2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-Dec-1997 to 12-Mar-1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): CFG-C-1607
- Physical state: yellow solid
- Analytical purity: >98%
- Lot/batch No.: 6
- Expiration date of the lot/batch: 30 Jul 1999
- Stability under test conditions: stable in PEG 400 for at least 2 hours
- Storage condition of test material: in well-closed container at room temperature away from direct sunlight.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: 4 weeks
- Fasting period before study: none
- Weight at study initiation: Males: 60-91g Females: 45-69 g
- Housing: Groups of five in Makrolon type-3 cages with standard softwood bedding.
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 rat maintenance diet available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itengen, available ad libitum
- Acclimation period: 7 days under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degrees centigrade
- Humidity (%): 40 - 70%
- Air changes (per hr):10 - 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From (Delivery): 1-Dec-1997; To (Termination): 13-March-1998

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was placed into a glass beaker on a tared Mettler PM 460 balance and the vehicle was added. A weight by volume dilution was prepared using a magnetic stirrer as homogenizer. Homogeneity of the test article in the vehicle was maintained during treatment. Test preparations made daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility and stability
- Concentration in vehicle: 20, 100, and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg-bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article / vehicle mixtures were determined in samples taken during the first week of treatment. Intercurrent samples for homogeneity and concentration analyses were taken during the second and third month of treatment.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Once daily, 7 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of 14 day range-finding study

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, weekly

FOOD EFFICIENCY:- Relative food consumption (g/kg-bw) determined: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to first exposure and at week 13
- Dose groups that were examined: pre-test: 10 rats, per sex, per dose; at week 13: 10 rats, per sex, from control and high dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 5, 9, and 13
- Anaesthetic used for blood collection: light ether anesthesia
- Animals fasted: Yes, 18 hours
- How many animals: 10 per sex per dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5, 9, and 13
- Animals fasted: Yes, 18 hours
- How many animals: 10 per sex per dose

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5, 9, and 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-test and at weeks 6 and 12.
- Dose groups that were examined: all control and dose groups
- Battery of functions tested: modified Irwin screen test incl. sensory activity and motor activity; and hind-forelimb grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Five animals died spontaneously during the study, one male died accidentally after blood sampling and one animal died accidentally during anesthesia; all of them were necropsied imrnediately.

ORGAN WEIGHTS: Yes
Adrenals, Spleen, Brain, Testes, Kidneys, Thyroid, Liver, Thymus

HISTOPATHOLOGY: Yes
Adrenal glands
Aorta
Brain - including section of medulla/pons, cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides
Esophagus
Exorbital lacrimal glands
Eyes with optic nerve and Harderian gland
Femur with bone marrow
Heart
Ileum
Jejunum including Peyer's patches
Kidneys
Liver
Lungs, infused with formalin
Lymph nodes - mesenteric, mandibular
Mammary gland area
Nasal turbinates
Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Semina! vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid gland / parathyroid gland
Trachea
Urinary bladder, infused with formalin
Uterus
Vagina
Zymbal's gland
All gross lesions

Statistics:

The following statistical methods will be used to analyze the body weight, food consumption, organ weights and clinical laboratory data: If the variables can be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate will be applied for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) will be applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution. The exact Fisher-test will be applied to the ophthalmoscopy data and to macroscopical findings. Additional methods of statistical analysis will be used at the discretion of the statistician. The methods and the results will be described in the report.

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:

no effects observed

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:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test substance related adverse effects observed

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mortality

No test article-related cases of death occurred during the conduct of the study.

There were some cases of death that are considered to be not test article-related:

-      1 (control)/2 (low dose) male rats and 1 (control) / 1 (high dose) female rat died spontaneously. According to the findings seen at necropsy, these animals are assumed to have died after accidental administration of the test article into the respiratory tract.

-      1 male rat (mid dose) died accidentally after blood sampling.

-      1 female rat (mid dose) died accidentally during anesthesia.

Clinical Signs

There were no test article-related clinical signs noted in any dose group.

 

Functional Observational Battery

At 6 and 12 weeks neither the functional observational battery nor the evaluation of hind- and forelimb grip strength revealed test article-related findings.

 

Body Weight / Body weight gain

There was no effect on body weight / body weight gain.

 

Food Consumption / Relative Food Consumption

There were no effects on food consumption or relative food consumption.

 

Ophthalmoscopic Examinations

There were no test article-related ophthalmologic findings.

 

Hematology

No test article-related remarkable findings were noted at 5, 9 and 13 weeks of treatment. In addition, there were no adverse effects on the immune system, i.e. no changes with regard to the total leucocyte count or to peripheral blood lymphocytes, monocytes and polymorphonuclear leucocyte populations in the differential leucocyte count.

 

Clinical Biochemistry

There were no toxicologically relevant effects on clinical biochemistry data at 5, 9 and 13 weeks of treatment. Any intergroup variation was considered to be within the range of the background data seen commonly in rats of this strain and age.

 

Urinalysis

There were no toxicologically relevant effects on urinalysis data at 5, 9 and 13 weeks of treatment. Any intergroup variation was considered to be within the range of the background data seen commonly in rats of this strain and age.

 

Organ Weights and Organ Weight Ratios

There were no test article-related changes in organ weights and ratios.

 

Macroscopical Findings

Necropsy revealed no pathomorphologic lesions considered to be related to the treatment with the test article.

 

Microscopical Findings

No microscopical findings considered to be related to the treatment with the test article were recorded. In particular, no adverse effect on the immune system was recorded and there was no evidence of any neurotoxic effect of the test article.

Applicant's summary and conclusion

Conclusions:

The oral administration of CGF-C-1607 to Wistar rats at doses of 100, 500 or 1000 mg/kg/day, for at least 92 days, resulted in no evidence, either on the in-life or pathomorphologic parameters, of toxic effects of the test article. In particular, no adverse effect on the immune system was recorded and there was no evidence of any neurotoxic effect of the test article. The few changes noted in clinical biochemistry and urinalysis parameters were considered to be typical findings within the range of biological variation and the historical control data. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for CGF-C-1607 was >= 1000 mg/kg.

Executive summary:

In a subchronic toxicity study, CGF-C-1607 was administered orally by gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 500 or 1000 mg/kg body weight/day, daily for a period of at least 92 days. Each group comprised 20 animals per sex. A similarly constituted group received the vehicle only and served as a control. Mortality/viability, clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment period. Ophthalmoscopic examinations were performed during acclimatization and in the last week of the treatment period (control and high dose animals only). A functional observational battery including an evaluation of hind- and forelimb grip strength was performed during pretest and at weeks 6 and 12. At weeks 5, 9 and 13, blood samples were withdrawn from the 10 rats with the lowest identification numbers per sex and group for hematology and clinical biochemistry analyses, and urine samples were collected for urinalysis. All animals were killed, necropsied and examined post mortem after at least 92 days of treatment. The weights of the adrenals, brain, kidneys, liver, spleen, testes, thyroid and thymus were recorded. Organs and tissues from all animals of any groups, as well as gross lesions from all animals, were processed as hematoxylin and eosin stained slides and examined by light microscopy.

The oral administration of CGF-C-1607 to Wistar rats at doses of 100, 500 or 1000 mg/kg/day, for at least 92 days, resulted in no evidence, either on the in-life or pathomorphologic parameters, of toxic effects of the test article. In particular, no adverse effect on the immune system was recorded and there was no evidence of any neurotoxic effect of the test article. The few changes noted in clinical biochemistry and urinalysis parameters were considered to be typical findings within the range of biological variation and the historical control data. Based on the results of this study, the no-observed-effect-level (NOEL) for CGF-C-1607 was defined as 1000 mg/kg.