Registration Dossier
Registration Dossier
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EC number: 500-734-6 | CAS number: 162492-01-5
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 May - 17 Jul 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- 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:
- 10 May - 17 Jul 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- adopted in 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)
- Version / remarks:
- adopted in 2000
- GLP compliance:
- yes (incl. QA statement)
- 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 or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 98 - 103 days (males and females)
- Weight at study initiation: 367 - 439 g (males), 221 - 267 g (females); The weight variation did not exceed ± 20% 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: 48 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
- 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:
- polyethylene glycol
- Remarks:
- polyethylene glycol 400 (PEG 400)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL. The weighed amount of the test item was heated up to 60°C in a water bath (protected from light) and filled up to the final volume with the vehicle. Formulations were prepared in the formulation laboratory of the Test Facility not longer than three days before administration and was stored in a refrigerator until use.
VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 16I284004 - 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 1 mL of each formulation (20, 60 and 200 mg/mL) and five aliquots of 1 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 97% to 102% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 96 and 99% at ~1 and ~200 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 96% of starting concentrations at 1 mg/mL and 99% at 200 mg/mL) and at 5 ± 3°C for 3 days (recovery was 104% of starting concentrations at 1 mg/mL and 100% at 200 mg/mL).
- Duration of treatment / exposure:
- 54 - 55 days (males, control and test groups)
54 - 56 days (females, control and test groups, depending on the effectiveness of mating)
54 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.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day) - 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 tets material in rats. The high and mid-high doses were 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 (once daily)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; 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); weekly during treatment and post-treatment observation period (recovery groups)
FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 0, 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 (Day 54) 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 Days 54, 55; dams not selected for toxicology examinations: on post-partum days 14 - 20 or shortly thereafter (Days 55, 56 or 57); dams selected for toxicology examinations: shortly after post-partum days 15 - 18 (Day 54); recovery animals after 14-day post-treatment observation period (Day 68). Examination of the external appearance and the appearance of the tissues and organs was performed. 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 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.
Additionally, these organs were processed and examined histologically in one non-pregnant female animal and male animal this female cohabited with at 300 mg/kg bw/day, and in four dams at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose including recovery groups. Histological examinations were performed on organs with macroscopic findings (uterus) in the low and mid dose groups.. - 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. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: alopecia on the skin of the left forelimb in 1/12 animal
FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: dyspnea in 1/17 animal in the control on Day 34 (gestation day 19; animal died on the same day)
100 mg/kg bw/day: alopecia in 1/12 animal between lactation days 0 and 12 - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- FEMALES
Control: 1/12 animal died on gestation day 19 (Day 34) - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- FEMALES
100 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant decreased mean body weight gain on week 2; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
300 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
1000 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant increased summarized body weight gain in the recovery group; statistically significant slightly decreased mean body weight gain during the second week of the recovery period - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- FEMALES
100 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
300 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
1000 mg/kg bw/day: statistically significant slightly increased food consumption during the first week of the premating period - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
100, 300 and 1000 mg/kg bw/day: statistically significant increased percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes
1000 mg/kg bw/day: statistically significant increased mean percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes at the end of the recovery period; statistically significant increased mean percentage of reticulocytes at the end of the recovery period
FEMALES
100, 300 and 1000 mg/kg bw/day: slightly increased percentage of neutrophil granulocytes (not statistically significant)
1000 mg/kg bw/day: statistically significant slightly decreased activated partial thromboplastin time at the end of the recovery period - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
100 mg/kg bw/day: statistically significant decreased mean activity of aspartate aminotransferase and sodium concentration
300 mg/kg bw/day: statistically significant decreased sodium concentration
1000 mg/kg bw/day: statistically significant decreased mean urea and sodium concentration; statistically significant slightly decreased sodium concentration at the end of the recovery period
FEMALES
100 mg/kg bw/day: statistically significant slightly decreased mean creatinine concentration - 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
1000 mg/kg bw/day: statistically significant increased mean liver weight (relative to body and brain weight) at the end of the recovery period
FEMALES
100 mg/kg bw/day: statistically significant increased mean liver weights (absolute and relative to body and brain weights)
300 mg/kg bw/day: statistically significant decreased brain weight relative to body weight; statistically significant increased body weight relative to brain weight; statistically significant decreased kidney weights (relative to body weight)
1000 mg/kg bw/day: statistically significant decreased kidney and spleen weights (relative to body weight) - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
Control: pyelectasia of both kidneys in 1/12 animal; thymic hemorrhage in 2/5 animals at the end of the recovery period
1000 mg/kg bw/day: thymic hemorrhage in 1/5 animal at the end of the recovery period
FEMALE
Control: hydrometra in 1/12 animal; hemorrhage in the lungs in 1/12 animal (animal died on Day 34); hydrometra in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: hydrometra in 6/12 animal
1000 mg/kg bw/day: hydrometra in 2/5 animals at the end of the recovery period - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
Control: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; hyperplasia of bronchus associated lymphoid tissue in1/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in1/5 animal and in 1/5 animal at the end of the recovery period
FEMALES
Control: dilatation of uterine horns in 1/11 animal and in 1/5 animal at the end of the recovery period; pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; alveolar emphysema (moderate degree) and acute hemorrhage (moderate degree) in the lung in one animal which dies on Day 34 (gestation day 19)
300 mg/kg bw/day: dilatation of uterine horns in 5/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; dilatation of uterine horns in 2/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period - 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
Soft stool was considered to be caused by the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats, which occurred in a single animal of low dose group in this study. This was considered to be an individual sign and not related to the test item.
MORTALITY
Histological examination of the dead female of the control group revealed alveolar emphysema and acute hemorrhage (moderate degree) in the lungs, as the cause of death, in connection with a probably shock. No other lesions were observed in the investigated organs of this animal.
BODY WEIGHT AND BODY WEIGHT GAIN
The differences in body weight and body weight gain observed in females as associated with increased food and was not judged to be of toxicological relevance. The differences in the recovery groups were of minor degree and judged to be toxicologically not relevant.
HAEMATOLOGICAL FINDING
Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males of all dose groups at the end of the treatment period were related to doses but were not proportional to doses. The differences with respect to the control were of minor degree and all values remained within the historical control range. Moreover, the changes were not accompanied with changes in white blood cell count and therefore were considered to have little or no biological significance. Effects on the percentage of neutrophil granulocytes observed infemale animals at all dose groups at the end of the treatment period were not related to doses. Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males at 1000 mg/kg bw/day at the end of the recovery period was partially originated from the relative low value of the control group (13.74 ± 5.5% vs. 19.2 ± 6.5% of historical control mean). The increased mean percentage of reticulocytes in the male animals at 1000 mg/kg bw/day at the end of the recovery period was of minor degree and was not observed at the end of the treatment period; therefore, was considered to have no toxicological relevance. The slightly lower activated partial thromboplastin time in females at 1000 mg/kg bw/day at the end of the recovery period is a result of a relatively high control mean value. This difference was not observed at the end of the treatment period and was considered to have no toxicological relevance.
CLINICAL BIOCHEMISTRY FINDINGS
The slight, but statistically significant differences with respect to their controls in aspartate aminotransferase activity, urea and sodium concentration observed in males were considered to have little or no toxicological importance. Lower enzyme activity of aspartate aminotransferase and creatinine concentration was only seen in the low dose group of males and females, respectively, and has no toxicological meaning. Thus, the slight, statistically significant differences with respect to control were considered toxicologically not relevant due to the minor degree of changes or due to the lack of dose relevance. All values were within the historical control ranges.
GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. The pyelectasia was an individual change in a control animal. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.
HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
The dilatation of uterine horns observed in females of the control and mid-dose group is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance. Pulmonary alveolar emphysema and acute hemorrhage observed in males and females of control and high-dose groups were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue is a physiological immune-morphological phenomenon, without toxicological significance. - Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no treatment-related effects at highest dose tested
- Critical effects observed:
- no
- 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 1000 mg/kg bw/day.
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 |
1000 mg/kg bw/day |
||
Treatment period* |
Recovery period |
Treatment period* |
Recovery period |
|||
Normal |
17/17 |
5/5 |
12/12 |
12/12 |
17/17 |
5/5 |
Soft stool |
17/17 |
5/5 |
12/12 |
12/12 |
17/17 |
5/5 |
Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined
* Including animals of recovery group
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 |
12/12 |
12/12 |
Soft stool |
12/12 |
12/12 |
12/12 |
12/12 |
Post-mating periods |
||||
Normal |
1/1 |
- |
1/1 |
- |
Soft stool |
1/1 |
- |
1/1 |
- |
Gestation period |
||||
Normal |
10/11 |
12/12 |
11/11 |
12/12 |
Soft stool |
11/11 |
12/12 |
11/11 |
12/12 |
Dyspnea |
1/11 |
0/12 |
0/11 |
0/12 |
Died |
1/11 |
0/12 |
0/11 |
0/12 |
Lactation period |
||||
Normal |
10/10 |
11/12 |
11/11 |
12/12 |
Soft stool |
10/10 |
12/12 |
11/11 |
12/12 |
Skin: Alopecia |
0/10 |
1/12 |
0/11 |
0/12 |
Recovery animals |
||||
Normal |
5/5 |
5/5 |
5/5 |
5/5 |
Soft stool |
5/5 |
5/5 |
5/5 |
5/5 |
Table 3: Summary of body weight gain in females
Group |
|
Body weight gain (g) between |
|||||||||
Pre-mating days |
Treatment days |
Total |
|||||||||
0 – 7 |
7 – 13 |
0 – 13 |
13 – 20 |
20 – 27 |
27 – 34 |
34 – 41 |
41 – 48 |
48 - 53 |
0 – 53 |
||
Control |
Mean |
-0.1 |
7.6 |
7.5 |
2.0 |
9.2 |
2.6 |
0.6 |
1.6 |
3.4 |
24.5 |
SD |
4.5 |
6.6 |
5.7 |
2.1 |
5.0 |
4.4 |
7.9 |
7.8 |
7.2 |
5.0 |
|
n |
17 |
17 |
17 |
5 |
5 |
5 |
5 |
5 |
5 |
6 |
|
100 mg/kg bw/day |
Mean |
3.8* |
2.3* |
6.1 |
- |
- |
- |
- |
- |
|
- |
SD |
6.9 |
8.2 |
8.3 |
- |
- |
- |
- |
- |
|
- |
|
n |
12 |
12 |
12 |
- |
- |
- |
- |
- |
|
- |
|
300 mg/kg bw/day |
Mean |
8.5** |
6.3 |
14.8** |
- |
- |
- |
- |
- |
|
- |
SD |
5.2 |
5.2 |
4.9 |
- |
- |
- |
- |
- |
|
- |
|
n |
12 |
12 |
12 |
- |
- |
- |
- |
- |
|
- |
|
1000 mg/kg bw/day |
Mean |
7.6** |
8.1 |
15.7** |
0.8 |
8.6 |
-0.6 |
4.8 |
4.2 |
2.4 |
35.8 |
SD |
3.8 |
4.1 |
4.3 |
5.1 |
5.0 |
5.3 |
5.0 |
5.8 |
5.0 |
2.3 |
|
n |
17 |
17 |
17 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
|
DN |
NS |
DN |
NS |
NS |
NS |
NS |
NS |
NS |
** |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
Table 4: 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 |
-1.2 |
3.6 |
2.4 |
SD |
2.6 |
2.3 |
1.1 |
|
n |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
1.4 |
-1.4 |
0.0 |
SD |
7.4 |
4.2 |
4.6 |
|
n |
5 |
5 |
5 |
|
|
NS |
* |
NS |
|
* p < 0.05
NS = Not Significant
Table 5: Summary of food consumption in females
Group |
|
Daily mean food consumption (g/animal/day) |
|||||||||
Pre-mating days |
Post-mating days |
Recovery period |
|||||||||
0 – 7 |
7 – 13 |
13 – 20 |
20 – 27 |
27 – 34 |
34 – 41 |
41 – 48 |
48 – 53 |
Day 0 – 6 |
Day 6 – 13 |
||
Control |
Mean |
15.7 |
15.7 |
17.4 |
17.5 |
17.1 |
16.8 |
16.5 |
17.2 |
17.5 |
18.0 |
SD |
1.70 |
0.86 |
0.17 |
0.45 |
0.44 |
0.67 |
1.03 |
0.35 |
1.36 |
0.15 |
|
n |
8 |
8 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
100 mg/kg bw/day |
Mean |
16.3 |
15.5 |
- |
- |
- |
- |
- |
- |
- |
- |
SD |
1.67 |
1.73 |
- |
- |
- |
- |
- |
- |
- |
- |
|
n |
6 |
6 |
- |
- |
- |
- |
- |
- |
- |
- |
|
±% |
4 |
-1 |
- |
- |
- |
- |
- |
- |
- |
- |
|
300 mg/kg bw/day |
Mean |
17.3 |
16.9 |
- |
- |
- |
- |
- |
- |
- |
- |
SD |
1.07 |
0.88 |
- |
- |
- |
- |
- |
- |
- |
- |
|
n |
6 |
6 |
- |
- |
- |
- |
- |
- |
- |
- |
|
±% |
11 |
7 |
- |
- |
- |
- |
- |
- |
- |
- |
|
1000 mg/kg bw/day |
Mean |
17.4* |
16.9 |
18.1 |
18.2 |
18.4 |
18.9 |
17.3 |
19.2 |
18.4 |
18.4 |
SD |
1.30 |
1.18 |
1.20 |
1.35 |
1.94 |
2.12 |
1.41 |
2.22 |
1.87 |
1.08 |
|
n |
8 |
8 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
±% |
11 |
8 |
4 |
4 |
8 |
13 |
5 |
12 |
6 |
2 |
|
|
DN |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
|
* p < 0.05
NS = Not Significant
DN = Duncan´s multiple range test
Table 6: Summary of hematology
Group |
|
Neutrophil [%] |
Lymphocyte [%] |
Reticulocyte [%] |
Activated partial thromboplastin time [sec] |
MALES - TREATMENT PERIOD |
|||||
Control |
Mean |
18.26 |
78.00 |
3.11 |
20.94 |
SD |
2.25 |
2.29 |
0.69 |
1.54 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
24.60** |
70.82** |
2.94 |
20.96 |
SD |
1.87 |
1.94 |
0.43 |
1.14 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
35 |
-9 |
-5 |
0 |
|
300 mg/kg bw/day |
Mean |
27.14* |
67.06* |
3.24 |
20.18 |
SD |
7.86 |
8.84 |
0.44 |
1.70 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
49 |
-14 |
4 |
-4 |
|
1000 mg/kg bw/day |
Mean |
28.96** |
66.34** |
3.28 |
21.76 |
SD |
6.07 |
7.11 |
0.10 |
1.58 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
59 |
-15 |
5 |
4 |
|
|
U |
U |
NS |
NS |
|
MALES - RECOVERY PERIOD |
|||||
Control |
Mean |
13.74 |
81.94 |
2.58 |
26.96 |
SD |
5.05 |
5.84 |
0.33 |
4.48 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
21.76 |
72.90 |
3.09 |
29.30 |
SD |
4.47 |
5.43 |
0.12 |
5.04 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
58 |
-11 |
20 |
9 |
|
|
* |
* |
* |
NS |
|
FEMALES - TREATMENT PERIOD |
|||||
Control |
Mean |
17.78 |
79.00 |
8.14 |
20.70 |
SD |
7.08 |
6.90 |
2.82 |
1.63 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
25.36 |
70.80 |
8.37 |
21.22 |
SD |
11.96 |
12.43 |
1.76 |
1.27 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
43 |
-10 |
3 |
3 |
|
300 mg/kg bw/day |
Mean |
22.00 |
74.18 |
8.33 |
19.32 |
SD |
3.69 |
4.36 |
2.92 |
0.50 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
24 |
-6 |
2 |
-7 |
|
1000 mg/kg bw/day |
Mean |
22.60 |
74.00 |
6.79 |
20.26 |
SD |
10.57 |
10.89 |
1.54 |
0.81 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
27 |
-6 |
-17 |
-2 |
|
|
NS |
NS |
NS |
NS |
|
FEMALES - RECOVERY PERIOD |
|||||
Control |
Mean |
16.08 |
79.60 |
3.73 |
28.76 |
SD |
3.28 |
3.09 |
0.49 |
3.13 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
14.82 |
81.44 |
3.40 |
23.92 |
SD |
3.62 |
4.34 |
0.67 |
1.09 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-8 |
2 |
-9 |
-17 |
|
|
NS |
NS |
NS |
* |
|
* p < 0.05, **p < 0.01
NS = Not Significant
U = Mann-Whitney U-test vs. Control
Table 7: Summary of clinical biochemistry findings
Group |
|
Aspartate aminotransferase activity [U/L] |
Urea [mmol/L] |
Creatinine [µmol/L] |
Sodium [mmol/L] |
MALES - TREATMENT PERIOD |
|||||
Control |
Mean |
117.36 |
7.88 |
26.44 |
142.20 |
SD |
12.24 |
1.58 |
1.65 |
1.10 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
97.58* |
7.67 |
25.50 |
140.40* |
SD |
11.21 |
1.02 |
2.57 |
1.14 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-17 |
-3 |
-4 |
-1 |
|
300 mg/kg bw/day |
Mean |
126.70 |
7.54 |
29.30 |
139.40** |
SD |
15.05 |
0.82 |
2.65 |
0.89 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
8 |
-4 |
11 |
-2 |
|
1000 mg/kg bw/day |
Mean |
123.74 |
6.13* |
27.92 |
138.00** |
SD |
6.36 |
0.96 |
2.43 |
1.00 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
5 |
-22 |
6 |
-3 |
|
|
DN |
DN |
NS |
DN |
|
MALES - RECOVERY PERIOD |
|||||
Control |
Mean |
102.94 |
6.65 |
25.00 |
142.20 |
SD |
24.99 |
1.29 |
1.12 |
0.45 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
98.82 |
6.66 |
25.82 |
141.20 |
SD |
6.72 |
1.12 |
3.44 |
0.84 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-4 |
0 |
3 |
-1 |
|
|
NS |
NS |
NS |
* |
|
FEMALES - TREATMENT PERIOD |
|||||
Control |
Mean |
127.00 |
11.30 |
26.06 |
135.80 |
SD |
18.29 |
1.35 |
2.89 |
1.30 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
120.28 |
12.48 |
21.86* |
135.00 |
SD |
24.17 |
1.86 |
1.81 |
1.22 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-5 |
10 |
-16 |
-1 |
|
300 mg/kg bw/day |
Mean |
115.62 |
12.23 |
28.26 |
135.00 |
SD |
15.47 |
3.29 |
1.73 |
0.71 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-9 |
8 |
8 |
-1 |
|
1000 mg/kg bw/day |
Mean |
101.04 |
11.42 |
24.92 |
134.80 |
SD |
13.17 |
2.09 |
2.71 |
1.30 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-20 |
1 |
-4 |
-1 |
|
|
NS |
NS |
DN |
NS |
|
FEMALES - RECOVERY PERIOD |
|||||
Control |
Mean |
88.68 |
7.29 |
30.36 |
141.20 |
SD |
6.05 |
1.13 |
3.71 |
1.48 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
92.96 |
7.75 |
31.98 |
140.20 |
SD |
18.69 |
1.01 |
4.83 |
1.10 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
5 |
6 |
5 |
-1 |
|
|
NS |
NS |
NS |
NS |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
Table 8: Summary of organ weights of males
MALES – MAIN GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
450.6 |
2.16 |
10.20 |
2.62 |
0.65 |
SD |
38.40 |
0.11 |
0.99 |
0.21 |
0.08 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
449.0 |
2.17 |
10.37 |
2.59 |
0.66 |
SD |
23.69 |
0.08 |
0.92 |
0.17 |
0.05 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
2 |
-1 |
1 |
|
300 mg/kg bw/day |
Mean |
449.4 |
2.15 |
10.81 |
2.63 |
0.63 |
SD |
28.72 |
0.15 |
0.65 |
0.22 |
0.09 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
6 |
0 |
-3 |
|
1000 mg/kg bw/day |
Mean |
451.7 |
2.16 |
11.11 |
2.67 |
0.65 |
SD |
27.77 |
0.09 |
1.22 |
0.21 |
0.05 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
9 |
2 |
-1 |
|
|
NS |
NS |
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
464.4 |
2.24 |
11.41 |
2.54 |
0.73 |
SD |
27.55 |
0.08 |
1.01 |
0.14 |
0.07 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
476.4 |
2.21 |
12.84 |
2.77 |
0.78 |
SD |
26.28 |
0.15 |
1.15 |
0.23 |
0.06 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
3 |
-1 |
13 |
9 |
7 |
|
|
NS |
NS |
NS |
NS |
NS |
|
MALES – MAIN GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.482 |
2.302 |
0.592 |
0.148 |
SD |
NA |
0.041 |
0.101 |
0.024 |
0.014 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
NA |
0.485 |
2.360 |
0.591 |
0.150 |
SD |
NA |
0.037 |
0.151 |
0.040 |
0.011 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
1 |
2 |
0 |
1 |
|
300 mg/kg bw/day |
Mean |
NA |
0.480 |
2.409 |
0.585 |
0.141 |
SD |
NA |
0.042 |
0.153 |
0.038 |
0.014 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
0 |
5 |
-1 |
-5 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.480 |
2.462 |
0.593 |
0.144 |
SD |
NA |
0.034 |
0.178 |
0.041 |
0.015 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
0 |
7 |
0 |
-2 |
|
|
|
NS |
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.483 |
2.455 |
0.547 |
0.157 |
SD |
NA |
0.029 |
0.132 |
0.016 |
0.015 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.464 |
2.692 |
0.581 |
0.165 |
SD |
NA |
0.029 |
0.137 |
0.036 |
0.018 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-4 |
10 |
6 |
5 |
|
|
|
NS |
* |
NS |
NS |
|
MALES – MAIN GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
20874.9 |
NA |
468.94 |
120.48 |
30.06 |
SD |
1701.64 |
NA |
38.24 |
6.99 |
3.14 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
20747.8 |
NA |
482.91 |
120.64 |
30.65 |
SD |
1577.94 |
NA |
50.77 |
10.34 |
2.98 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
-1 |
NA |
3 |
0 |
2 |
|
300 mg/kg bw/day |
Mean |
20963.0 |
NA |
502.37 |
122.02 |
29.37 |
SD |
1773.58 |
NA |
57.17 |
13.79 |
4.36 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
0 |
NA |
7 |
1 |
-2 |
|
1000 mg/kg bw/day |
Mean |
20925.5 |
NA |
518.51 |
124.96 |
30.25 |
SD |
1509.23 |
NA |
65.83 |
16.95 |
3.30 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
0 |
NA |
11 |
4 |
1 |
|
|
NS |
|
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
20763.1 |
NA |
509.21 |
113.55 |
32.53 |
SD |
1309.90 |
NA |
32.90 |
6.56 |
2.73 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
21619.1 |
NA |
582.79 |
125.87 |
35.64 |
SD |
1244.43 |
NA |
55.10 |
12.42 |
4.72 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
4 |
NA |
14 |
11 |
10 |
|
|
NS |
|
* |
NS |
NS |
|
* p < 0.05
NS = Not Significant
NA = Not Applicable
Table 9: Summary of organ weights of females
FEMALES – MAIN GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
263.8 |
1.97 |
8.73 |
1.88 |
0.66 |
SD |
14.34 |
0.06 |
0.59 |
0.15 |
0.07 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
272.4 |
1.96 |
10.02* |
1.87 |
0.71 |
SD |
20.84 |
0.05 |
1.07 |
0.04 |
0.11 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
3 |
-1 |
15 |
-1 |
7 |
|
300 mg/kg bw/day |
Mean |
284.8 |
1.92 |
8.81 |
1.84 |
0.62 |
SD |
11.58 |
0.11 |
0.56 |
0.11 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
8 |
-2 |
1 |
-2 |
-7 |
|
1000 mg/kg bw/day |
Mean |
277.6 |
1.94 |
8.95 |
1.79 |
0.57 |
SD |
12.78 |
0.08 |
0.55 |
0.10 |
0.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
5 |
-2 |
3 |
-5 |
-14 |
|
|
NS |
NS |
DN |
NS |
NS |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
262.2 |
2.05 |
7.74 |
1.71 |
0.59 |
SD |
6.38 |
0.04 |
1.10 |
0.16 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
266.4 |
2.01 |
7.83 |
1.70 |
0.56 |
SD |
12.03 |
0.07 |
0.24 |
0.06 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
2 |
-2 |
1 |
-1 |
-6 |
|
|
NS |
NS |
NS |
NS |
NS |
|
FEMALES – MAIN GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.750 |
3.315 |
0.714 |
0.252 |
SD |
NA |
0.056 |
0.251 |
0.067 |
0.019 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
NA |
0.723 |
3.676* |
0.689 |
0.260 |
SD |
NA |
0.051 |
0.252 |
0.049 |
0.036 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-4 |
11 |
-4 |
3 |
|
300 mg/kg bw/day |
Mean |
NA |
0.676* |
3.094 |
0.647* |
0.218 |
SD |
NA |
0.038 |
0.197 |
0.019 |
0.021 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-10 |
-7 |
-9 |
-13 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.698 |
3.226 |
0.646* |
0.206* |
SD |
NA |
0.024 |
0.158 |
0.020 |
0.025 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-7 |
-3 |
-10 |
-18 |
|
|
|
DN |
DN |
DN |
DN |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.780 |
2.951 |
0.653 |
0.226 |
SD |
NA |
0.012 |
0.417 |
0.050 |
0.017 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
600 mg/kg bw/day |
Mean |
NA |
0.758 |
2.940 |
0.639 |
0.209 |
SD |
NA |
0.049 |
0.059 |
0.024 |
0.017 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-3 |
0 |
-2 |
-7 |
|
|
|
NS |
NS |
NS |
NS |
|
FEMALES – MAIN GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
13393.6 |
NA |
442.45 |
95.26 |
33.73 |
SD |
945.10 |
NA |
21.58 |
5.54 |
3.80 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
13883.8 |
NA |
510.19* |
95.22 |
36.02 |
SD |
1006.61 |
NA |
46.31 |
1.40 |
4.98 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
4 |
NA |
15 |
0 |
7 |
|
300 mg/kg bw/day |
Mean |
14830.5* |
NA |
459.23 |
95.99 |
32.42 |
SD |
820.56 |
NA |
44.96 |
6.08 |
4.45 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
11 |
NA |
4 |
1 |
-4 |
|
1000 mg/kg bw/day |
Mean |
1434.1 |
NA |
463.02 |
92.53 |
29.50 |
SD |
493.85 |
NA |
34.50 |
2.05 |
3.73 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
7 |
NA |
5 |
-3 |
-13 |
|
|
DN |
|
DN |
NS |
NS |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
12815.8 |
NA |
378.06 |
83.72 |
28.90 |
SD |
196.57 |
NA |
52.61 |
6.77 |
2.11 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
13245.5 |
NA |
389.19 |
84.55 |
27.62 |
SD |
885.93 |
NA |
22.07 |
3.07 |
2.35 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
3 |
NA |
3 |
1 |
-4 |
|
|
NS |
|
NS |
NS |
NS |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
NA = Not Applicable
Table 10: Summary of necropsy findings
Organs |
Observations |
Frequency of observations per group |
|||||
Control |
100 mg/kg bw/day |
300 mg/kg bw/day |
1000 mg/kg bw/day |
||||
Main group |
Recovery group |
|
|
Main group |
Recovery group |
||
MALES |
|||||||
|
No macroscopic finding |
11/12 |
3/5 |
12/12 |
12/12 |
12/12 |
4/5 |
Thymus |
Hemorrhages |
0/12 |
2/5 |
0/12 |
0/12 |
0/12 |
1/5 |
Kidney |
Pyelectasia – both sides |
1/12 |
0/5 |
0/12 |
0/12 |
0/12 |
0/5 |
FEMALES - DAMS |
|||||||
|
No macroscopic finding |
10/10 |
4/5 |
12/12 |
6/11 |
12/12 |
3/5 |
Uterus |
Hydrometra |
0/10 |
1/5 |
0/12 |
5/11 |
0/12 |
2/5 |
FEMALES – NON-PREGNANT |
|||||||
Uterus |
Hydrometra |
1/1 |
NA |
NA |
1/1 |
NA |
NA |
FEMALES – DEAD ANIMAL |
|||||||
Lungs |
Hemorrhages |
1/1 |
NA |
NA |
NA |
NA |
NA |
Frequency of observation =number of animals with observations / number of animals examined
NA = not applicable
Table 11: Summary of histopathological findings
Organs |
Observations |
Incidence of observations per group |
||||||
Control |
100 mg/kg bw/day |
300 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
Main group |
Dead animal |
Recovery group |
|
|
Main group
|
Recovery group |
||
MALES |
||||||||
Kidneys |
Pyelectasia (both side) |
1/5 |
NA |
0/5 |
- |
- |
0/5 |
0/5 |
Lungs |
Alveolar emphysema |
1/5 |
NA |
0/5 |
- |
- |
1/5 |
1/5 |
Acute hemorrhage |
1/5 |
NA |
0/5 |
- |
- |
0/5 |
0/5 |
|
Hyperplasia of BALT |
1/5 |
NA |
0/5 |
- |
- |
1/5 |
1/5 |
|
Thymus |
Acute hemorrhage |
0/5 |
NA |
1/5 |
- |
- |
0/5 |
1/5 |
FEMALES |
||||||||
Lungs |
Alveolar emphysema |
1/5 |
1/1 |
1/5 |
- |
- |
1/5 |
0/5 |
Acute hemorrhage |
0/5 |
1/1 |
0/5 |
- |
- |
1/5 |
0/5 |
|
Hyperplasia of BALT |
0/5 |
0/1 |
0/5 |
- |
- |
0/5 |
1/5 |
|
Uterus |
Dilatation |
1/11 |
0/1 |
1/5 |
- |
5/5 |
0/12 |
2/5 |
Incidence of observation =number of animals with observations / number of animals examined
BALT = bronchus associated lymphoid tissue
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- Adopted in 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)
- Version / remarks:
- adopted in 2000
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
- Limit test:
- no
Test material
- Reference substance name:
- Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate
- EC Number:
- 500-734-6
- EC Name:
- Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate
- Cas Number:
- 162492-01-5
- Molecular formula:
- C39 H60 N6 O15
- IUPAC Name:
- Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate
Constituent 1
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 or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 98 - 103 days (males and females)
- Weight at study initiation: 367 - 439 g (males), 221 - 267 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: 48 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
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- polyethylene glycol
- Remarks:
- polyethylene glycol 400 (PEG 400)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL. The weighed amount of the test item was heated up to 60°C in a water bath (protected from light) and filled up to the final volume with the vehicle. Formulations were prepared in the formulation laboratory of the Test Facility not longer than three days before administration and was stored in a refrigerator until use.
VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 16I284004 - 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 1 mL of each formulation (20, 60 and 200 mg/mL) and five aliquots of 1 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 97% to 102% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 96 and 99% at ~1 and ~200 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 96% of starting concentrations at 1 mg/mL and 99% at 200 mg/mL) and at 5 ± 3°C for 3 days (recovery was 104% of starting concentrations at 1 mg/mL and 100% at 200 mg/mL).
- Duration of treatment / exposure:
- 54 - 55 days (males, control and test groups)
54 - 57 days (females, control and test groups, depending on the effectiveness of mating)
54 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.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day) - 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 tets material in rats. The high and mid-high doses were 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
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 (once daily)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; 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); weekly during treatment and post-treatment observation period (recovery groups)
FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 0, 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 (Day 54) 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. - 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. 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 all parental males of the control and high dose group:
testis weight, epididymis weight, other: quantity and morphology of spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa) representing different stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - 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, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: runts (pups that are significantly smaller than normal 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; on the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups) - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals after the optionally extended post-mating period on Days 54, 55.
- Maternal animals: All surviving animals not selected for toxicology examinations on post-partum days 14 - 20 or shortly thereafter (Days 55, 56 or 57). All surviving animals selected for toxicology examinations shortly after post-partum days 15 - 18 (Day 54).
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations of the tissues and organs.
HISTOPATHOLOGY / ORGAN WEIGHTS
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. 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. These organs were processed and examined histologically in one non-pregnant female animal and male animal this female cohabited with at 300 mg/kg bw/day, and in four dams at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day) including recovery groups. Histological examinations were performed on organs with macroscopic findings (uterus) in the low and mid dose groups. - 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. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
- 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 liveborns 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, non-treatment-related
- Description (incidence and severity):
- MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: alopecia on the skin of the left forelimb in 1/12 animal
FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: dyspnea in 1/17 animal in the control on Day 34 (gestation day 19; animal died on the same day)
100 mg/kg bw/day: alopecia in 1/12 animal between lactation days 0 and 12 - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- FEMALES
Control: 1/12 animal died on gestation day 19 (Day 34) - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- FEMALES
100 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant decreased mean body weight gain on week 2; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
300 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
1000 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant increased summarized body weight gain in the recovery group; statistically significant slightly decreased mean body weight gain during the second week of the recovery period - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- FEMALES
100 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
300 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
1000 mg/kg bw/day: statistically significant slightly increased food consumption during the first week of the premating period - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
100, 300 and 1000 mg/kg bw/day: statistically significant increased percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes
1000 mg/kg bw/day: statistically significant increased mean percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes at the end of the recovery period; statistically significant increased mean percentage of reticulocytes at the end of the recovery period
FEMALES
100, 300 and 1000 mg/kg bw/day: slightly increased percentage of neutrophil granulocytes (not statistically significant)
1000 mg/kg bw/day: statistically significant slightly decreased activated partial thromboplastin time at the end of the recovery period - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
100 mg/kg bw/day: statistically significant decreased mean activity of aspartate aminotransferase and sodium concentration
300 mg/kg bw/day: statistically significant decreased sodium concentration
1000 mg/kg bw/day: statistically significant decreased mean urea and sodium concentration; statistically significant slightly decreased sodium concentration at the end of the recovery period
FEMALES
100 mg/kg bw/day: statistically significant slightly decreased mean creatinine concentration - 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, non-treatment-related
- Description (incidence and severity):
- MALES
Control: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; hyperplasia of bronchus associated lymphoid tissue in1/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in1/5 animal and in 1/5 animal at the end of the recovery period
FEMALES
Control: dilatation of uterine horns in 1/11 animal and in 1/5 animal at the end of the recovery period; pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; alveolar emphysema (moderate degree) and acute hemorrhage (moderate degree) in the lung in one animal which dies on Day 34 (gestation day 19)
300 mg/kg bw/day: dilatation of uterine horns in 5/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; dilatation of uterine horns in 2/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period - 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:
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
100 mg/kg bw/day: statistically significant increased fertility index
1000 mg/kg bw/day: statistically significant increased fertility index
FEMALES
100 mg/kg bw/day: statistically significant increased fertility index
1000 mg/kg bw/day: statistically significant increased fertility index
Details on results (P0)
Soft stool was considered to be caused by the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats, which occurred in a single animal of low dose group in this study. This was considered to be an individual sign and not related to the test item.
MORTALITY
Histological examination of the dead female of the control group revealed alveolar emphysema and acute hemorrhage (moderate degree) in the lungs, as the cause of death, in connection with a probably shock. No other lesions were observed in the investigated organs of this animal.
BODY WEIGHT AND BODY WEIGHT GAIN
The differences in body weight and body weight gain observed in females as associated with increased food and was not judged to be of toxicological relevant. The differences in the recovery groups were of minor degree and judged to be toxicologically not relevant.
HAEMATOLOGICAL FINDING
Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males of all dose groups at the end of the treatment period were related to doses but were not proportional to doses. The differences with respect to the control were of minor degree and all values remained within the historical control range. Moreover, the changes were not accompanied with changes in white blood cell count and therefore were considered to have little or no biological significance. Effects on the percentage of neutrophil granulocytes observed infemale animals at all dose groups at the end of the treatment period were not related to doses. Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males at 1000 mg/kg bw/day at the end of the recovery period was partially originated from the relative low value of the control group (13.74 ± 5.5% vs. 19.2 ± 6.5% of historical control mean). The increased mean percentage of reticulocytes in the male animals at 1000 mg/kg bw/day at the end of the recovery period was of minor degree and was not observed at the end of the treatment period; therefore, was considered to have no toxicological relevance. The slightly lower activated partial thromboplastin time in females at 1000 mg/kg bw/day at the end of the recovery period is a result of a relatively high control mean value. This difference was not observed at the end of the treatment period and was considered to have no toxicological relevance.
CLINICAL BIOCHEMISTRY FINDINGS
The slight, but statistically significant differences with respect to their controls in aspartate aminotransferase activity, urea and sodium concentration observed in males were considered to have little or no toxicological importance. Lower enzyme activity of aspartate aminotransferase and creatinine concentration was only seen in the low dose group of males and females, respectively, and has no toxicological meaning. Thus, the slight, statistically significant differences with respect to control were considered toxicologically not relevant due to the minor degree of changes or due to the lack of dose relevance. All values were within the historical control ranges.
GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. The pyelectasia was an individual change in a control animal. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.
HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
The dilatation of uterine horns observed in females of the control and mid-dose group is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance. Pulmonary alveolar emphysema and acute hemorrhage observed in males and females of control and high-dose groups were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue is a physiological immune-morphological phenomenon, without toxicological significance.
REPRODUCTIVE PERFORMANCE
Because one male animal in the control mated but, however, its pair was not pregnant an increased fertiliy index was observed in males and females at 100 and 1000 mg/kg bw/day. These differences with respect to the control were considered to be indicative of biological variation.
Effect levels (P0)
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no treatment-related effects at highest dose tested
Target system / organ toxicity (P0)
- Critical effects observed:
- no
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 100 and 300 mg/kg bw/day: pale, bitten smaller than normal size, hemorrhage and cyanotic body
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- Control: two stillborn offspring
100 mg/kg bw/day: one stillborn offspring
300 mg/kg bw/day: two dead pups on postnatal day 0, one dead pup on postnatal day 1
1000 mg/kg bw/day: five stillborn offspring - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day: statistically significant slightly increased mean pup weight between postnatal days 0 and 4
- 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):
- Control: 1/2 stillborn offspring was uncleaned and its umbilical cord was intact
300 mg/kg bw/day: 1/3 dead pup was partially cannibalized; 1/3 dead pup had autolyzed visceral organs - Histopathological findings:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- ANOGENITAL DISTANCE
100 mg/kg bw/day: statistically significant longer anogenital distance in males
Details on results (F1)
Clinical signs were only observed in pups of low and mid dose groups but not in the high dose group. Therefore, these signs were judged to be incidental rather than treatment related or toxicologically not relevant.
Effect levels (F1)
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects at highet dose tested
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 |
1000 mg/kg bw/day |
||
Treatment period* |
Recovery period |
Treatment period* |
Recovery period |
|||
Normal |
17/17 |
5/5 |
12/12 |
12/12 |
17/17 |
5/5 |
Soft stool |
17/17 |
5/5 |
12/12 |
12/12 |
17/17 |
5/5 |
Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined
* Including animals of recovery group
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 |
12/12 |
12/12 |
Soft stool |
12/12 |
12/12 |
12/12 |
12/12 |
Post-mating periods |
||||
Normal |
1/1 |
- |
1/1 |
- |
Soft stool |
1/1 |
- |
1/1 |
- |
Gestation period |
||||
Normal |
10/11 |
12/12 |
11/11 |
12/12 |
Soft stool |
11/11 |
12/12 |
11/11 |
12/12 |
Dyspnea |
1/11 |
0/12 |
0/11 |
0/12 |
Died |
1/11 |
0/12 |
0/11 |
0/12 |
Lactation period |
||||
Normal |
10/10 |
11/12 |
11/11 |
12/12 |
Soft stool |
10/10 |
12/12 |
11/11 |
12/12 |
Skin: Alopecia |
0/10 |
1/12 |
0/11 |
0/12 |
Recovery animals |
||||
Normal |
5/5 |
5/5 |
5/5 |
5/5 |
Soft stool |
5/5 |
5/5 |
5/5 |
5/5 |
Table 3: Summary of body weight gain in females
Group |
|
Body weight gain (g) between |
|||||||||
Pre-mating days |
Treatment days |
Total |
|||||||||
0 – 7 |
7 – 13 |
0 – 13 |
13 – 20 |
20 – 27 |
27 – 34 |
34 – 41 |
41 – 48 |
48 - 53 |
0 – 53 |
||
Control |
Mean |
-0.1 |
7.6 |
7.5 |
2.0 |
9.2 |
2.6 |
0.6 |
1.6 |
3.4 |
24.5 |
SD |
4.5 |
6.6 |
5.7 |
2.1 |
5.0 |
4.4 |
7.9 |
7.8 |
7.2 |
5.0 |
|
n |
17 |
17 |
17 |
5 |
5 |
5 |
5 |
5 |
5 |
6 |
|
100 mg/kg bw/day |
Mean |
3.8* |
2.3* |
6.1 |
- |
- |
- |
- |
- |
|
- |
SD |
6.9 |
8.2 |
8.3 |
- |
- |
- |
- |
- |
|
- |
|
n |
12 |
12 |
12 |
- |
- |
- |
- |
- |
|
- |
|
300 mg/kg bw/day |
Mean |
8.5** |
6.3 |
14.8** |
- |
- |
- |
- |
- |
|
- |
SD |
5.2 |
5.2 |
4.9 |
- |
- |
- |
- |
- |
|
- |
|
n |
12 |
12 |
12 |
- |
- |
- |
- |
- |
|
- |
|
1000 mg/kg bw/day |
Mean |
7.6** |
8.1 |
15.7** |
0.8 |
8.6 |
-0.6 |
4.8 |
4.2 |
2.4 |
35.8 |
SD |
3.8 |
4.1 |
4.3 |
5.1 |
5.0 |
5.3 |
5.0 |
5.8 |
5.0 |
2.3 |
|
n |
17 |
17 |
17 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
|
DN |
NS |
DN |
NS |
NS |
NS |
NS |
NS |
NS |
** |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
Table 4: 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 |
-1.2 |
3.6 |
2.4 |
SD |
2.6 |
2.3 |
1.1 |
|
n |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
1.4 |
-1.4 |
0.0 |
SD |
7.4 |
4.2 |
4.6 |
|
n |
5 |
5 |
5 |
|
|
NS |
* |
NS |
|
* p < 0.05
NS = Not Significant
Table 5: Summary of food consumption in females
Group |
|
Daily mean food consumption (g/animal/day) |
|||||||||
Pre-mating days |
Post-mating days |
Recovery period |
|||||||||
0 – 7 |
7 – 13 |
13 – 20 |
20 – 27 |
27 – 34 |
34 – 41 |
41 – 48 |
48 – 53 |
Day 0 – 6 |
Day 6 – 13 |
||
Control |
Mean |
15.7 |
15.7 |
17.4 |
17.5 |
17.1 |
16.8 |
16.5 |
17.2 |
17.5 |
18.0 |
SD |
1.70 |
0.86 |
0.17 |
0.45 |
0.44 |
0.67 |
1.03 |
0.35 |
1.36 |
0.15 |
|
n |
8 |
8 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
100 mg/kg bw/day |
Mean |
16.3 |
15.5 |
- |
- |
- |
- |
- |
- |
- |
- |
SD |
1.67 |
1.73 |
- |
- |
- |
- |
- |
- |
- |
- |
|
n |
6 |
6 |
- |
- |
- |
- |
- |
- |
- |
- |
|
±% |
4 |
-1 |
- |
- |
- |
- |
- |
- |
- |
- |
|
300 mg/kg bw/day |
Mean |
17.3 |
16.9 |
- |
- |
- |
- |
- |
- |
- |
- |
SD |
1.07 |
0.88 |
- |
- |
- |
- |
- |
- |
- |
- |
|
n |
6 |
6 |
- |
- |
- |
- |
- |
- |
- |
- |
|
±% |
11 |
7 |
- |
- |
- |
- |
- |
- |
- |
- |
|
1000 mg/kg bw/day |
Mean |
17.4* |
16.9 |
18.1 |
18.2 |
18.4 |
18.9 |
17.3 |
19.2 |
18.4 |
18.4 |
SD |
1.30 |
1.18 |
1.20 |
1.35 |
1.94 |
2.12 |
1.41 |
2.22 |
1.87 |
1.08 |
|
n |
8 |
8 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
±% |
11 |
8 |
4 |
4 |
8 |
13 |
5 |
12 |
6 |
2 |
|
|
DN |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
NS |
|
* p < 0.05
NS = Not Significant
DN = Duncan´s multiple range test
Table 6: Summary of hematology
Group |
|
Neutrophil [%] |
Lymphocyte [%] |
Reticulocyte [%] |
Activated partial thromboplastin time [sec] |
MALES - TREATMENT PERIOD |
|||||
Control |
Mean |
18.26 |
78.00 |
3.11 |
20.94 |
SD |
2.25 |
2.29 |
0.69 |
1.54 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
24.60** |
70.82** |
2.94 |
20.96 |
SD |
1.87 |
1.94 |
0.43 |
1.14 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
35 |
-9 |
-5 |
0 |
|
300 mg/kg bw/day |
Mean |
27.14* |
67.06* |
3.24 |
20.18 |
SD |
7.86 |
8.84 |
0.44 |
1.70 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
49 |
-14 |
4 |
-4 |
|
1000 mg/kg bw/day |
Mean |
28.96** |
66.34** |
3.28 |
21.76 |
SD |
6.07 |
7.11 |
0.10 |
1.58 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
59 |
-15 |
5 |
4 |
|
|
U |
U |
NS |
NS |
|
MALES - RECOVERY PERIOD |
|||||
Control |
Mean |
13.74 |
81.94 |
2.58 |
26.96 |
SD |
5.05 |
5.84 |
0.33 |
4.48 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
21.76 |
72.90 |
3.09 |
29.30 |
SD |
4.47 |
5.43 |
0.12 |
5.04 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
58 |
-11 |
20 |
9 |
|
|
* |
* |
* |
NS |
|
FEMALES - TREATMENT PERIOD |
|||||
Control |
Mean |
17.78 |
79.00 |
8.14 |
20.70 |
SD |
7.08 |
6.90 |
2.82 |
1.63 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
25.36 |
70.80 |
8.37 |
21.22 |
SD |
11.96 |
12.43 |
1.76 |
1.27 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
43 |
-10 |
3 |
3 |
|
300 mg/kg bw/day |
Mean |
22.00 |
74.18 |
8.33 |
19.32 |
SD |
3.69 |
4.36 |
2.92 |
0.50 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
24 |
-6 |
2 |
-7 |
|
1000 mg/kg bw/day |
Mean |
22.60 |
74.00 |
6.79 |
20.26 |
SD |
10.57 |
10.89 |
1.54 |
0.81 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
27 |
-6 |
-17 |
-2 |
|
|
NS |
NS |
NS |
NS |
|
FEMALES - RECOVERY PERIOD |
|||||
Control |
Mean |
16.08 |
79.60 |
3.73 |
28.76 |
SD |
3.28 |
3.09 |
0.49 |
3.13 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
14.82 |
81.44 |
3.40 |
23.92 |
SD |
3.62 |
4.34 |
0.67 |
1.09 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-8 |
2 |
-9 |
-17 |
|
|
NS |
NS |
NS |
* |
|
* p < 0.05, **p < 0.01
NS = Not Significant
U = Mann-Whitney U-test vs. Control
Table 7: Summary of clinical biochemistry findings
Group |
|
Aspartate aminotransferase activity [U/L] |
Urea [mmol/L] |
Creatinine [µmol/L] |
Sodium [mmol/L] |
MALES - TREATMENT PERIOD |
|||||
Control |
Mean |
117.36 |
7.88 |
26.44 |
142.20 |
SD |
12.24 |
1.58 |
1.65 |
1.10 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
97.58* |
7.67 |
25.50 |
140.40* |
SD |
11.21 |
1.02 |
2.57 |
1.14 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-17 |
-3 |
-4 |
-1 |
|
300 mg/kg bw/day |
Mean |
126.70 |
7.54 |
29.30 |
139.40** |
SD |
15.05 |
0.82 |
2.65 |
0.89 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
8 |
-4 |
11 |
-2 |
|
1000 mg/kg bw/day |
Mean |
123.74 |
6.13* |
27.92 |
138.00** |
SD |
6.36 |
0.96 |
2.43 |
1.00 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
5 |
-22 |
6 |
-3 |
|
|
DN |
DN |
NS |
DN |
|
MALES - RECOVERY PERIOD |
|||||
Control |
Mean |
102.94 |
6.65 |
25.00 |
142.20 |
SD |
24.99 |
1.29 |
1.12 |
0.45 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
98.82 |
6.66 |
25.82 |
141.20 |
SD |
6.72 |
1.12 |
3.44 |
0.84 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-4 |
0 |
3 |
-1 |
|
|
NS |
NS |
NS |
* |
|
FEMALES - TREATMENT PERIOD |
|||||
Control |
Mean |
127.00 |
11.30 |
26.06 |
135.80 |
SD |
18.29 |
1.35 |
2.89 |
1.30 |
|
n |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
120.28 |
12.48 |
21.86* |
135.00 |
SD |
24.17 |
1.86 |
1.81 |
1.22 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-5 |
10 |
-16 |
-1 |
|
300 mg/kg bw/day |
Mean |
115.62 |
12.23 |
28.26 |
135.00 |
SD |
15.47 |
3.29 |
1.73 |
0.71 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-9 |
8 |
8 |
-1 |
|
1000 mg/kg bw/day |
Mean |
101.04 |
11.42 |
24.92 |
134.80 |
SD |
13.17 |
2.09 |
2.71 |
1.30 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
-20 |
1 |
-4 |
-1 |
|
|
NS |
NS |
DN |
NS |
|
FEMALES - RECOVERY PERIOD |
|||||
Control |
Mean |
88.68 |
7.29 |
30.36 |
141.20 |
SD |
6.05 |
1.13 |
3.71 |
1.48 |
|
n |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
92.96 |
7.75 |
31.98 |
140.20 |
SD |
18.69 |
1.01 |
4.83 |
1.10 |
|
n |
5 |
5 |
5 |
5 |
|
±% |
5 |
6 |
5 |
-1 |
|
|
NS |
NS |
NS |
NS |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
Table 8: Summary of organ weights of males
MALES – MAIN GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
450.6 |
2.16 |
10.20 |
2.62 |
0.65 |
SD |
38.40 |
0.11 |
0.99 |
0.21 |
0.08 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
449.0 |
2.17 |
10.37 |
2.59 |
0.66 |
SD |
23.69 |
0.08 |
0.92 |
0.17 |
0.05 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
2 |
-1 |
1 |
|
300 mg/kg bw/day |
Mean |
449.4 |
2.15 |
10.81 |
2.63 |
0.63 |
SD |
28.72 |
0.15 |
0.65 |
0.22 |
0.09 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
6 |
0 |
-3 |
|
1000 mg/kg bw/day |
Mean |
451.7 |
2.16 |
11.11 |
2.67 |
0.65 |
SD |
27.77 |
0.09 |
1.22 |
0.21 |
0.05 |
|
n |
12 |
12 |
5 |
5 |
5 |
|
±% |
0 |
0 |
9 |
2 |
-1 |
|
|
NS |
NS |
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
464.4 |
2.24 |
11.41 |
2.54 |
0.73 |
SD |
27.55 |
0.08 |
1.01 |
0.14 |
0.07 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
476.4 |
2.21 |
12.84 |
2.77 |
0.78 |
SD |
26.28 |
0.15 |
1.15 |
0.23 |
0.06 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
3 |
-1 |
13 |
9 |
7 |
|
|
NS |
NS |
NS |
NS |
NS |
|
MALES – MAIN GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.482 |
2.302 |
0.592 |
0.148 |
SD |
NA |
0.041 |
0.101 |
0.024 |
0.014 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
NA |
0.485 |
2.360 |
0.591 |
0.150 |
SD |
NA |
0.037 |
0.151 |
0.040 |
0.011 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
1 |
2 |
0 |
1 |
|
300 mg/kg bw/day |
Mean |
NA |
0.480 |
2.409 |
0.585 |
0.141 |
SD |
NA |
0.042 |
0.153 |
0.038 |
0.014 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
0 |
5 |
-1 |
-5 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.480 |
2.462 |
0.593 |
0.144 |
SD |
NA |
0.034 |
0.178 |
0.041 |
0.015 |
|
n |
NA |
12 |
5 |
5 |
5 |
|
±% |
NA |
0 |
7 |
0 |
-2 |
|
|
|
NS |
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.483 |
2.455 |
0.547 |
0.157 |
SD |
NA |
0.029 |
0.132 |
0.016 |
0.015 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.464 |
2.692 |
0.581 |
0.165 |
SD |
NA |
0.029 |
0.137 |
0.036 |
0.018 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-4 |
10 |
6 |
5 |
|
|
|
NS |
* |
NS |
NS |
|
MALES – MAIN GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
20874.9 |
NA |
468.94 |
120.48 |
30.06 |
SD |
1701.64 |
NA |
38.24 |
6.99 |
3.14 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
20747.8 |
NA |
482.91 |
120.64 |
30.65 |
SD |
1577.94 |
NA |
50.77 |
10.34 |
2.98 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
-1 |
NA |
3 |
0 |
2 |
|
300 mg/kg bw/day |
Mean |
20963.0 |
NA |
502.37 |
122.02 |
29.37 |
SD |
1773.58 |
NA |
57.17 |
13.79 |
4.36 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
0 |
NA |
7 |
1 |
-2 |
|
1000 mg/kg bw/day |
Mean |
20925.5 |
NA |
518.51 |
124.96 |
30.25 |
SD |
1509.23 |
NA |
65.83 |
16.95 |
3.30 |
|
n |
12 |
NA |
5 |
5 |
5 |
|
±% |
0 |
NA |
11 |
4 |
1 |
|
|
NS |
|
NS |
NS |
NS |
|
MALES – RECOVERY GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
20763.1 |
NA |
509.21 |
113.55 |
32.53 |
SD |
1309.90 |
NA |
32.90 |
6.56 |
2.73 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
21619.1 |
NA |
582.79 |
125.87 |
35.64 |
SD |
1244.43 |
NA |
55.10 |
12.42 |
4.72 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
4 |
NA |
14 |
11 |
10 |
|
|
NS |
|
* |
NS |
NS |
|
* p < 0.05
NS = Not Significant
NA = Not Applicable
Table 9: Summary of organ weights of females
FEMALES – MAIN GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
263.8 |
1.97 |
8.73 |
1.88 |
0.66 |
SD |
14.34 |
0.06 |
0.59 |
0.15 |
0.07 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
272.4 |
1.96 |
10.02* |
1.87 |
0.71 |
SD |
20.84 |
0.05 |
1.07 |
0.04 |
0.11 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
3 |
-1 |
15 |
-1 |
7 |
|
300 mg/kg bw/day |
Mean |
284.8 |
1.92 |
8.81 |
1.84 |
0.62 |
SD |
11.58 |
0.11 |
0.56 |
0.11 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
8 |
-2 |
1 |
-2 |
-7 |
|
1000 mg/kg bw/day |
Mean |
277.6 |
1.94 |
8.95 |
1.79 |
0.57 |
SD |
12.78 |
0.08 |
0.55 |
0.10 |
0.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
5 |
-2 |
3 |
-5 |
-14 |
|
|
NS |
NS |
DN |
NS |
NS |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
Body weight [g] |
Organ weight [g] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
262.2 |
2.05 |
7.74 |
1.71 |
0.59 |
SD |
6.38 |
0.04 |
1.10 |
0.16 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
266.4 |
2.01 |
7.83 |
1.70 |
0.56 |
SD |
12.03 |
0.07 |
0.24 |
0.06 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
±% |
2 |
-2 |
1 |
-1 |
-6 |
|
|
NS |
NS |
NS |
NS |
NS |
|
FEMALES – MAIN GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.750 |
3.315 |
0.714 |
0.252 |
SD |
NA |
0.056 |
0.251 |
0.067 |
0.019 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
NA |
0.723 |
3.676* |
0.689 |
0.260 |
SD |
NA |
0.051 |
0.252 |
0.049 |
0.036 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-4 |
11 |
-4 |
3 |
|
300 mg/kg bw/day |
Mean |
NA |
0.676* |
3.094 |
0.647* |
0.218 |
SD |
NA |
0.038 |
0.197 |
0.019 |
0.021 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-10 |
-7 |
-9 |
-13 |
|
1000 mg/kg bw/day |
Mean |
NA |
0.698 |
3.226 |
0.646* |
0.206* |
SD |
NA |
0.024 |
0.158 |
0.020 |
0.025 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-7 |
-3 |
-10 |
-18 |
|
|
|
DN |
DN |
DN |
DN |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
|
Organ weight relative to body weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
NA |
0.780 |
2.951 |
0.653 |
0.226 |
SD |
NA |
0.012 |
0.417 |
0.050 |
0.017 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
600 mg/kg bw/day |
Mean |
NA |
0.758 |
2.940 |
0.639 |
0.209 |
SD |
NA |
0.049 |
0.059 |
0.024 |
0.017 |
|
n |
NA |
5 |
5 |
5 |
5 |
|
±% |
NA |
-3 |
0 |
-2 |
-7 |
|
|
|
NS |
NS |
NS |
NS |
|
FEMALES – MAIN GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
13393.6 |
NA |
442.45 |
95.26 |
33.73 |
SD |
945.10 |
NA |
21.58 |
5.54 |
3.80 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
100 mg/kg bw/day |
Mean |
13883.8 |
NA |
510.19* |
95.22 |
36.02 |
SD |
1006.61 |
NA |
46.31 |
1.40 |
4.98 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
4 |
NA |
15 |
0 |
7 |
|
300 mg/kg bw/day |
Mean |
14830.5* |
NA |
459.23 |
95.99 |
32.42 |
SD |
820.56 |
NA |
44.96 |
6.08 |
4.45 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
11 |
NA |
4 |
1 |
-4 |
|
1000 mg/kg bw/day |
Mean |
1434.1 |
NA |
463.02 |
92.53 |
29.50 |
SD |
493.85 |
NA |
34.50 |
2.05 |
3.73 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
7 |
NA |
5 |
-3 |
-13 |
|
|
DN |
|
DN |
NS |
NS |
|
FEMALES – RECOVERY GROUP |
||||||
|
|
Body weight relative to brain weight [%] |
Organ weight relative to brain weight [%] |
|||
Group |
|
|
Brain |
Liver |
Kidney |
Spleen |
Control |
Mean |
12815.8 |
NA |
378.06 |
83.72 |
28.90 |
SD |
196.57 |
NA |
52.61 |
6.77 |
2.11 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
1000 mg/kg bw/day |
Mean |
13245.5 |
NA |
389.19 |
84.55 |
27.62 |
SD |
885.93 |
NA |
22.07 |
3.07 |
2.35 |
|
n |
5 |
NA |
5 |
5 |
5 |
|
±% |
3 |
NA |
3 |
1 |
-4 |
|
|
NS |
|
NS |
NS |
NS |
|
* p < 0.05, **p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
NA = Not Applicable
Table 10: Summary of necropsy findings
Organs |
Observations |
Frequency of observations per group |
|||||
Control |
100 mg/kg bw/day |
300 mg/kg bw/day |
1000 mg/kg bw/day |
||||
Main group |
Recovery group |
|
|
Main group |
Recovery group |
||
MALES |
|||||||
|
No macroscopic finding |
11/12 |
3/5 |
12/12 |
12/12 |
12/12 |
4/5 |
Thymus |
Hemorrhages |
0/12 |
2/5 |
0/12 |
0/12 |
0/12 |
1/5 |
Kidney |
Pyelectasia – both sides |
1/12 |
0/5 |
0/12 |
0/12 |
0/12 |
0/5 |
FEMALES - DAMS |
|||||||
|
No macroscopic finding |
10/10 |
4/5 |
12/12 |
6/11 |
12/12 |
3/5 |
Uterus |
Hydrometra |
0/10 |
1/5 |
0/12 |
5/11 |
0/12 |
2/5 |
FEMALES – NON-PREGNANT |
|||||||
Uterus |
Hydrometra |
1/1 |
NA |
NA |
1/1 |
NA |
NA |
FEMALES – DEAD ANIMAL |
|||||||
Lungs |
Hemorrhages |
1/1 |
NA |
NA |
NA |
NA |
NA |
Frequency of observation =number of animals with observations / number of animals examined
NA = not applicable
Table 11: Summary of histopathological findings
Organs |
Observations |
Incidence of observations per group |
||||||
Control |
100 mg/kg bw/day |
300 mg/kg bw/day |
1000 mg/kg bw/day |
|||||
Main group |
Dead animal |
Recovery group |
|
|
Main group
|
Recovery group |
||
MALES |
||||||||
Kidneys |
Pyelectasia (both side) |
1/5 |
NA |
0/5 |
- |
- |
0/5 |
0/5 |
Lungs |
Alveolar emphysema |
1/5 |
NA |
0/5 |
- |
- |
1/5 |
1/5 |
Acute hemorrhage |
1/5 |
NA |
0/5 |
- |
- |
0/5 |
0/5 |
|
Hyperplasia of BALT |
1/5 |
NA |
0/5 |
- |
- |
1/5 |
1/5 |
|
Thymus |
Acute hemorrhage |
0/5 |
NA |
1/5 |
- |
- |
0/5 |
1/5 |
FEMALES |
||||||||
Lungs |
Alveolar emphysema |
1/5 |
1/1 |
1/5 |
- |
- |
1/5 |
0/5 |
Acute hemorrhage |
0/5 |
1/1 |
0/5 |
- |
- |
1/5 |
0/5 |
|
Hyperplasia of BALT |
0/5 |
0/1 |
0/5 |
- |
- |
0/5 |
1/5 |
|
Uterus |
Dilatation |
1/11 |
0/1 |
1/5 |
- |
5/5 |
0/12 |
2/5 |
Incidence of observation =number of animals with observations / number of animals examined
BALT = bronchus associated lymphoid tissue
Table 12: Summary data of reproductive ability
Values |
|
Group [mg/kg bw/day] |
|
|
|
|
|
Control |
100 |
300 |
1000 |
MALES |
|||||
Fertility index |
% |
92 |
100** |
92 |
100** |
FEMALES |
|||||
Fertility index |
% |
91 |
100** |
92 |
100** |
* p < 0.05 CH2, ** p < 0.01 CH2
Table 13: Summary of clinical observation and fate of offspring
Observations/Fate of pup |
Group [mg/kg bw/day] |
||||
Control |
100 |
300 |
1000 |
||
Number of pups examined (liveborns) |
119 |
137 |
130 |
136 |
|
No signs |
N |
100 |
83 |
75 |
104 |
% |
84 |
61 |
58 |
76 |
|
Cold |
N |
15 |
50 |
42 |
36 |
% |
13 |
36 |
32 |
26 |
|
No milk in the stomach |
N |
19 |
33 |
33 |
32 |
% |
16 |
24 |
25 |
24 |
|
Hemorrhage |
N |
0 |
1 |
1 |
0 |
% |
0 |
1 |
1 |
0 |
|
Cianotic |
N |
0 |
1 |
0 |
0 |
% |
0 |
1 |
0 |
0 |
|
Pale |
N |
0 |
0 |
1 |
0 |
% |
0 |
0 |
1 |
0 |
|
Partially cannibalized |
N |
0 |
0 |
1 |
0 |
% |
0 |
0 |
1 |
0 |
|
Smaller than normal |
N |
0 |
0 |
1 |
0 |
% |
0 |
0 |
1 |
0 |
|
Found dead |
N |
0 |
0 |
3 |
0 |
% |
0 |
0 |
2 |
0 |
|
Missing |
N |
0 |
4 |
1 |
1 |
% |
0 |
3 |
1 |
1 |
|
Interim euthanasia |
N |
39 |
34 |
40 |
39 |
% |
33 |
25 |
31 |
29 |
|
Terminal euthanazia |
N |
80 |
99 |
86 |
96 |
% |
67 |
72 |
66 |
71 |
|
Table 14: Summary of body weight gain of offspring
Group |
|
Body weight gain (g) of offspring between postnatal days |
||
0 – 4 |
4 – 13 |
0 – 13 |
||
Control |
Mean |
4.0 |
13.0 |
23.0 |
SD |
0.8 |
1.9 |
2.1 |
|
n |
10 |
10 |
10 |
|
100 mg/kg bw/day |
Mean |
4.6 |
14.5 |
24.0 |
SD |
0.7 |
1.8 |
2.6 |
|
n |
12 |
12 |
12 |
|
300 mg/kg bw/day |
Mean |
4.3 |
14.3 |
24.7 |
SD |
1.0 |
2.9 |
2.1 |
|
n |
11 |
11 |
11 |
|
1000 mg/kg bw/day |
Mean |
4.8* |
14.3 |
24.7 |
SD |
0.6 |
2.0 |
1.8 |
|
n |
12 |
12 |
12 |
|
|
DN |
NS |
NS |
|
* p < 0.05
NS = Not Significant
DN = Duncan´s multiple range test
Table 15: Summary of anogenital distance of offspring
Group |
|
Body weight (g) on PN 4 |
Anogenital distance (AGD) on post-natal day 4 |
|
|
Absolute (mm) |
Normalized (AGD/3√bw) |
||
MALES |
||||
Control |
Mean |
10.10 |
6.2 |
2.86 |
SD |
1.17 |
0.51 |
0.21 |
|
n |
56 |
56 |
56 |
|
N |
10 |
10 |
10 |
|
100 mg/kg bw/day |
Mean |
10.98** |
6.5** |
2.93 |
SD |
1.17 |
0.57 |
0.26 |
|
n |
61 |
61 |
61 |
|
N |
12 |
12 |
12 |
|
300 mg/kg bw/day |
Mean |
10.18 |
6.3 |
2.92 |
SD |
1.16 |
0.54 |
0.22 |
|
n |
57 |
57 |
57 |
|
N |
11 |
11 |
11 |
|
1000 mg/kg bw/day |
Mean |
11.02** |
6.2 |
2.79 |
SD |
0.91 |
0.51 |
0.21 |
|
n |
66 |
66 |
66 |
|
N |
12 |
12 |
12 |
|
|
DN |
DN |
NS |
|
FEMALES |
||||
Control |
Mean |
10.00 |
3.4 |
1.58 |
SD |
1.12 |
0.55 |
0.24 |
|
n |
63 |
63 |
63 |
|
N |
10 |
10 |
10 |
|
100 mg/kg bw/day |
Mean |
10.66** |
3.5 |
1.61 |
SD |
0.90 |
0.53 |
0.24 |
|
n |
72 |
72 |
72 |
|
N |
12 |
12 |
12 |
|
300 mg/kg bw/day |
Mean |
10.08 |
3.4 |
1.59 |
SD |
1.18 |
0.50 |
0.23 |
|
n |
69 |
69 |
69 |
|
N |
11 |
11 |
11 |
|
1000 mg/kg bw/day |
Mean |
10.65** |
3.4 |
1.53 |
SD |
0.87 |
0.48 |
0.21 |
|
n |
69 |
69 |
69 |
|
N |
12 |
12 |
12 |
|
|
U |
NS |
NS |
|
* p < 0.05, ** p < 0.01
NS = Not Significant
DN = Duncan´s multiple range test
U = Mann-Whitney U-Test Versus Control
n = number of offspring
N = Number of litters
Applicant's summary and conclusion
- Conclusions:
- The test material had no effect on reproductive performance.
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