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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-16 to 2014-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: Rat, Hsd.Brl.Han: of Wistar origin
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90, Budapest, Hungary
- Age at study initiation: 44-49 days
- Weight at study initiation: males: 170-205 g, females: 123-155 g
- Housing: 2 or 3 animals of the same sex/cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice, ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 400)
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore Polyethylene glycol 400 was used for preparing formulations appropriate for oral administration. Polyethylene glycol 400 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: The test item was formulated in the vehicle (PEG 400) in concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations (concentration and homogeneity) in the vehicle in the conditions employed on the study was performed in the Analytical Laboratory of the Test Facility. The test item was analyzed using reverse phase HPLC method with UV detection. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on 2 occasions. Similarly, five samples were taken from the control solution (group 1) and analyzed.
Date of samplings and analysis: October 21, 2013 and January 13, 2014
Duration of treatment / exposure:
Dosing of both sexes began after the acclimatization and was continued up to and including the day before the necropsy for the main groups.
Duration of treatment: 90 or 91 days (depending on day of necropsy). Animals were not treated on the day of gross pathology.
Animals assigned to the recovery group were treated identically up to Day 90 then they were only observed without administration for four weeks.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
0 and 1000 mg/kg bw/day: 15 males and 15 females each (10 animals allocated to main groups and 5 animals allocated to recovery groups each)
100 and 300 mg/kg bw/day: 10 males and 10 females each
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 100, 300 and 1000 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study with SIKA Hardener LI in the Rat (28-day oral toxicity study in rats, Report no. 08/732-100P). Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and once weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed with an accuracy of 1 g on Day 0, then weekly during the course of the treatment and recovery periods.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During acclimation period (all animals), prior to test termination on all control and high dose test animals

HAEMATOLOGY: Yes
- How many animals: all animals
- Parameters: WBC (White Blood Cell (leukocyte) count), RBC (Red Blood Cell (erythrocyte) count), HGB (Hemoglobin concentration), HCT (Hematocrit), MCV (Mean Corpuscular (erythrocyte) Volume), MCH (Mean Corpuscular (erythrocyte) Hemoglobin), MCHC (Mean Corpuscular (erythrocyte) Hemoglobin Concentration), Reticulocytes, Platelet (thrombocyte) count

CLINICAL CHEMISTRY: Yes
- How many animals: all animals
- Parameters: ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), GGT (Gamma Glutamyltransferase activity), ALP (Alkaline Phosphatase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration), Urea concentration, BUN (Blood Urea Nitrogen Concentration), Glucose concentration, Cholesterol concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organs: adrenal glands, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes ((lachrymal gland and Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with cervix and vagina), gross lesions, heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular and 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
HISTOPATHOLOGY: Yes
Organs: Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Thymus of one male animal of 300 mg/kg bw/day groups was also processed and examined histologically due to necropsy finding.

Oran weights: With precision of 0.01g: liver, kidneys, testes, epididymides, uterus, thymus, spleen, brain and heart, With precision of 0.001g: adrenals, ovaries
Statistics:
Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- food consumption
- estrous cycle
- hematology
- blood coagulation
- clinical chemistry
- organ weight data
- sperm parameters
The heterogeneity 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) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. For the evaluation of data in the recovery group, 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.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated number of the white blood cells was observed in female animals administered with 1000 or 300 mg/kg bw/day with respect to the control at the termination of the treatment.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item related higher mean spleen weights were detected in female animals of 1000 mg/kg bw/day group and in 300 mg/kg bw/day at the termination of the treatment.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Treatment period
Test item related salivation was observed in male and female animals administered with 1000 or 300 mg/kg bw/day with variable frequency within a group but in a dose related manner regarding the incidence and onset (male: 15/15, 6/10; female 15/15, 6/10; respectively to doses). One male animal of the 100 mg/kg bw/day group (1/10) salived between Days 73 and 77. The behavior and physical condition of animals were considered to be normal at each dose level (1000, 300 and 100 mg/kg bw/day) during the treatment period.
Individual dermal alterations were observed in three male animals of 300 mg/kg bw/day group (3/10; scar on the neck) and in one male animal of 100 mg/kg bw/day group (1/10; scar on the neck). Scar is a common clinical sign in this strain of experimental rats of this age and was only present in animals of the low and the middle dose groups.
Recovery period
Clinical signs were not detected in the male or female animals of 1000 mg/kg bw/day group or in the control group during the recovery period.
The general physical condition and behavior of animals were considered to be normal at the detailed weekly observations throughout the entire observation period (treatment and recovery periods). Scars were also recorded in animals mentioned above: 2/10 male animals of 300 mg/kg bw/day and in one male animal (1/10) of 100 mg/kg bw/day group on the days of detailed weekly clinical observations.

FUNCTIONAL OBSERVATION BATTERY
Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 1000, 300 or 100 mg/kg bw/day).
The behavior and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

WEIGHT ASSESSMENT
Treatment period
The body weight and body weight gain of the male and female animals were unaffected in all test item treated groups (1000, 300 and 100 mg/kg bw/day) during the entire observation period.
Statistical significance was only noted for the slightly less mean body weight gain of female animals in group of 1000 mg/kg bw/day between Days 84 and 89 and for the slightly higher mean body weight gain of female animals of 100 mg/kg bw/day group between Days 63 and 70. Although differences with respect to the control reached statistical significances these were transient and did not influence the mean body weight or the summarized body weight gain. Therefore these slight changes were considered to be without any toxicological relevance.
Recovery period
The body weight and body weight gain were similar in the male and female animals of 1000 mg/kg bw/day and control groups during the recovery period.

FOOD CONSUMPTION
Test item related effects on the mean daily food consumption were not detected.
Treatment period
The daily mean food consumption was comparable in the control and all test item treated groups (1000, 300 or 100 mg/kg bw/day).
The mean daily food consumption exceeded the control value in female animals at 100 mg/kg bw/day on weeks 4, 7, 9 and 10. These slight but statistically significant differences were not considered to be biologically or toxicologically relevant due to the low degree and sporadic occurrence.
Recovery period
There were no significant differences in the mean daily food consumption between the control and 1000 mg/kg bw/day groups during the four weeks post-treatment period.

OPHTHALMOLOGICAL EXAMINATION
The eyes were without any detected abnormalities in all animals before treatment and in the high dose group at termination of the treatment.

EXAMINATION OF ESTROUS CYCLE
A test item influence on the estrous cycle was not detected.
The percent of animals with regular and irregular estrous cycle was similar in the control and test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of cycles, days in estrous or diestrous at the end of the treatment period or recovery period. Statistically significant difference was detected in the higher mean number of days in pro-estrous in animals of 300 mg/kg bw/day. This slight change was considered to be indicative of biological variation and not related to the test item as similar findings was not found at the higher dose and value was within the historical control ranges.

HAEMATOLOGY
Hematological investigations revealed test item related changes in the white blood cell count in female animals administered with 1000 mg/kg bw/day at termination of the treatment and at the end of the recovery period.
Main groups
At termination of the treatment, statistical significance was noted in male animals for a slightly shorter prothrombin time (PT) at 300 mg/kg bw/day.
The mean white blood cell count (WBC) was significantly higher and the mean percentage of eosinophil granulocytes (EOS) was significantly less in female animals of 1000 and 300 mg/kg bw/day with respect to the control group. The mean percentage of basophil granulocytes (BASO) and reticulocytes (RET), as well as the prothrombin time was higher than in the control group in female animals of 1000 mg/kg bw/day group.
Recovery groups
In the male animals of 1000 mg/kg bw/day recovery group, all examined hematological parameters were similar to values in the control group.
In the female recovery group of 1000 mg/kg bw/day dose, the mean white blood cell count, mean percentage of lymphocytes (LYM) and reticulocytes were higher than in the control females. The mean percentage of neutrophil granulocytes (NEU), monocytes (MONO) and eosinophil granulocytes were less with respect to the appropriate value in the control group.
The higher count of the white blood cells in female animals of 300 mg/kg bw/day, and the statistically significant differences between the control and treated groups in the mentioned parameters (NEU, LYM, EOS, BASO, MONO and RET) were judged to be of little or no biological significance as the mean values were well within the historical control ranges and there were no related pathologic histological changes.

CLINICAL CHEMISTRY
No pathologic test item effect was detected at the evaluation of clinical chemistry parameters.
Main groups
In male animals, statistically significant higher mean activity of alkaline phosphatase (ALP; 1000 mg/kg bw/day) and slightly higher mean inorganic phosphorous concentration (Pi; 1000, 300 and 100 mg/kg bw/day) were observed with respect to the control at the termination of the treatment. Furthermore, in the male animals, activity of alanine aminotransferase (ALT) was slightly less than in the control group at 1000 and 300 mg/kg bw/day. Less mean concentration of glucose (GLUC) was noted for males of the 300 mg/kg bw/day group and total protein (TPROT) and higher albumin/globulin ratio (A/G) were noted for male animals administered with 300 and 100 mg/kg bw/day doses. Statistical significances in ALT activity, total protein and inorganic phosphorous concentrations and A/G ratio were without dose response. The mean activity of alkaline phosphatase in the high dose group exceeded the value of control group however was well within the historical control range. Therefore these findings were not considered to be of toxicological relevance.
In female animals, statistical significances were noted for the higher mean concentration of chloride (Cl-) and mean inorganic phosphorous concentration (Pi) at 300 mg/kg bw/day group and for less mean concentration of bile acids (BAC) at 100 mg/kg bw/day.
The mentioned differences between the control and test item treated groups were statistically significant but were only slight and all values remained within the historical control ranges for these parameters. Therefore these findings were considered to be of little or no biological relevance.
Recovery groups
The mean activity of alkaline phosphatase was slightly higher in female animals of 1000 mg/kg bw/day with respect to the control. Although the difference from the control reached statistical significance the slight change was considered to be indicative of biological variation as values remained well within the historical control range and similar findings were not observed at the termination of the treatment.
All other examined parameters were comparable in male and female animals in the control or 1000 mg/kg bw/day groups.

NECROPSY
Specific macroscopic findings related to the test item were not detected at the necropsy of male or female animals of 1000, 300 or 100 mg/kg bw/day groups.
Main groups
Sporadic point-like hemorrhages in the thymus was found in one male animal of 300 mg/kg bw/day (1/10).
In female animals, enlargement of the spleen was noted for one animal of the 1000 mg/kg bw/day group (1/10) and 2 mm dense formation in the uterine horn in one animal of the 300 mg/kg bw/day group (1/10).
Slight, moderate or marked hydrometra was observed in several female animals in each group: 4/10 at 1000 mg/kg bw/day, 2/10 at 300 mg/kg bw/day, 2/10 at 100 mg/kg bw/day and 1/10 in the control group.
Recovery groups
Four weeks after the termination of the treatment, enlarged submandibular salivary glands with point-like hemorrhages were detected in one male animal administered with 1000 mg/kg bw/day dose (1/5) and slight or moderate hydrometra was observed in the female animals of 1000 mg/kg bw/day (4/5) and control (2/5) groups.
All these necropsy findings were considered to be individual findings and not related to the test item. Hemorrhages in the thymus might be caused by the exsanguination procedure. Hydrometra related to the female sexual cycle is a frequent observation in experimental rats and was present in all groups. Enlargement of the spleen, 2 mm dense formation in the uterine horn and changes in salivary glands occurred only in single animals, were not related to doses and were without pathologic histological lesion.

ORGAN WEIGHTS
Test item related spleen weight changes were observed in female animals of 1000 mg/kg bw/day at the end of the treatment and recovery periods.
Main groups
Slight, but statistically significant difference was detected in the higher mean kidney weights relative to body weight in male animals in 1000 mg/kg bw/day group.
The mean liver and spleen weights (absolute and relative to body and brain weights) and kidney weights (relative to body and brain weights) of female animals of 1000 mg/kg bw/day group were slightly, but statistically significantly higher with respect to controls. Similar findings were also noted for higher mean spleen weight relative to the body weight in female animals at 300 mg/kg bw/day.
Recovery groups
The mean testes weight relative to body weight slightly exceeded the control value in male animals of 1000 mg/kg bw/day recovery group.
The mean spleen weights (absolute and relative to body and brain weights) of female animals in 1000 mg/kg bw/day group were statistically significantly higher than in the control group at the end of the recovery period. Statistically significant difference with respect to the control was detected in the body weight relative to the brain weight in the female animals of the 1000 mg/kg bw/day group.
Statistically significant changes in the mean liver or kidneys weights in high dose animals and in spleen weights of 300 mg/kg bw/day group were considered to be of little or no biological significance because the values were within the historical control range and thus, considered to be of no toxicological relevance.

SPERM EXAMINATIONS
Sperm examinations did not point out any test item related influence on the sperm cells at 1000 mg/kg bw/day.
The total sperm count, sperm motility and percentage of sperms with not normal morphology (separated head and tail) were similar in 1000 mg/kg bw/day and in the control groups.

HISTOPATHOLOGY
Histopathological investigation did not reveal test item related changes in the organs or tissues of animals administered with 1000 mg/kg bw/day dose (male or female).
Minimal or mild alveolar emphysema (2/10 male and 2/10 female in the control group; 1/10 male at 1000 mg/kg bw/day) and acute hemorrhages (1/10 male in the control group; 2/10 male at 1000 mg/kg bw/day) were detected in the lungs. These findings were also present in some animals of the recovery group: alveolar emphysema in 2/5 control male and in 2/5 male at 1000 mg/kg bw/day. Acute hemorrhages were observed in the thymus (1/1 male at 300 mg/kg bw/day), in the salivary gland and in the submandibular lymph node (1/5 male in the recovery group of 1000 mg/kg bw/day). Pulmonary emphysema, acute hemorrhages in the lungs, thymus, salivary gland or submandibular lymph node were considered to be the consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
The hyperplasia of bronchus associated lymphoid tissue (BALT) in some control and treated animals (4/10 male and 3/10 female control; 3/10 male and 1/10 female at 1000 mg/kg bw/day; recovery control 1/5 male and 2/5 female; 1000 mg/kg bw/day recovery 1/5 male) was considered to be a physiological phenomenon.
The dilatation of the uterine horns in some female animals (1/10 control; 4/10 at 1000 mg/kg bw/day; recovery: 2/5 control and 3/5 in the 1000 mg/kg bw/day; group) is a slight neuro-hormonal phenomenon in connection with the sexual function – estrous phase – of the inner genital organs.
The hyperplasia in the spleen (1/10 female at 1000 mg/kg bw/day) and the myoma in the uterus (1/1 female at 300 mg/kg bw/day) were individual disorders. No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the digestive system, cardiovascular system, urinary system, immune system, hematopoietic system, the skeleton, the muscular system, the male reproductive system or the central, or peripheral nervous system was observed.
There were no differences between the test item treated and control animals regarding the quantity or cytomorphology of lymphoid cells in the spleen, thymus, lymph nodes, Peyer’s patches and of the myeloid and erythroid blast cells in the bone marrow (in the sternum and femur) by light microscopic investigation.
The structure and the cell morphology of the endocrine glands was the same in the control and treated animals.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: haematology; organ weights

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, under the test conditions chosen the NOAEL for SIKA Hardener LI was determined to be 1000 mg/kg bw/day in male rats and 300 mg/kg bw/day in female rats.
Executive summary:

A 90-day oral (gavage) toxicity study was performed with SIKA Hardener LI in male and female Hsd.Brl.Han: Wistar according to OECD Gudeline 408. The test item was administered orally to the test animals (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day doses corresponding to concentrations of 200 mg/mL, 60 mg/mL and 20 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 days. 5 animals/sex in the control and high dose groups were observed without administration for four weeks (recovery observations). Polyethylene glycol 400 was used as vehicle.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were determined. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Thymus showing macroscopic changes was examined histologically in one male animal of 300 mg/kg bw/day group.

SIKA Hardener LI caused salivation (male and female), changes in white blood cell count (females) and in spleen weight (females) at 1000 mg/kg bw/day in Hsd.Brl.Han: Wistar rats after the consecutive 90-day oral (gavage) administration. At 300 mg/kg bw/day, salivation was observed in some male and female animals during the treatment period. Slight changes in the white blood cell count and spleen weight relative to body weight in female animals were judged to be toxicologically not relevant. At 100 mg/kg bw/day, there was no test item related effect. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows: NOAEL: 1000 mg/kg bw/day for male animals; NOAEL: 300 mg/kg bw/day for female animals.