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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Principles of method if other than guideline:
- purity of test substance not mentioned (reference to analytical data sheet)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-020-3
EC Name:
-
Cas Number:
446824-06-2
Molecular formula:
C43H55N3O10 - C65H95N3O14
IUPAC Name:
1-isoctyloxycarbonyl ethylated 2,4,6 tris (2,4- hydroxyphenyl) -1,3,5 triazine derivatives
Details on test material:
- Substance type: Organic
- Physical state: Viscous liquid
- Storage condition of test material: Room temperature (approx. 20°C), away from direct sunlight

Test animals

Species:
rat
Strain:
other: HanBrLWIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Biotechnology & Animal Breeding Division, CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 130-161 grams (mean 143 grams), Females: 112 -133 grams (mean 125 grams)
- Fasting period before study:
- Housing: In groups of five
- Diet: ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 5d

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

IN-LIFE DATES:
Delivery of Animals 14 February 2002
Acclimatization/Pretest 15 to 20 February 2002
Administration/Treatment 21 February to 20 March 2002
Recovery 21 March to 03 April 2002
Termination (Necropsy) 21 March 2002 (Allocation A), 04 April 2002 (Allocation B)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly.
Test item was weighed into a glass beaker on a tared Mettler balance and the
vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer and
stored at room temperature (17-23°C).
Homogeneity of the test item in the vehicle was maintained during the daily administration
period using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): mixed with vehicle (corn oil)
- Storage temperature of food: RT

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 5 ml(kg bw
- Lot/batch no. (if required): 07939204 and 02938667
- expiry date: 2005 and 2004
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations
were determined in samples taken after experimental start. Concentration and homogeneity
of the dose formulations were determined in samples taken during week 3 of the treatment.
The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics
Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
Basis: actual ingested
Dose / conc.:
50 mg/kg bw (total dose)
Remarks:
Basis: actual ingested
Dose / conc.:
200 mg/kg bw (total dose)
Remarks:
Basis: actual ingested
Dose / conc.:
800 mg/kg bw (total dose)
Remarks:
Basis: actual ingested
No. of animals per sex per dose:
- 0 mg/kg bw/day (10 male and 10 female)
- 50 mg/kg bw/day (5 male and 5 female)
- 200 mg/kg bw/day (5 male and 5 female)
- 800 mg/kg bw/day (10 male and 10 female)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based upon the results of a non-GLP 5-day dose range-finding study, the test article was administered by gavage to 2 rats per group and sex. Test item-related changes were noted in animals treated with 600 mg/kg/day or 1000 mg/kg/day, whereas the findings noted in the latter group were more clearly defined. Based on the results of the five-day dose range-finding study, dose levels of 50, 200 or 800 mg/kg body weight/day are proposed for this study. In this study, test item-related findings are expected at 800 mg/kg/day, and 200 mg/kg/day is considered, likely to be the no-effect level. The highest dose of 1000 mg/kg/day used in the preliminary study was considered inappropriate for the main study as it was above the Maximum Tolerated Dose.
- Rationale for animal assignment (if not random): Computer-generated random algorithm
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

FUNCTIONAL OBSERVATION: Yes
- During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks, after 6 weeks
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes, 18h
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks, after 6 weeks
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks, after 6 weeks
- Metabolism cages used for collection of urine: Urine was collected during the 18-hour fasting period into a specimen vial.
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4, modified Irwin screen test were evaluated in all animals
- Dose groups that were examined: all
- Battery of functions tested: grip strength, locomotor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, after 4 weeks at 21 March 2002 (Allocation A) and after 6 weeks at 04 April 2002 (Allocation B)

Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (cerebrum, cerebellum, brain stem), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions

HISTOPATHOLOGY: Yes, slides of all organs and tissues listed in boldface type (see Necropsy, above) which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.
Other examinations:
ABSOLUTE AND RELATIVE ORGAN WEIGHTS:
The following organ weights were recorded on the scheduled dates of necropsy:
Brain, Thymus, Spleen, Ovaries, Heart, Liver. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,
body weight, organ weights and ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was
applied if the variables could be assumed to follow a normal distribution for the
comparison ofthe treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test
when the data could not be assumed to follow a normal distribution.
• Student's t-test was applied to grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test item related clinical signs of relevance for toxicological evaluation were observed during daily or weekly observations (weeks 1-3).
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and mean body weight gain of test item treated animals were comparable with those of the control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily and relative food consumption of test item treated animals compared well with those of the control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Increased (statistically significant, p<0.05) mean relative segmented neutrophils were noted in males treated with 200 mg/kg/day after four weeks when compared with controls. This was considered to be incidental because the value lies within the range of the historical control data.
Decreased (statistically significant, p<0.05) mean relative lymphocytes and absolute lymphocytes were noted in males treated with 200 mg/kg/day or 800 mg/kg/day, respectively, after four weeks when compared with controls. This was also considered to be incidental because the values lie within the range of the historical control data.
Increased (statistically significant, p<0.05) mean high reticulocyte fluorescence ratio as well as decreased (statistically significant, p<0.05) low reticulocyte fluorescence ratio were noted in females treated with 800 mg/kg/day after four weeks when compared with controls. This was considered to be incidental because no dose response relationship could be observed and it was seen in females only.
No other relevant changes in parameters of hematology were evident in animals after four weeks.
Increased (statistically significant, p<0.05) level of mean relative methemoglobin was noted in males treated with 800 mg/kg/day after six weeks when compared with controls. This was considered to be incidental because the value lies within the range of the historical control data.
Increased mean hemoglobin (statistically significant, p<0.01) and mean hematocrit (statistically significant, p<0.05) were noted in females treated with 800 mg/kg/day after six weeks when compared with controls. These were considered to be incidental because the values lie within the range of the historical control data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Decreased (statistically significant, p<0.05) mean phosphorus values were noted in males and decreased (statistically significant, p<0.01) mean values of triglycerides were seen in females treated with 800 mg/kg/day after four weeks when compared with controls. These were considered to be incidental because the values lie within the range of the historical control data.
Decreased (statistically significant, p<0.05) mean activity of alanine aminotransferase was noted in females treated with 50 mg/kg/day after four weeks when compared with controls. This was considered to be incidental because no dose response relationship was evident.
Decreased (statistically significant, p<0.01) mean activity of lactate dehydrogenase was noted in all test item treated females when compared with controls after four weeks. This was considered to be incidental because it was due to a relative high control value, no dose response relationship could be observed and it was not seen in males.
Decreased (statistically significant, p<0.01) activity of creatine kinase was noted in all test item treated females when compared with controls after four weeks. This was considered to be incidental because it was due to a relative high control value, no dose response relationship could be observed and it was not seen in males.
Increased (statistically significant, p<0.05) mean levels of calcium were noted in females treated with 800 mg/kg/day after six weeks when compared with controls. This was considered to be incidental because the value lies within the range of the historical control data.
Increased (statistically significant, p<0.01) mean phosphorus levels were noted in females treated with 800 mg/kg/day when compared with controls after six weeks. This was considered to be incidental because the value lies within the range of the historical control data.
The mean level of protein was increased (statistically significant, p<0.05) in females after six weeks when compared with controls. This was considered to be incidental because the value lies within the range of the historical control data. No other relevant differences were noted in test item treated animals after four or six weeks when compared with controls.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
Decreased (statistically significant, p<0.05) specific gravity and osmolality were noted in males treated with 800 mg/kg/day after four weeks when compared with controls. These were considered to be incidental because the values lie within the range of the historical data.
No other significant differences in parameters of urinalysis were noted in test item treated animals after four or six weeks when compared with controls.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Functional observational battery: No test item related findings of relevance for toxicological evaluation were observed during daily or weekly observations (week 4). Grip Strength: No test item related differences in fore- or hindlimb grip strength were evident. Locomotor Activity: Decreased (statistically significant, p<0.05) mean locomotor activity recorded from 0 to 15 minutes was observed in males and females treated with 800 mg/kg/ when compared with controls. This was considered to be a possible test item-related effect of uncertain toxicological relevance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
After 4 Weeks:
The mean organ weights, organ to body weight ratios and organ to brain weight ratios of test item treated males were comparable with those of controls. Increased mean liver weights (statistically significant, p<0.05), liver to body weight ratios (statistically significant, p<0.01) and liver to brain weight ratios (statistically significant, p<0.05) were noted in females treated with 50 mg/kg/day. The liver to body weight ratio of
the females treated with 800 mg/kg/day was increased (statistically significant, p<0.01) when compared with the controls. This was considered to be incidental because a dose response relationship was not observed at 200 mg/kg/day and it was not seen in males. Increased adrenals to body weight ratios (statistically significant, p<0.05) were observed in females at 800 mg/kg/day. This was considered to be incidental because it was due to a relative low body weight.
After 6 Weeks: Increased mean heart to body weight ratios (statistically significant, p<0.05) were noted in males treated with 800 mg/kg/day. This was considered to be incidental because it was seen
in males only and was not accompanied by macroscopic changes. No differences in mean organ weights, mean organ to body weight ratios or organ to brain weight ratios were evident in females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the end of treatment and following the recovery period, no test item-related macroscopic findings were observed. The few observed findings corresponded to those naturally occurring in the untreated control rats of this colony, age, and type of study and were considered incidental, reflecting the usual individual variability.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following microscopical changes were noted: In the liver of females a slight diminution of fatty change at doses of 800 mg/kg/day and in the adrenals of males a slight diminution of cortical fatty change at doses of 200 and 800 mg/kg/day at the end of treatment. Although test item-related, these findings are not considered to be of adverse character. After a 14-day treatment free recovery period, no test item related changes were observed.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
histopathology: neoplastic
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 50 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) and 800 mg/kg body weight/day as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

GENERAL

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 800 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 800 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

 

MORTALITY / VIABILITY: All animals survived until scheduled necropsy.

CLINICAL SIGNS: No test item related clinical signs of relevance for toxicological evaluation were observed during daily or weekly observations (weeks 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY: No test item related findings of relevance for toxicological evaluation were observed during daily or weekly observations (week 4).

- Grip Strength: No test item related differences in fore- or hindlimb grip strength were evident.

- Locomotor Activity: The observed significantly decreased mean locomotor activity in males and females, treated with 800 mg/kg/day, recorded from 0 to 15 minutes was considered to be a test item related effect.

FOOD CONSUMPTION: The mean daily and relative food consumption of test item treated animals compared well with those of the control animals.

BODY WEIGHT: The mean body weights and mean body weight gain of test item treated animals were comparable with those of the control animals.

CLINICAL LABORATORY INVESTIGATIONS

- Hematology: No test item related differences in parameters of hematology were evident after four or six weeks when compared with the controls.

- Clinical Biochemistry: No test item related differences in parameters of clinical biochemistry were evident after four or six weeks when compared with the controls.

- Urinalysis: No test item related differences in parameters of urinalysis were evident after four or six weeks when compared with controls.

ORGAN WEIGHTS: No test item related differences in mean organ weights, organ to body weight- and organ to brain weight ratios were evident after four or six weeks when compared with the controls.

MACROSCOPIC FINDINGS: At necropsy performed at the end of treatment period and following the recovery period, no test item-related macroscopic findings were observed.

HISTOPATHOLOGICAL FINDINGS: In the liver of females a slight increase of fatty change at doses of 800 mg/kg/day and in the adrenals of males a slight increase of cortical fatty change at doses of 200 and 800 mg/kg/day at the end of treatment. After 14-day treatment-free recovery period, no test item-related changes were observed.