"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 27, 1995

Deviations: no

Test guideline 2

Qualifier: according to guideline

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

Version / remarks: September 30, 1996 Toxicological information Repeated dose toxicity: oral REACH 3,4,5-trifluorophenol

Deviations: no

Test guideline 3

Qualifier: according to guideline

Guideline: other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances

Version / remarks: December 5, 1986

Deviations: no

GLP compliance: yes

Limit test: no

Test material

Test material information

Constituent 1

Reference substance name: 3,4,5-trifluorophenol

EC Number: 436-120-9

EC Name: 3,4,5-trifluorophenol

Cas Number: 99627-05-1

Molecular formula: C6H3F3O

IUPAC Name: 3,4,5-trifluorophenol

Test animals

Species: rat

Strain: Wistar

Remarks: HanBrl:WIST

Sex: male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: RCC Ltd, Laboratory Animal Services CH-4414 Fullinsdorf / Switzerland

- Age at study initiation: 6 weeks

- Weight at study initiation: Males: 132.9 - 152.0 grams (mean 139.8 grams) Females: 115.0 -129.5 grams (mean 120.2 grams)

- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding.

- Diet: ad libitum, Pelleted standard Provimi Kliba 3433 rat maintenance diet

- Water: ad libitum, community tap-water

- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

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 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (19-25°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations: yes

Details on analytical verification of doses or concentrations:

First analysis: The mean concentrations of the homogeneity samples taken during pretest preparations were found to be 92.2%, 110.3% and 112.9% of the nominal concentrations of dose group 2 (10 mg/mL), dose group 3 (30 mg/mL), and dose group 4 (90 mg/mL), respectively. The individual concentrations varied in the range from -8% to +7% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle. Second analysis: The mean concentrations of the homogeneity samples were found to be 109.0%, 108.0%, and 95.8% of the nominal concentrations of dose group 2 (10 mg/mL), dose group 3 (30 mg/mL), and dose group 4 (90 mg/mL), respectively. The individual concentrations varied in the range from -3% to +2% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Duration of treatment / exposure: 28 days (+ 14 days recovery)

Frequency of treatment: daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.: 50 mg/kg bw/day (nominal)

Doses / concentrations 2 Dose / conc.: 150 mg/kg bw/day (nominal)

Doses / concentrations 3 Dose / conc.: 450 mg/kg bw/day (nominal)

No. of animals per sex per dose: 10

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 (RCC Study Number 854323) in which the test item was administered by gavage to 2 rats per group and sex.

GENERAL

In the dose range-finding toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 30, 150 or 750 mg/kg body weight for a period of five days. A control group received a similar dose volume (5 mL/kg body weight) of the vehicle, PEG 300. The study comprised two animals per group and sex which were sacrificed after five days of treatment. Clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. At the end of the dosing period, all animals were killed, necropsied and examined post mortem. The results of the study are summarized as follows:

Mortality

Female no. 16 (750 mg/kg/day) was found dead on treatment day 3. All other animals survived until scheduled necropsy.

Clinical Signs

There were no clinical signs evident at any dose level.

Food Consumption

The mean daily food consumption of the test item-treated males compared favorably with that of the control males. The mean daily food consumption of the females treated with 750 mg/kg/day was markedly reduced from days 3-5 of treatment.

Body Weights

No test item-related changes in mean body weights were noted at any dose level.

Organ Weights

Elevated liver-to-body weight ratios were noted in males and females treated with 750 mg/kg/day when compared with the controls. Elevated kidney-to-body weight ratios were noted in males treated with 750 mg/kg/day, whereas elevated spleen-to-body weights were seen in the remaining female treated with 750 mg/kg/day. No organ weight changes of toxicological relevance were noted at any other dose level.

Macroscopical Findings

Red thymic foci were noted in one female treated with 30 mg/kg/day and one female treated with 150 mg/kg/day. The one female treated with 750 mg/kg/day which was found dead (no.16) on treatment day 3 was without macroscopical changes; a cause of death could not be established.

ASSESSMENT

Based on the results of the five-day dose range-finding study, dose levels of 50, 150 or 450 mg/kg body weight/day are proposed for the 28-day study (RCC Study Number 854324).

Positive control: none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

- Time schedule: daily

The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29- 42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: weekly

The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1- 3) thereafter.

BODY WEIGHT: Yes

- Time schedule for examinations: weekly

Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes

The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes

- Time schedule for collection of blood: after 4 weeks and after 6 weeks

- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia

- Animals fasted: Yes

- How many animals: all

- Parameters examined: Erythrocyte count, Reticulocyte count, Hemoglobin, Reticulocyte maturity index, Hematocrit, Methemoglobin, Mean corpuscular volume, Total leukocyte count, Red cell volume distribution with Differential leukocyte count, Mean corpuscular hemoglobin, Coagulation, Mean corpuscular hemoglobin concentration, Thromboplastin time, Hemoglobin concentration, distribution width, Activated partial thromboplastin time, Platelet (thrombocyte) count

CLINICAL CHEMISTRY: Yes

- Time schedule for collection of blood: after 4 weeks and after 6 weeks

- Animals fasted: Yes, light isoflurane anesthesia

- How many animals: all

- Parameters examined: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic Protein, total Albumin, Globulin, Albumin/Globulin ratio, Bile acids

URINALYSIS: Yes

Urine was collected during the 18-hour fasting period into a specimen vial after 4 weeks and after 6 weeks. The following urinalysis parameters were determined: Volume (18 hours), Glucose, Specific gravity (relative density), Ketones, Color, Urobilinogen, Appearance, Bilirubin, pH, Erythrocytes, Nitrite, Leukocytes, Protein

NEUROBEHAVIOURAL EXAMINATION: Yes

- Time schedule for examinations: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.

- Dose groups that were examined: all

- Battery of functions tested: grip strength / locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (3 levels), 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

ABSOLUTE AND RELATIVE ORGAN WEIGHTS

The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides

HISTOPATHOLOGY: Yes

Slides of organs and tissues that were collected at scheduled sacrifice from the animals of control and high-dose groups, as well as livers of low and middle dose groups, were examined.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:

• The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.

• Fisher's exact-test were applied to the macroscopic findings. The following statistical methods were used for statistical analysis of clinical laboratory data:

• Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p<0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05)."

Male HanBrl:WIST Rats