (17/17 animals) with variable incidence and duration; prone position and closed eye ceased between Day 13 and 24

FEMALES
Control: piloerection and paleness at parturition (1/12 animal)
300 mg/kg bw/day: salivation and nuzzling up the bedding material during the pre-mating (8/12 and 12/12 animals, respectively), gestation (2/12 and 12/12 animals, respectively) and lactation (1/12 and 12/12 animals, respectively) periods
600 mg/kg bw/day: salivation (7/17 animals), nuzzling up the bedding material (17/17 animals), compulsive biting of the bedding material (17/17 animals), prone position (17/17 animals) and closed eyes (17/17 animals) during the pre-mating period (main groups) or during the treatment period (recovery groups); compulsive biting of the bedding material and nuzzling up the bedding material during gestation (12/12 animals) and salivation during gestation and lactation (3/12 and 2/12, respectively) for a short period of time after the treatment; alopecia of the abdomen in 1/12 dam between gestation days 19 and lactation day 8
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
600 mg/kg bw/day: 1/17 males euthanized
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistical signigicant increased mean body weight gain between Days 7 and 13 (non-treatment related)
600 mg/kg bw/day: statistical signigicant decreased mean body weight gain between Days 34 and 41 and for the whole treatment period (between Days 0 and 48); increased mean body weight gain at each occasion of the recovery period; slightly increased mean body weight during recovery period (not statistically significant)

FEMALES
100 mg/kg bw/day: statistical significant decreased mean body weight gain between Days 7 and 13 (non-treatment related)
300 mg/kg bw/day: statistical significant increased mean body weight gain between Days 0 and 7 (non-treatment related); statistical significant decreased mean body weight gain between Days 7 and 13 (non-treatment related)
600 mg/kg bw/day: statistical significant increased mean body weight gain between Days 0 and 7 (non-treatment related)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
MALES
600 mg/kg bw/day: statistical significant decreased mean daily food consumption during the entire treatment period with statistical significance during the premating period and last week of the treatment period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: slightly longer activated thromboplastin time (not significant)
600 mg/kg bw/day: statistical significant increased mean white blood cell count and mean percentage of neutrophil granulocytes; statistical significant decreased percentage of lymphocytes

FEMALES
600 mg/kg bw/day: statistical significant increased mean white blood cell count; slightly shorter prothrombin time; statistical significant increased percentage of neutrophil granulocytes and decreased percentage of lymphocytes (both not statistically significant); slightly increased mean white blood cell count at the end of the recovery period
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
600 mg/kg bw/day: statistical significant slightly increased mean concentrations of urea and bile acids at the end of the recovery period (not significant)

FEMALES
100 mg/kg bw/day: statistical significant increased mean total protein concentration
600 mg/kg bw/day: statistical significant slightly increased mean cholesterol concentration; statistical significant slightly increased concentrations of total bilirubin, cholesterol and bile acids at the end of the recovery period; statistical significant slightly decreased albumin level at the end of the recovery period
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES
Control: alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (2/5 animals; non-treatment related); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period (1/5 animal; non-treatment related)
300 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals)
600 mg/kg bw/day: serious deep ulceration and necrosis in the forestomach and purulent inflammation in the kidney (purulent nephritis) and brain (purulent leptomeningitis), as well as centrilobular vacuolation in the liver of the prematurely euthanized animal; squamous cell hyperplasia in the forestomach (12/12 animals); ulceration in the forestomach (10/12 animals); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period (1/4 animal; non-treatment related)

FEMALES
Control: alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); acute hemorrhage in the thymus (1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (1/5 animal; non-treatment related); dilatation of uterine horns (1/12 animal; non-treatment related); dilatation of uterine horns at the end of the recovery period (1/5 animal; non-treatment related); alveolar emphysema at the end of the recovery period (1/5 animal; non-treatment related)
300 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals)
600 mg/kg bw/day: squamous cell hyperplasia in the forestomach (12/12 animals); ulceration in the forestomach (10/12 animals); alveolar emphysema (minimal degree; 1/5 animal; non-treatment related); hyperplasia of bronchus associated lymphoid tissue (1/5 animal; non-treatment related); dilatation of uterine horns (1/12 animal; non-treatment related); dilatation of uterine horns at the end of the recovery period (2/5 animals; non-treatment related); hyperplasia of bronchus associated lymphoid tissue at the end of the recovery period(1/5 animal; non-treatment related)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS
Dermal changes (scar in 1/12 male of the mid dose group and alopecia in 1/12 female of the high dose group) are common spontaneous finding in this strain of experimental rats and are seen also in untreated rats. These were considered to be individual signs and not related to the test item.

MORTALITY
The male of the high dose group was euthanized due to its moribund condition and subjected to necropsy on Day 9. Nuzzling up the bedding material, compulsive biting of the bedding material, prone position, closed eyes, piloerection, decreased muscle tone, sanguineous fur around the nose and decreased activity were noted for this animal between Days 1 and 9. The body weight of this animal decreased significantly during the first week of study and animal became moribund on Day 9. Histological examinations revealed test item related serious deep ulceration and necrosis in the forestomach accompanied by purulent inflammation in the kidney (purulent nephritis) and brain (purulent leptomeningitis) as well as centrilobular vacuolation in the liver in accordance with necropsy findings (thickened mucous membrane in the stomach).

BODY WEIGHT AND WEIGHT CHANGES
The slight changes in the body weight gain at the low dose in males and at all doses in females had no influence on the mean body weight. These changes were not considered treatment-related.

FOOD CONSUMPTION
The effect on the food consumption in the high dose males was reversible during the recovery period.

CLINICAL BIOCHEMISTRY FINDINGS
The slight, but statistically significant differences with respect to controls in total protein and cholesterol concentrations in females at 100 and 600 mg/kg bw/day, respectively, were considered to have little or no toxicological importance because all these changes were with low degree, values remained within the historical control ranges or there were no dose relevance. The differences with respect to the control in urea and bile acids in males at the highest dose as well as in total bilirubin and albumin levels in females at the highest dose were detected at the end of the recovery period but not at the termination of the treatment. Moreover, there were no histological alterations to substantiate the relevance of these changes.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus in one control female was due to circulatory disturbance developed during exsanguination. Hydrometra in one control females, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) it was judged not to be toxicologically relevant.
Scars on the skin, as observed in 1/12 male at 300 mg/kg bw/day, is a common spontaneous finding in this strain of experimental rats and was considered to be individual sign and not related to the test item.

ORGAN WEIGHT FINDINGS
The differences in thymus weights in high dose females were minor and not related to doses. In the lack of histological changes, thymus weight alterations were not considered to be related to the test item.
All changes in organ weights were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
Treatment related findings: The purulent lesions observed in prematurely euthanized male probably were in connection with bacterial infection, which was a consequence of the deep ulceration and necrosis in the forestomach. The centrilobular vacuolation in the liver of this animal could be developed due to inanition during the disease. The development of squamous cell hyperplasia and ulceration was considered to be a consequence of the local effect of the test item. Spontaneous occurrence of erosion or ulceration of the stomach is uncommon in rats. In the animals belonging to the 300 mg/kg bw/day treated groups, the intensity of squamous cell hyperplasia was milder than in the high dose group and ulceration was not detected. In the 100 mg/kg bw/day treated male and female animals the squamous cell hyperplasia and ulceration were not detectable. No histopathological changes were present in the stomach of recovery animals at 600 mg/kg bw/day (male or female) therefore these were considered to be reversible.
Non-treatment related findings: Acute changes in the lungs and thymus in control or high dose treated animals (alveolar emphysema, acute hemorrhage in the thymus) were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations. Hyperplasia of bronchus associated lymphoid tissue as observed in control animals and high dose treated females is a physiological immune-morphological phenomenon, without toxicological significance. The dilatation of uterine horns observed in control and high dose treated females - without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.

REPRODUCTIVE FUNCTION / PERFORMANCE
There were no test item related differences in the evaluated delivery parameters of dams between the control and test item treated groups. The number of pregnant females and dams delivered, the mean number of implantations, post-implantation loss, duration of pregnancy, and pups births (total, live, still birth, viable) and live birth index were comparable in all groups. Statistical significance was noted with respect to the control for the lower mean post-implantation loss at 600 mg/kg bw/day dams. This deviation had no toxicological meaning but was indicative of biological variation. All examined parameters of reproductive performance were comparable in the control and treated animals. There were no difference between the control and test item treated groups in copulatory and fertility indices (male and female animals), in the pre-coital interval, conceiving days and gestation indices.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
Dose descriptor:
NOAEL
Remarks:
local effetcs
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: statistical significant slightly decreased mean body weight gain between postnatal days 0 and 4

600 mg/kg bw/day: statistical significant slightly decreased mean litter weight on postnatal day 13; statistical significant slightly decreased mean litter weight gain between postnatal days 0 and 4 and 4 and 13 as well as for the whole observation period (between postnatal days 0 and 13); statistical significant decreased mean body weight of pups on postnatal days 4 and 13; statistical significant decreased mean body weight gain in pups between postnatal days 0 and 4, 4 and 13 as well as for the whole observation period (between postnatal days 0 and 13)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in Day 13 pups compared to the control.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day: autolyzed visceral organs in dead pups (6/7) on postnatal day 1 or 2

600 mg/kg bw/day: autolyzed visceral organs in dead pups (1/1) on postnatal day 1 or 2
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
SEX DISTRIBUTION
600 mg/kg bw/day: slightly decreased number and percentage of male pups per litter and increased mean number and percentage of female pups per litter

ANOGENITAL DISTANCE
100, 300 and 600 mg/kg bw/day: slightly longer anogenital distance in female pups

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

BODY WEIGHT AND BODY WEIGHT GAIN
The minor change in body weight gain in pups of dams at 300 mg/kg bw/day did not result in significant change in the mean body weight or in summarized mean body weight gain of pups therefore was not considered to be toxicologically relevant.

SEX DISTRIBUTION
A test substance relation with regard to difference in sex distribution at the highest dose cannot be excluded and cannot be verified by the results of this study. However, taking into consideration the historical control ranges of sex distribution (43-61 % for male and 39-60 % for female pups) this slight deviation from the control was judged to be indicative of biological variation.

ANOGENITAL DISTANCE
In the lack of dose dependency and due to the minor degree, slightly longer anogenital distance in female pups with respect to the control was not considered to be toxicologically relevant.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

17/17

5/5

12/12

0/12

0/17

4/4

Salivation

0/17

0/5

0/12

6/12

16/17

0/4

Compulsive biting of the bedding material

0/17

0/5

0/12

0/12

17/17

0/4

Nuzzling up the bedding material

0/17

0/5

0/12

12/12

17/17

0/4

Prone position

0/17

0/5

0/12

0/12

17/17

0/4

Closed eyes

0/17

0/5

0/12

0/12

17/17

0/4

Skin: scar

0/17

0/5

0/12

1/12

0/17

0/4

Piloerectiona

0/17

0/5

0/12

0/12

1/17

0/4

Decreased activitya

0/17

0/5

0/12

0/12

1/17

0/4

Decreased muscle tonea

0/17

0/5

0/12

0/12

1/17

0/4

Sanguineous fur around the nosea

0/17

0/5

0/12

0/12

1/17

0/4

Moribund

0/17

0/5

0/12

0/12

1/17

0/4

One animal of the high dose recovery group was re-located to main group due to early euthanazia of moribund animal.

* Including animals of recovery group

aNoted for moribund animal only

 

Table 2: Summary of clinical signs in females

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Pre-mating and mating periods

Normal

12/12

12/12

0/12

0/12

Salivation

0/12

0/12

8/12

6/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Prone position

0/12

0/12

0/12

12/12

Closed eyes

0/12

0/12

0/12

12/12

Gestation period

Normal

12/12

12/12

0/12

0/12

Salivation

0/12

0/12

2/12

3/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Alopecia

0/12

0/12

0/12

1/12

Lactation period

Normal

11/12

12/12

0/12

0/12

Salivation

0/12

0/12

1/12

2/12

Nuzzling up the bedding material

0/12

0/12

12/12

12/12

Compulsive biting of the bedding material

0/12

0/12

0/12

12/12

Pale

1/12

0/12

0/12

0/12

Piloerection

1/12

0/12

0/12

0/12

Alopecia

0/12

0/12

0/12

1/12

 

 

Table 3: Summary of clinical signs in females (recovery)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

5/5

5/5

-

-

0/5

5/5

Salivation

0/5

0/5

-

-

1/5

0/5

Nuzzling up the bedding material

0/5

0/5

-

-

5/5

0/5

Compulsive biting of the bedding material

0/5

0/5

-

-

5/5

0/5

Prone position

0/5

0/5

-

-

5/5

0/5

Closed eyes

0/5

0/5

-

-

5/5

0/5

* Including animals of recovery group

- no data

 

Table 4: Summary of body weight gain in males

Group

 

Body weight gain (g) between

Pre-mating days

Mating days

Post-mating days

Total

0 – 7

7 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 48

Control

Mean

29.2

16.9

19.6

21.1

18.5

14.5

14.2

134.2

SD

9.4

5.0

5.4

5.3

3.7

4.3

4.0

25.3

n

17

17

17

17

17

17

17

17

100 mg/kg bw/day

Mean

29.8

22.5**

22.0

19.5

18.7

16.3

12.1

140.8

SD

6.6

4.7

4.6

4.5

4.1

6.1

4.0

25.9

n

12

12

12

12

12

12

12

12

300 mg/kg bw/day

Mean

24.2

18.3

17.4

18.9

17.9

16.9

12.8

126.4

SD

6.1

4.3

8.3

6.1

4.0

7.1

4.6

21.8

n

12

12

12

12

12

12

12

12

600 mg/kg bw/day

Mean

11.0**

10.7*

17.6

19.0

18.2

8.4**

10.1

97.9**

SD

13.1

9.4

5.6

7.1

6.0

2.8

10.5

28.4

n

17

16

16

16

16

16

16

16

 

U

U

NS

NS

NS

U

NS

DN

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 5: Summary of body weight gain in males of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

7.2

0.8

8.0

SD

4.2

1.9

5.3

n

5

5

5

600 mg/kg bw/day

Mean

16.0*

9.8**

25.8**

SD

6.0

2.1

4.7

n

4

4

4

 

T

T

T

* p < 0.05, **p < 0.01

T = t-test vs. Control

 

Table 6: Summary of body weight gain in females

Group

 

Body weight gain (g) between

Pre-mating days

Total

Treatment days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 48

Control

Mean

2.6

8.4

11.0

6.8

1.8

3.2

8.2

2.0

34.8

SD

4.2

6.0

5.3

2.5

3.3

3.1

4.4

2.3

8.3

n

17

17

17

5

5

5

5

5

5

100 mg/kg bw/day

Mean

7.3

3.6*

10.9

-

-

-

-

-

-

SD

9.4

4.4

10.1

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

300 mg/kg bw/day

Mean

7.3*

3.5*

10.8

-

-

-

-

-

-

SD

4.8

4.7

4.1

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

600 mg/kg bw/day

Mean

6.8*

4.8

11.5

10.8

4.0

8.2

8.4

6.6

53.6

SD

4.9

5.4

6.9

4.5

5.4

5.5

5.9

4.2

27.2

n

17

17

17

5

5

5

5

5

5

 

U

DN

NS

NS

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 7: Summary of body weight gain in females of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

0.8

2.0

2.8

SD

4.7

4.8

2.9

n

5

5

5

600 mg/kg bw/day

Mean

2.4

0.0

2.4

SD

7.1

6.5

4.8

n

5

5

5

 

NS

NS

NS

NS = Not Significant

 

 

Table 8: Summary of food consumption in males

Group

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

20 – 27

27 – 34

34 – 41

41 – 48

Day 0 – 6

Day 6 – 13

Control

Mean

23.1

23.4

21.9

21.9

22.4

22.7

23.9

26.2

SD

1.6

1.2

2.2

2.0

1.9

2.3

1.9

2.4

n

8

8

8

8

8

8

2

2

100 mg/kg bw/day

Mean

22.6

24.1

22.3

21.9

22.4

21.5

-

-

SD

0.9

1.2

1.8

1.5

2.0

1.5

-

-

n

6

6

6

6

6

6

-

-

±%

-2

3

2

0

0

-5

-

-

300 mg/kg bw/day

Mean

21.8

22.3

21.8

21.6

22.4

21.9

-

-

SD

1.2

0.6

1.0

1.2

1.5

1.7

-

-

n

6

6

6

6

6

6

-

-

±%

-6

-5

0

-2

0

-4

-

-

600 mg/kg bw/day

Mean

18.7**

19.3**

21.4

20.7

20.6

19.7**

24.5

23.4

SD

1.6

2.1

2.4

2.5

1.6

1.5

0.5

3.9

n

8

8

8

8

8

8

2

2

±%

-19

-18

-2

-6

-8

-13

3

-11

 

 

DN

DN

NS

NS

DN

NS

NS

**p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 9: Summary of hematology

Group

 

White blood cell count [×10E9/L]

Neutrophil [%]

Lymphocyte [%]

Prothrombin time [sec]

Activated partial thromboplastin time [sec]

MALES – TREATMENT PERIOD

Control

Mean

9.39

18.56

77.68

20.30

21.30

SD

2.46

4.02

3.60

1.81

1.24

n

5

5

5

5

5

100 mg/kg bw/day

Mean

8.99

20.32

75.36

22.94

24.64*

SD

1.68

6.74

7.03

0.99

2.09

n

5

5

5

5

5

±%

-4

9

-3

13

16

300 mg/kg bw/day

Mean

9.83

21.66

74.10

21.78

23.46

SD

1.19

8.11

9.04

1.92

2.70

n

5

5

5

5

5

±%

5

17

-5

7

10

600 mg/kg bw/day

Mean

13.33*

39.50**

55.60**

22.12

22.34

SD

2.55

4.09

5.08

1.60

1.00

n

5

5

5

5

5

±%

42

113

-28

9

5

 

DN

DN

DN

NS

DN

MALES – RECOVERY PERIOD

Control

Mean

9.37

19.30

75.00

22.10

20.25

SD

1.19

3.49

3.87

1.49

1.59

n

5

5

5

4

4

600 mg/kg bw/day

Mean

11.00

20.45

73.70

23.78

22.65

SD

2.39

5.97

6.52

0.99

1.52

n

4

4

4

4

4

±%

17

6

-2

8

12

 

NS

NS

NS

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

5.00

32.14

63.82

21.98

19.26

SD

1.81

19.16

21.56

1.61

0.98

n

5

5

5

5

5

100 mg/kg bw/day

Mean

5.26

26.40

70.14

21.42

19.48

SD

0.94

12.80

12.49

1.33

1.19

n

5

5

5

5

5

±%

5

-18

10

-3

1

300 mg/kg bw/day

Mean

4.60

24.60

72.36

22.68

20.48

SD

1.01

7.24

7.22

1.59

1.39

n

5

5

5

5

5

±%

-8

-23

13

3

6

600 mg/kg bw/day

Mean

8.84**

41.82

54.72

17.66*

17.96

SD

2.68

3.72

4.45

4.91

2.95

n

5

5

5

5

5

±%

77

30

-14

-20

-7

 

DN

NS

NS

U

NS

FEMALES – RECOVERY PERIOD

Control

Mean

5.67

19.44

75.82

22.32

22.50

SD

0.54

4.27

4.48

0.50

1.47

n

5

5

5

5

5

600 mg/kg bw/day

Mean

7.32*

17.74

76.36

21.24

20.80

SD

1.29

5.28

5.51

1.35

0.83

n

5

5

5

5

5

±%

29

-9

1

-5

-8

 

*

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of necropsy findings

Organs

Observations

Frequency of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Main group

Recovery group

 

 

Main group - survivors

Main group - moribund

Recovery group

MALES

 

No macroscopic finding

12/12

5/5

12/12

4/12

0/12

0/1

0/4

Stomach

Thickened mucous membrane

0/12

0/5

0/12

6/12

12/12

1/1

4/4

Adhesion to liver

0/12

0/5

0/12

0/12

1/12

0/1

0/4

Thymus

Hemorrhages

0/12

0/5

0/12

1/12

0/12

0/1

0/4

Skin

Scar

0/12

0/5

0/12

1/12

0/12

0/1

0/4

FEMALES

 

No macroscopic finding

11/12

4/5

12/12

10/12

0/12

NA

3/5

Stomach

Thickened mucous membrane

0/12

0/5

0/12

2/12

12/12

NA

0/5

Thymus

Hemorrhages

1/12

0/5

0/12

0/12

0/12

NA

0/5

Uterus

Hydrometra

1/12

1/5

0/12

0/12

1/12

NA

2/5

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 11: Summary of organ weights of males

MALES – MAIN GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

386.1

9.96

0.43

0.63

0.077

SD

33.94

0.89

0.06

0.06

0.012

n

12

5

5

5

5

100 mg/kg bw/day

Mean

402.4

2.27

0.46

0.63

0.076

SD

37.96

0.32

0.14

0.10

0.010

n

12

5

5

5

5

±%

4

0

8

0

-2

300 mg/kg bw/day

Mean

387.5

2.41

0.43

0.70

0.077

SD

29.46

0.26

0.09

0.06

0.009

n

12

5

5

5

5

±%

0

6

0

11

0

600 mg/kg bw/day

Mean

354.0*

2.26

0.30*

0.67

0.077

SD

40.18

0.14

0.05

0.07

0.013

n

12

5

5

5

5

±%

-8

-1

-29

6

0

 

DN

NS

DN

NS

NS

MALES – RECOVERY GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

433.8

11.68

0.37

0.74

0.081

SD

28.99

1.78

0.04

0.12

0.010

n

5

5

5

5

5

600 mg/kg bw/day

Mean

400.8

10.54

0.44*

0.68

0.072

SD

23.26

0.75

0.03

0.13

0.017

n

4

4

4

4

4

±%

-8

-10

18

-8

-11

 

NS

NS

*

NS

NS

MALES – MAIN GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.519

0.109

0.161

0.020

SD

NA

0.074

0.023

0.025

0.004

n

NA

5

5

5

5

100 mg/kg bw/day

Mean

NA

2.545

0.118

0.163

0.020

SD

NA

0.148

0.032

0.027

0.002

n

NA

5

5

5

5

±%

NA

1

8

1

-1

300 mg/kg bw/day

Mean

NA

2.638

0.109

0.180

0.020

SD

NA

0.172

0.026

0.022

0.001

n

NA

5

5

5

5

±%

NA

5

0

12

0

600 mg/kg bw/day

Mean

NA

2.861**

0.083

0.187

0.021

SD

NA

0.111

0.013

0.035

0.003

n

NA

5

5

5

5

±%

NA

14

-24

16

7

 

 

DN

NS

NS

NS

MALES – RECOVERY GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.694

0.086

0.170

0.0188

SD

NA

0.379

0.011

0.024

0.0023

n

NA

5

5

5

5

600 mg/kg bw/day

Mean

NA

2.631

0.109**

0.168

0.0181

SD

NA

0.138

0.005

0.030

0.0045

n

NA

4

4

4

4

±%

NA

-2

27

-1

-4

 

 

NS

**

NS

NS

MALES – MAIN GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

18489.8

477.27

20.33

30.19

3.68

SD

1862.54

50.70

2.52

1.85

0.45

n

12

5

5

5

5

100 mg/kg bw/day

Mean

19297.8

466.17

21.75

29.95

3.57

SD

2093.34

47.69

6.88

6.16

0.34

n

12

5

5

5

5

±%

4

-2

7

-1

-3

300 mg/kg bw/day

Mean

18597.0

489.87

20.21

33.31

3.66

SD

1177.17

44.17

4.17

3.06

0.39

n

12

5

5

5

5

±%

1

3

-1

10

-1

600 mg/kg bw/day

Mean

17266.1

499.26

14.40

32.09

3.67

SD

1634.65

63.52

1.99

3.48

0.50

n

12

5

5

5

5

±%

-7

5

-29

6

0

 

NS

NS

NS

NS

NS

MALES – RECOVERY GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

19765.2

532.95

16.85

33.47

3.71

SD

1437.51

90.11

1.81

4.87

0.44

n

5

5

5

5

5

600 mg/kg bw/day

Mean

18984.2

498.58

20.63*

31.89

3.42

SD

1026.01

11.25

1.85

5.66

0.79

n

4

4

4

4

4

±%

-4

-6

22

-5

-8

 

NS

NS

*

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

NA = Not Applicable

 

Table 12: Summary of organ weights of females

FEMALES – MAIN GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

240.2

8.39

0.31

0.53

0.077

SD

15.63

0.58

0.04

0.07

0.012

n

5

5

5

5

5

100 mg/kg bw/day

Mean

244.2

9.04

0.24*

0.64

0.079

SD

12.76

0.76

0.05

0.08

0.016

n

5

5

5

5

5

±%

2

8

-22

20

2

300 mg/kg bw/day

Mean

245.0

8.72

0.21**

0.56

0.083

SD

14.98

0.93

0.04

0.07

0.022

n

5

5

5

5

5

±%

2

4

-30

5

7

600 mg/kg bw/day

Mean

240.4

9.89*

0.23*

0.70**

0.109**

SD

5.86

0.90

0.04

0.11

0.009

n

5

5

5

5

5

±%

0

18

-26

32

41

 

NS

DN

DN

DN

DN

FEMALES – RECOVERY GROUP

 

 

Body weight [g]

Organ weight [g]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

244.6

7.30

0.34

0.51

0.086

SD

18.16

0.65

0.03

0.08

0.018

n

5

5

5

5

5

600 mg/kg bw/day

Mean

260.0

8.50

0.31

0.58

0.073

SD

34.20

1.54

0.08

0.10

0.012

n

5

5

5

5

5

±%

6

16

-9

15

-15

 

NS

NS

NS

NS

NS

FEMALES – MAIN GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

3.497

0.128

0.22

0.0322

SD

NA

0.225

0.020

0.037

0.0039

n

NA

5

5

5

5

100 mg/kg bw/day

Mean

NA

3.700

0.098*

0.261

0.0323

SD

NA

0.175

0.019

0.027

0.0060

n

NA

5

5

5

5

±%

NA

6

-24

18

1

300 mg/kg bw/day

Mean

NA

3.557

0.087**

0.226

0.0336

SD

NA

0.282

0.015

0.017

0.0068

n

NA

5

5

5

5

±%

NA

2

-32

2

4

600 mg/kg bw/day

Mean

NA

4.113**

0.094*

0.290**

0.0453**

SD

NA

0.315

0.017

0.043

0.0030

n

NA

5

5

5

5

±%

NA

18

-27

31

41

 

 

DN

DN

DN

DN

FEMALES – RECOVERY GROUP

 

 

 

Organ weight relative to body weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

NA

2.985

0.139

0.208

0.0351

SD

NA

0.175

0.013

0.037

0.0059

n

NA

5

5

5

5

600 mg/kg bw/day

Mean

NA

3.258*

0.122

0.224

0.0280*

SD

NA

0.190

0.040

0.024

0.0032

n

NA

5

5

5

5

±%

NA

9

-12

8

-20

 

 

*

NS

NS

*

FEMALES – MAIN GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

12217.7

426.56

15.58

27.07

3.94

SD

670.13

23.66

1.99

4.52

0.64

n

5

5

5

5

5

100 mg/kg bw/day

Mean

12460.5

461.21

43.80

32.46

4.00

SD

650.10

34.91

3.52

3.15

0.62

n

5

5

5

5

5

±%

2

8

1

20

2

300 mg/kg bw/day

Mean

12309.8

438.67

43.56

27.93

4.15

SD

568.78

48.81

6.67

3.24

0.99

n

5

5

5

5

5

±%

1

3

1

3

5

600 mg/kg bw/day

Mean

12954.2

532.84**

45.80

37.46**

5.88**

SD

607.70

48.28

3.06

4.63

0.55

n

5

5

5

5

5

±%

6

25

6

38

49

 

NS

DN

NS

DN

DN

FEMALES – RECOVERY GROUP

 

 

Body weight relative to brain weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Thymus

Spleen

Adrenal glands

Control

Mean

12557.0

374.60

17.49

26.03

4.41

SD

754.69

26.79

1.85

4.49

0.84

n

5

5

5

5

5

600 mg/kg bw/day

Mean

13902.4

455.15

16.63

31.15

3.90

SD

1803.32

85.56

4.30

4.94

0.62

n

5

5

5

5

5

±%

11

22

-5

20

-12

 

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

NA = Not Applicable

 

 

Table 13: Summary of histopathological findings

Organs

Observations

Incidence of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Main group

Recovery group

 

 

Main group - survivors

Main group - moribund

Recovery group

MALES

Brain

Purulent leptomeningitis

0/5

0/5

/

/

0/5

1/1

0/4

Epididymides

Storage of mature spermatozoa

12/12

5/5

/

/

12/12

1/1

4/4

Kidneys

Purulent nephritis

0/5

0/5

/

/

0/5

1/1

0/4

Liver

Centrilobular vacuolation

0/5

0/5

/

/

0/5

1/1

0/4

Lungs

Alveolar emphysema

1/5

0/5

/

/

0/5

0/1

0/4

Hyperplasia of BALT

2/5

1/5

/

/

0/5

0/1

1/4

Stomach - forestomach

Squamous cell hyperplasia

0/12

0/5

0/12

12/12

12/12

1/1

0/4

Ulceration

0/12

0/5

0/12

0/12

10/12

1/1

0/4

FEMALES

Lungs

Alveolar emphysema

1/5

1/5

/

/

1/5

NA

0/5

Hyperplasia of BALT

2/5

1/5

/

/

1/5

NA

1/5

Stomach - forestomach

Squamous cell hyperplasia

0/12

0/5

0/12

12/12

12/12

NA

0/5

Ulceration

0/12

0/5

0/12

0/12

10/12

NA

0/5

Thymus

Acute hemorrhage

1/12

0/5

/

/

0/5

NA

0/5

Uterus

Dilatation

1/12

1/5

/

/

1/12

NA

2/5

Incidence of observation =number of animals with observations / number of animals examined

/ = not examined

BALT = bronchus associated lymphoid tissue

NA = not applicable

 


 

Table 14: Summary of litter weight and litter weight gain of offspring

Group

 

Litter weight (g) on post-natal days

Litter weight gain (g) between post-natal days

0

4

13

0 - 4

4 - 13

0 - 13

Control

Mean

57.6

108.9

232.0

51.3

147.6

187.5

SD

11.6

14.7

18.6

11.7

15.9

19.8

n

11

11

11

11

11

11

100 mg/kg bw/day

Mean

65.1

114.9

231.2

50.2

146.3

184.3

SD

13.3

22.6

51.8

11.9

32.1

42.3

n

12

12

12

12

12

12

300 mg/kg bw/day

Mean

63.3

104.7

230.4

44.2

145.4

181.6

SD

11.2

16.3

10.8

5.5

9.4

9.9

n

12

12

12

12

12

12

600 mg/kg bw/day

Mean

58.0

92.7

184.9**

35.2**

115.6**

141.7**

SD

15.7

27.0

41.6

12.1

31.6

36.9

n

12

12

12

12

12

12

 

NS

NS

U

DN

U

U

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

U = Mann-Whitney U-test vs. Control

 

 

Table 15: Summary of body weight and body weight gain of offspring

Group

 

Body weight (g) of offspring on post-natal days

Body weight gain (g) between post-natal days

0

4

13

0 - 4

4 - 13

0 - 13

Control

Mean

5.8

11.0

30.1

5.2

15.1

24.3

SD

0.9

1.1

2.3

1.1

2.9

2.4

n

11

11

11

11

11

11

100 mg/kg bw/day

Mean

6.1

10.9

30.3

4.8

14.3

24.2

SD

0.4

1.3

3.1

1.1

3.9

2.9

n

12

12

12

12

12

12

300 mg/kg bw/day

Mean

6.1

10.6

28.8

4.3*

15.0

22.7

SD

0.3

0.5

1.4

0.6

2.6

1.5

n

12

12

12

12

12

12

600 mg/kg bw/day

Mean

5.8

9.3**

24.7**

3.5**

11.9*

18.9**

SD

0.5

0.9

3.4

0.6

2.7

3.4

n

12

12

12

12

12

12

 

 

U

U

DN

DN

DN

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

U = Mann-Whitney U-test vs. Control

 

 

Table 16: Summary of viability and sex distribution of offsprings

Values

 

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

 

Number of viable pups

 

 

 

 

 

on post-natal day 0

Total

N

111

128

125

122

NS

%

100

99

99

100

Male

N

58

69

50

46

*

%

52

54

40

38

Female

N

53

59

75

76

*

%

48

46

60

62

on post-natal day 4

Total

N

111

127

119

121

NS

%

100

98

94

99

Male

N

58

68

50

46

*

%

52

54

42

38

Female

N

53

59

69

75

*

%

48

46

58

62

on post-natal day 13

Total

N

85

92

96

90

NS

%

77

71

76

74

Male

N

44

48

45

39

NS

%

52

52

47

43

Female

N

41

44

51

51

NS

%

48

48

53

57

* p < 0.05 CH2, **p < 0.01 CH2

NS = Not Significant

 

Table 17: Summary of viability and sex distribution of offsprings – litter data

Values

 

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

 

Number of viable pups

 

 

 

 

 

on post-natal day 0

Total Mean

N

10.1

10.7

10.4

10.2

NS

SD

2.1

2.1

2.0

3.2

n

11

12

12

12

Male

N

5.3

5.8

4.2

3.8

* DN

SD

1.4

1.8

1.5

1.6

n

11

12

12

12

Female

N

4.8

4.9

6.3

6.3

NS

SD

1.6

1.8

2.0

2.6

n

11

12

12

12

on post-natal day 4

Total

N

10.1

10.6

9.9

10.1

NS

SD

2.1

2.1

1.8

3.2

n

11

12

12

12

Male

N

5.3

5.7

4.2

3.8

*

DN

SD

1.4

1.7

1.5

1.6

n

11

12

12

12

Female

N

4.8

4.9

5.8

6.3

NS

SD

1.6

1.8

2.0

2.5

n

11

12

12

12

on post-natal day 13

Total

N

7.7

7.7

8.0

7.5

NS

SD

0.6

1.6

0.4

1.4

n

11

12

12

12

Male

N

4.0

4.0

3.8

3.3

* U

SD

0.4

1.5

1.3

0.8

n

11

12

12

12

Female

N

3.7

3.7

4.3

4.3

NS

SD

0.6

1.0

1.3

1.4

n

11

12

12

12

* p < 0.05 CH2, **p < 0.01 CH2

NS = Not Significant

DN = Duncan´s multiple range test

U = Mann-Whitney U-test vs. Control

 

 

Table 18: Summary of body weight and anogenital distance of offspring

Group

 

Body weight (g) on post-natal day 4

Anogenital distance (AGD) on post-natal day 4

Absolute (mm)

Normalized (AGD/3√bw)

MALES

Control

Mean

10.9

5.7

2.6

SD

1.2

0.8

0.3

n

58

58

58

N

11

11

11

100 mg/kg bw/day

Mean

11.1

5.7

2.6

SD

1.2

0.7

0.3

n

68

68

68

N

12

12

12

300 mg/kg bw/day

Mean

10.8

5.7

2.6

SD

0.6

0.5

0.2

n

50

50

50

N

12

12

12

600 mg/kg bw/day

Mean

9.5**

5.6

2.6

SD

1.0

0.6

0.2

n

46

46

46

N

12

10

12

 

U

NS

NS

FEMALES

Control

Mean

10.6

2.6

1.2

SD

1.3

0.6

0.2

n

53

53

53

N

12

12

12

100 mg/kg bw/day

Mean

10.6

2.9*

1.3*

SD

1.7

0.7

0.3

n

59

59

59

N

12

12

12

300 mg/kg bw/day

Mean

10.4

3.1**

1.4**

SD

0.7

0.4

0.2

n

69

69

69

N

12

12

12

600 mg/kg bw/day

Mean

9.0**

2.9**

1.4**

SD

0.7

0.4

0.2

n

74

74

74

N

12

10

12

 

U

U

U

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

n = number of offspring

N = Number of litters

 

Applicant's summary and conclusion

Conclusions:
The test material had no effect on reproductive performance.