"Observations and examinations performed and frequency: Daily observations

All animals were observed especially for the following symptoms daily:

General condition

Visible mucous membranes

Behaviour and locomotor activity

Central nervous symptoms

Body weight, Food and Water consumption

Individual body weights, food and water consumption were recorded weekly.

Haematology and Clinical Chemistry

Haematological, clinico-chemical and urine analyses were performed before final sacrifice. Blood samples were collected from each animal prior to necropsy following an overnight fasting period. Blood was obtained by puncture of the retrobulbar venous plexus under light halothane anaesthesia and collected in tubes coated with K2-EDTA (haematology) and heparin lithium salt (clinical chemistry). For determination of prothrombin time and partial thromboplastin time, blood was drawn at necropsy from the vena cava using a syringe with 10% (v/v) of 0.11 mmol/l citric acid sodium salt as the anticoagulant. The samples were analysed in a randomised sequence.

Sacrifice and pathology: Each animal was subjected to a full necropsy. During the study period, any animal judged to be moribund by the veterinarian or his designate and all animals at terminal sacrifice were killed with an overdose of CO2, exsanguinated and necropsied immediately. The physical condition of the animal prior to euthanasia and the examination of the internal organs were described in detail on individual autopsy protocol sheets. Qualified prosectors under the supervision of a veterinarian / pathologist were available seven days a week, so that the necropsies were performed on a timely basis. In all cases, dead animals were identified by reference to each animal’s identification number.

The following organs and tissues were collected from each rat and fixed in 10% neutral buffered formalin (lungs were inflated at approximately 20 cm water pressure with formalin): Brain, pituitary, tongue, eyes (optic nerve), extra orbital lacrimal glands, Harderian glands, / nasal and paranasal cavities, larynx, pharynx, trachea, thyroid, parathyroids, lungs, thymus, heart, aorta, lung associated lymph nodes, salivary glands, mandibular lymph nodes, liver, pancreas, spleen, kidneys, adrenals, oesophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum, colon, rectum, mesenterium and lymph nodes, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina/cervix skeletal muscle, femur including joint, spinal cord, skin, peripheral nerve (N. ischiadicus), sternum and bone marrow. In addition to the terminal body weight, the lung, liver, kidneys, adrenals, testes were weighed (paired organs) separately). Relative organ weight data were also computed.

Histopathology

Tissues for histological examination were embedded in parafin, sectioned at 4 μm and stained with haematoxylin and eosin.

Complete histopathological examination (according to Bahnemann et al., 1995) were performed in all animals of the control and high exposure groups.

The slides were examined by light microscopy and the observations were recorded with an on-line computer programme.

Statistics: Statistical tests on the comparison of treatment groups were performed at the level of α = 0.05. Bodyweight, food and water consumption, and haematology data were analysed using analysis of variance as a global test. Pair-wise comparison of the means of treatment groups with the means of the clean air control group were performed using Dunnett’s modification of the t-test. Thus the experiment-wise error rate was controlled in this multiple testing procedure. For comparisons between two treatment groups, the two-sided t-test at a level of α = 0.05 was used. If applicable other statistical tests were used. Statistical evaluation for histopathological findings: significance of differences of the frequencies were evaluated as pair-wise comparison between clean air control and treatment groups using Fisher’s exact test. These tests were performed at the local significance level of α = 0.05."

Inflammatory Cell Infiltration, Submucosal

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