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148 B. Rihn et aUToxicologv 109 (1996) 147-156

undcrstood (Walker et al., 1992; Brody, 1993) while studies involving transgenic mice in toxi-cology are increasing.

Therefore, we undertook the construction of an inhalation chamber permitting inhalation studies on mice using a nose-only exposure. This chamber was validated with an adequate aerosol of crocidolite fibers to intoxicate mice during a short time. The uniformity of the aerosol was assessed by air sampling in the inhalation chamber. To validate further the fiber toxicity three toxicological endpoints were checked: cytological responses in bronchoalveolar lavages, lung his-tology and fiber burden in the lung.

We thereby demonstrate that short-term inhalation intoxications produce significant inflam-matory responses in the lungs of exposed mice and question the necessity of longer term expo-sure (Reeves et al., 1974).

2. Materials and methods

2.1.    Mice

Twenty seven week-old Balb/c małe mice (IfTa Credo, Arbrelesle, F-69210) were randomized and numbered by tattoo. Animals were housed in polycarbonate cages (1/cage) covered with spun-bonded polyester cage filters. Room temperaturę was 21 ± 1°C, the pressure was 5 mmH₂0 above the atmospheric pressure; humidity ranged from 40-60% and fluorescent light was on for 12 h/day. Animals were fed with pellet food and water ad libitum. Fifteen animals were exposed to the crocidolite aerosol whereas 5 control animals were housed in nose-only exposure tubes without crocidolite during the time of the experi-ment i.e. 6 h/day during 5 days. Four weeks after the end of the intoxication the experiment was terminated.

2.2.    Exposure techniąues

2.2.1. Crocidolite fibers and generator

The crocidolite used in this study came from a batch replacing the original UICC (Union Internationale Contrę le Cancer) sample when it ran out (gift of Dr. Rendall, National Center for

Occupational Health, Johannesburg, South Afri-ca). The fiber aerosol generator was purchased from CR Equipments (Tannay, Switzerland). It was similar in its design to the generator de-scribed by Bernstein et al. (1995), namely the fibers were packed in a cylinder and a Teflon-coated piston pushed them on a Steel brush. Clean air was provided by a six bar compressor delivering an air stream with a flow ratę of 100 1/min by an “inverted cyclone” device (Schreck et al., 1980). The tangential position of the air inlet pipę gave the air flow a helicoidal movement from the top to the bottom of the celi in order to ensure the aerosol homogeneity. The aerosol con-centration was monitóred on linę by photometry using a Sigrist KTN B2/F3™ model (Ennetbiir-gen, Switzerland).

2.2.2. Chamber operations

Fig. 1 is a sketch of the nose only chamber including the location inside the chamber of the various sensors: the humidity and temperaturę sensors were located on the upper part of the chamber and the pressure sensor was in the middle of the chamber.

Briefly, the process control system included measurements of (i) the air flow taken by differ-ential pressure sensors (Scheavitz™, PM Instru-mentation, Orgeval, France) set up on Venturi tubes, (ii) the depression using Scheavitz™ difler-ential pressure sensors, (iii) the temperaturę and humidity using a CH12 sensor (Lee Integer Ptd™, Agemip, Le Perreux). Ali output signals were transferred into a microcomputer (Umac 5000/Umac 4030™, Analog Devices, Norwood, MA, USA) which processed sensor information, calculated regulation loops and dispatched ana-logical tensions to the regulation valves. A sec-ond Computer RS232 connected to the first was used to monitor (i) the inhalation chamber con-ditions (air flow and relative humidity), (ii) the aerosol concentration and the adjustments of the aerosol generator parameters. The temperaturę of the chamber atmosphere was maintained be-tween 20 and 22°C and the humidity between 45-55%, the volume exchange (100 1) was 60 times/h.

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