Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethyl]-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-01-25 - 2005-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 27th 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

July 30th 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

479-940-2

EC Name:

-

Cas Number:

613246-75-6

Molecular formula:

NA: Multi-const. substance

IUPAC Name:

16-(dodecanoyloxy)-2,2,5,8,12,15,15-heptamethyl-7,10-dioxa-4,13-diazahexadeca-3,13-dien-1-yl dodecanoate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER (EUROPE) LABORATORIES INC.
- Age at study initiation: 50 - 61 days
- Weight at study initiation: male: 232 - 257g; female: 184 - 209g
- Housing: Group caging (5 animals/cage); III. type polypropylene/polycarbonate
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on oral exposure:

The test item was administered daily by gavage on a 7 days/week basis. Control animals were treated with vehicle only. Treatment volume was constant at 10 mL/kg bw throughout all groups. Actual treatment volume was calculated according to the most recent body weight. Animals were not treated on the day of gross pathology.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were chosen based on a previous 14-day oral toxicity study with Sika Hardener LJ. Dosing solution concentrations were controlled on the first and last treatment day using GC methodology in the Analytical Laboratory of LAB International Hungary. Three parallel samples were taken from the upper, middle and lower layers of each treatment concentration and control at both sampling times (72 samples total). Sika Hardener LJ content in dosing formulations ranged from 100% to 108% of the nominal concentration and was distributed homogeneously in sunflower oil.

Duration of treatment / exposure:

28 days

Frequency of treatment:

The test item was administered daily by gavage on a 7 days/week basis.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were chosen based on a 14-day oral toxicity study with Sika Hardener LJ.
- Rationale for selecting satellite groups: no satellite groups.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
- observation sites: skin, fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on day 0, then weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment and in week 4
- Dose groups that were examined: before treatment all test animals were examined. Examination in control group and group 4 on week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment by heart puncture
- Anaesthetic used for blood collection: Yes, pentobarbital (Nembutal) anaesthesia
- Animals fasted: Yes, overnight before blood sampling
- How many animals: all animals
- Parameters checked are as follows: RBC (Red Blood Cell count), WBC (White Blood Cell count), Hgb (Haemoglobin concentration), Htc (Haematocrit; relative volume of erythrocytes), MCV (Mean Corpuscular (erythrocyte) Volume), MCH (Mean Corpuscular (erythrocyte) Haemoglobin), MCHC (Mean Corpuscular (erythrocyte) Haemoglobin Concentration) Plt (Platelet (thrombocyte) count), PTT (Partial Thromboplastin Time), PT (Prothrombin Time), Differential white blood cell count, Reticulocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment by heart puncture
- Animals fasted: Yes, overnight.
- How many animals: all animals
- Parameters checked are as follows Glucose, T-BIL, Urea Nitrogen, Chol., Creat. Phos., Na+, K+, Ca++, Cl-, Total Protein, Albumin, A/G, AST/GOT, ALT/GPT, ALP, GGT

URINALYSIS: Yes
- Time schedule for collection of urine: Samples were collected over a period of 16 h from all animals on week 4
- Animals fasted: No
- Parameters checked are as follows: Leukocytes, Nitrite, pH, Protein, Glucose, UBG, Bilirubin, Ketone, Blood, Spec. gravity, Sediment, Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before first exposure and in the fourth exposure week
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to stimuli of different types (auditory, visual and proprioceptive), grip strength and motor activity

Sacrifice and pathology:

Gross pathology was performed on every animal after terminating treatment (one day after last dosing). Animals were sacrificed under pentobarbital (Euthanyl) anaesthesia. After exsanguination external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of tissues and organs were abserved macroscopically. Any abnormality was recorded with details of the location. colour, shape and size. The following organs/tissues were removed and preserved in 10% buffered formaldehyde solution:

Gross lesions, lymph nodes (submandibular, mesenteric), sternum, skin and female mammary gland, salivary glands (submandibular), femur and bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid and parathyroid, oesophagus, stomach, small and large intestines including Peyer´s patches (duodenum, ileum, jejunum, caecum, colon, rectum), urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum and ponds), eyes with optic nerve, Harderian glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve and aorta

Histopathology was performed on preserved organs and tissues of the control group and group 4 animals. Groups 2 and 3 showed no undiagnosed gross lesions at necropsy, so organs were not examined histologically.

The following organs were weighed and recorded:

With precision of 0.01 g: liver, kidneys (left + right), testes (left + right), epididymides (left + right), thymus, spleen, brain, heart
With precision of 0.001 g: adrenals (left + right)

Statistics:

Statistical analysis was done with SPSS PC software for the following data:

- body weight
- haematology
- clinical chemistry
- urine
- organ weight

The heterogeneity of variance between groups was checked by Bartlett´s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan´s Multiple Range test was used to assess 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 a 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.

Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

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):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality or clinical symptoms occured during the observation period. The behaviour, general state and reactivity to different types of stimuli of all animals were normal during the 28-day treatment period.

BODY WEIGHT AND WEIGHT GAIN
The body weight development was undisturbed during the 28 days. The mean body weight and the body weight gain were similar in all experimental groups and was similar to the expected values in untreated animals of the same age and strain.

OPHTHALMOSCOPIC EXAMINATION
No eye alterations were recorded at the end of the treatment period. The adnexa, conjunctiva, cornea, lens, vitrous body and fundus of eyes were intact in the control and high dose treated animals.

HAEMATOLOGY
Examined haematological parameters were comparable in control and treatment groups.

CLINICAL CHEMISTRY
Clinical chemistry revealed no biologically significant changes in parameters examined, even though some findings in treatment groups were statistically different when compared to the control group. No clear dose-response relationship was found and the degree of change was within the normal physiological range (and within historical control ranges), specific for animals of this species and strain with the same age.
Creatinine concentration (50mg/kg bw/day and 1000 mg/kg bw/day, male) and aspartate aminotransferase activity (1000 mg/kg bw/day, male) were decreased compared to control values. Sodium (50mg/kg bw/day and 1000 mg/kg bw/day, female), chloride (250mg/kg bw/day and 1000 mg/kg bw/day male, 50 mg/kg bw/day female), calcium (all dosed groups) and phosphorous (50 mg/kg bw/day, female) levels exceeded appropriate control values. Increased alanin aminotransferase activity was recorded for the male 250 mg/kg bw/day group. Total protein (1000 mg/kg bw/day) and albumin (50mg/kg bw/day, 1000 mg/kg bw/day) concentrations increased in female groups. The A/G ratio increased in both sexes ( 1000mg/kg bw/day male and 50 mg/kg bw/day female), due to the slight increase in albumin concentration.

URINALYSIS
Urine parameters varied within the normal (historical control) range. Control and treatment groups were not significantly different

NEUROBEHAVIOUR
The behaviour, general state and reactivity to different types of stimuli of all animals were normal during the 28-day treatment period.

ORGAN WEIGHTS
Spleen weight (absolute and referred to the body and brain weight) in group 4 (males) was statistically significant decreased when compared to the control group but were within the historical control range. Thus, changes in spleen weight were not considered biologically significant.Kidney weight referred to the brain weight, but not relative to the body weight or absolute kidney weight was statistically significant different in group 3 (males) compared to control animals. No dose dependent reduction in kidney weight was observed and the data was within the historical range. No statistically significant differences in organ weight were recorded for female groups. Clinical pathology, gross pathology and histopathology did not confirm treatment related effects. None of the statistically significant findings were indicative of treatment related adverse effects, but considered to be the result of biological variation.

GROSS PATHOLOGY
Gross pathology did not reveal macroscopic alterations related to treatment.
Pulmonary emphysema, point-like and pinprick-sized haemorrhages in the lungs were observed in the control and treated group. These alterations were caused by the termination procedures and exsanguination.
Pyelectasis occurred in one male animal. Taking into account the low incidence of this alteration and historical control data, pyelectasis has no toxicological significance in this study.
Hydrometra due to the sexual cycle was detected in one female rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histological examination of organs detected no lesions attributable to treatment. Observed changes were of equal frequency comparing control and group 4 animals (1000mg/kg bw/day) or were judged specific for species and strain of equal age.
The alveolar emphysema and focal haemorrhage in the lungs recorded for control and group 4 animals were considered a consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination.
Pyelectasis (one side) in the kidney occurred only in one mal animal without degenerative, inflammatory or fibrotic lesions in the renal tissues. This is a common disorder found in experimental rats of this strain.
The focal proliferation of cells belonging to the mononuclear phagocyte system (MPS) and focal vacuolisation of hepatocytes in the liver occurred in comparable incidences in control and group 4. Thus, these alterations were not considered to be of toxicological significance.
The dilatation of the uterine horn in one female animal is an individual slight neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs. This finding is not considered to be relevant taking into consideration of historical control.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Sika Hardener LJ caused no toxic effects in male and female CRL:(WI)BR rats after consecutive 28-day oral (gavage) administration of 50 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day doses. Therefore the No Observed Effect Level (NOEL) is 1000 mg/kg bw/day.

Executive summary:

Sika Hardener LJ was tested for repeated dose toxicity in a 28 day oral gavage test according to OECD guideline 407 and EU method B.7. Sika Hardener LJ was administered to CRL:(WI)BR rats (n=5/sex/dose) at dose levels of 0 mg/kg bw/day (vehicle only, control), 50 mg/kg bw/day, 250 mg/kg bw/day and 1000 mg/kg bw/day orally for 28 consecutive days. The stability and accurateness of concentrations of dosing solutions were analytically proven. Solutions were stable at room temperature for 4 hours and at 5±3 °C for 5 days. Test item content in dosing formulation samples ranged between 100 % and 108 % of nominal concentrations and was distributed homogeneously. Individual clinical observations were made on all animals daily. Detailed clinical observations were made outside the home cage on all animals once, prior to the first exposure and once on week 4. Body weight and food consumption were measured weekly. Blood and urine sampling for clinical pathology and gross pathology were conducted at the end of the treatment period. Selected organs were weighed. Histological examination was performed on the preserved organs or tissues of group 1 (control) and group 4 (1000 mg/kg bw/day).

Results were interpreted comparing treatment groups to the control group. The control group was treated in a similar way compared to treatment groups, but test compound was omitted and only vehicle (sunflower oil) administered.

No mortality or clinical symptoms were recorded during the 28-day treatment period. The general state, behaviour and reaction to different types of stimuli were similar in control and experimental groups.

The body weight, body weight gain and the mean daily food intake were comparable in control and experimental groups.

No eye alterations were recorded at the end of the treatment period.

Dosing animals with test item did not influence clinical pathology parameters. The slight changes in creatinine, sodium, chloride, calcium, phosphorous, total protein and albumin concentrations, in AST and ALT activity, in A/G ratio were within the physiological range and were judged to be without toxicological significance.

Pulmonary emphysema, point-like and pinprick-sized haemorrhages in the lungs (due to the termination procedures), pyelectasis and hydrometra (common alterations specific in experimental rats) were recorded in gross pathology. These findings were without toxicological significance as they were observed in comparable frequency between groups or in single animals only.

In group 4 (males, 1000 mg/kg bw/day), spleen weight was statistically significantly decreased on an absolute and relative basis compared to control animals, but within the historical control range. Kidney weight, relative to brain weight, was statistically significantly different in group 3 (250 mg/kg bw/day) animals when compared to control animals, but was within the historical control range. Clinical chemistry, gross pathology and histopathology do not support the conclusion of a treatment related effect with respect to organ weight. Overall, none of the statistically significant findings were indicative of treatment related adverse effects, but considered to be the result of biological variation.

In organs subjected to histopathological examination no test item related lesions were recorded. None of the histopathological findings were dose dependent. Alveolar emphysema and focal haemorrhage in the lungs (consequence of hypoxia, dyspnoea and circulatory disturbance developed during exsanguination), pyelectasis without degenerative, inflammatory or fibrotic lesions in the renal tissues (common alteration in this species), focal proliferation of cells belonging to the mononuclear phagocyte system (MPS) in the liver, focal vacuolisation of hepatocytes, (occurred in control group and in group 4 with similar frequency), dilatation of the uterine horns (due to the sexual function of the inner genital organs) were observed. These alterations are specific for this species, and are therefore not considered toxicologically significant.

The study was marked as reliable without restrictions. The No Observed Effect Level (NOEL) 1000 was mg/kg bw/day. (LAB, 2005)