Industrial experience with the detection of retroviruses.

An important aspect of the virological assessment of cell substrates used to produce biologicals is their retroviral status. In this presentation we will attempt to document some of the practical experiences of industry when testing cell substrates for retroviruses using infectivity tests, reverse transcriptase and electron microscopy. We will also explore some important aspects of the experimental design of these retrovirus assay systems and review some results from the application of these methods in model, experimental systems.

Assessment of environmental contamination associated with a mammalian cell transformation assay.

To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal an environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 microgram) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating particles and procedures provided adequate safeguards for personnel and the environment.

Design and testing of a biocontainment system for chemical carcinogens.

A biocontainment facility was built for studies in which the chemical carcinogen N-methyl-N-nitrosourea (MNU) was instilled intrarectally in guinea pigs. The system operated by constant flow of uncontaminated air into carcinogen-contaminated animal isolation chambers and filtration through a high-efficiency particulate air (HEPA) filter prior to release into the environment. The facility was tested for efficiency of carcinogen containment by substituting for the MNU a similar concentration of a fluorescent tracer, sodium fluorescein, under standard operating procedures for carcinogen administration to guinea pigs. Wipe samples from the floor, isolation chambers, animal handlers and clothing, and intake and exhaust air samples were analyzed for fluorescein before and after intrarectal instillation of the tracer. The recovery of very low concentrations of total and respirable suspended fluorescein from sampling points within the facility and the absence of detectable fluorescein in the air downstream from the HEPA filter indicated that the facility provided adequate protection against contamination of personnel or the environment.

Penetration of rat skin by N-nitrosodiethanolamine and N-nitrosomorpholine.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were painted on the clipped upper dorsal skin of male F344 rats. NDELA was applied undiluted, dissolved in water, and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. Aqueous solutions of the nitrosamines were used for gavage. Rats were housed individually. Blood and urine samples were analyzed for nitrosamines by chromatography combined with a Thermal Energy Analyzer. Maximum penetration of NMOR was approximately equal to 34% 2 hours after application of 5 mg to the skin or by gavage; less than 1% appeared in the urine in 24 hours. Skin painting with NDELA in water (20 mg/100 microliters) and in cutting oil (25 mg/25 microliters) yielded small concentrations of NDELA (always < 25 micrograms/ml blood). When 50 mg of undiluted NDELA was painted on the skin, 130 to 220 micrograms/ml of blood was recovered after 1 hour. Administering 50 mg NDELA in water by gavage yielded similar blood concentrations. Maximum skin penetration observed with NDELA was 78% 1 hour after application of 50 mg. From 20 to 30% of the NDELA applied undiluted and by gavage appeared in the urine in 24 hours. Although animals and humans differ, skin exposure to NMOR or NDELA represents a risk due to absorption.

The penetration of rat skin by some nitrosamines of environmental importance.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were applied to the clipped dorsal skin (about 3.5 X 3.5 cm) of adult male Fischer 344 rats. NDELA was applied undiluted, dissolved in water and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. To compare the extent of absorption through the skin with that from the stomach, aqueous solutions of the nitrosamines were used for gavage. Blood and urine samples were analysed for nitrosamines, using GC-TEA or HPLC-TEA. Maximum skin penetration observed with NMOR was 56%, following application of 5 mg, whereas a similar proportion of the dose was regularly present after gavage of 5 mg. Less than 1% was recovered from the urine. Skin painting of NDELA in water (20 mg in 100 microliter) and in cutting oil (25 mg in 25 microliter) yielded small concentrations of NDELA in the blood; less than 25 mg/l in all cases. However, when 50 mg NDELA was painted on the skin undiluted, from 130 to 220 mg/l of blood were recovered after one hour. fifty mg NDELA in water yielded similar blood concentrations when administered by gavage. From 20 to 30% of the NDELA applied undiluted and by gavage were recovered in the urine. It is concluded that, although there are differences between animals, exposure to NMOR or NDELA represents a risk due to absorption through the skin.

Potential hazard associated with scraping preparative thin layer chromatography plates.

Scraping TLC plates in a hood leads to contamination of the hood, equipment, worker, and the environment. The magnitude of contamination is presented as a fraction of the chemical delivered, and the effects of plate thickness, dryness, and adsorbent material are examined. Frequent clean-up and decontamination are recommended.

Sources and dissemination of contamination in material handling operations.

To assess the potential risks to personnel preparing feed for carcinogen bioassay research, a tracer was mixed with a meal diet to yield 10 kg batches of 100, 3,000 and 5,890 ppm. Wipe samples were obtained from horizontal and vertical surfaces, equipment and personnel before operations began, after they were completed, and following clean-up. Total and respirable suspended particulate matter samples were obtained. All operations led to contamination of clothing and equipment; cleaning did not remove all contamination. These results, obtained in a well controlled environment in which trained and well protected personnel were working, suggest that a higher level of process control may be required for adequate protection of personnel performing material handling operations with known or suspected carcinogens.