H.-B. Richter-Reichhelm

A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters.

SummaryUsing repeated cloning and treatment with cis-HPL (200 μg/ml), an analogue of a procollagen precursor inhibitory to the growth of collagen-synthesizing cells of mesenchymal origin, clonally premature epithelial cell lines were isolated from fetal SGH lungs cultured on the 15th day of gestation. One of the cell lines, M3E3/C3, which has been extensively studied for biological characterization, developed poorly differentiated carcinomas in injected hamsters after transformation by MNNG. Moreover, when grown on collagen gel, this cell line indicated an obvious potency for in vitro differentiation in response to vitamin A by developing activated Golgi regions, well developed rER and a number of mucus-like granules. Since such a differentiative responses is expected to be definable in the light of respiratory epithelium developing in utero, this cell line may be useful for studying mechanisms of differentiation-dependent sensitivity of fetal organs to transplacental carcinogen exposure.

Workshop report. Children as a special subpopulation: focus on immunotoxicity. Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), 15-16 November 2001, Berlin, Germany

Abstract. An international symposium on the impact of environmental hazards, chemicals and drugs on the developing immune system of children was held in Berlin (Germany) organized by the BgVV. Epidemiological evidence indicates that an immature immune system challenged early in life by bacterial antigens may prevent, to some extent, allergic reactions including asthma bronchiale triggered by environmental pollutants. However, the prevalence for infectious disease is increased in childhood, especially when exposure to contaminants takes place in the period of pregnancy and breast-feeding. The effects of chlorinated biphenyls, dioxin, endotoxins, hexachlorobenzene, and direct and indirect in utero tobacco smoke exposure are examples. All participants recommend comparative and follow-up epidemiological studies and clinical examination of infants and children at risk during upbringing. There is ample evidence from experimental studies that indicates adverse effects on the developing immune system after in utero and postnatal exposure to chemicals and drugs. The adverse reactions of aciclovir, benzodiazepines, hexachlorobenzene, organotins (di-n-octyltin dichloride, tributyltin oxide), pesticides (methoxychlor, heptachlor) and polyhalogenated aromatic hydrocarbons (2,3,7,8-tetrachlorodibenzo-p-dioxin) are presented and reviewed. To determine the predictive value of test data in risk assessment for neonates and children, development, differentiation and maturation of the immune system in humans and laboratory rodents is compared in their pre- and postnatal stages. Considering some differences in immunocompetence at birth and after lactation, and differences in the time frame for maturation of the immune system, reaction types are thought to be common, comparable and similar in human childhood and early adolescence and the postnatal lifetime of laboratory rodents. The participants of the symposium felt strongly that regulatory steps urgently need to be initiated to incorporate some relevant aspects into existing test guidelines for testing developmental immunotoxicity. In this context, it is recommended that animals culled otherwise in one- and two-generation studies be examined for developmental immunotoxicity according to the valid methods and parameters discussed. The majority of participants agreed that a safety factor of 10 is too low in risk assessment and management to protect a sensitive subpopulation of children against man-made environmental pollutants.

Results of a cyclosporin A ringstudy

The aim of the study was to find out whether an extended subacute toxicity study (additional organ weights, histopathology and immune functional tests), routinely employed in testing of chemicals, would shown indications of (adverse) effects on the immune system below general toxicity. Therefore, a five laboratory ring study on the basis of an oral 28-day repeated dose study in rats (OECD guideline 407) was carried out with 1, 5 and 25 mg/kg of cyclosporin A (CsA) per day by gavage. Besides some toxic effects such as reduced body weight gain and increased kidney calcification in the high-dose group, the results of the additional pathologic examinations revealed that CsA caused a pattern of specific morphological alterations of the lymphoid tissues in mid- and high-dose groups. Selected immune parameters such as immunoglobulin determination, plaque-forming assay, flow cytometry, activation status of macrophages and natural killer cells (NK), and proliferative response of spleenocytes and cells from mesenteric lymph nodes (concanvalin A (ConA) and pokeweed mitogen (PWM) stimulation) were also investigated. Some results compared to the controls revealed alterations down to the low-dose group. The extended methodology consistently indicated the potential detection of effects on the immune system below general systemic toxicity. The study will be continued by investigating a second compound with primarily immunostimulating effects. Results from those studies should further contribute to the current discussion of up-dating of repeated dose toxicity guidelines with respect to immunotoxicity.

Sensitivity of Syrian golden hamster fetal lung cells to benzo[a]pyrene and other polycyclic hydrocarbons in vitro.

Dose responses were compared of cultured fetal Syrian golden hamster lung cells (FSHL) to the toxic and transforming effects of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[B]F), benz[a]anthracene (B[a]A, indeno[1,2,3-c,d]pyrene (I[c,d]P), benzo[k]fluoranthene (B[k]F) and benzo[e]pyrene (B[e]P). Effort was first given to standardising the techniques for evaluating B[a]P dose-responses. These polycyclic aromatic hydrocarbons (PAH) were then tested at concentrations of up to 1 microgram/ml, and only B[a]P showed clear cytotoxicity. The transforming effects of B[b]F, B[a]A and I[c,d]P at 1 microgram/ml appeared comparable to those of B[a]P at 0.05 microgram/ml.

Toxic and transforming effects of polycyclic hydrocarbons in fetal hamster lung-cell cultures. I. benzo(a)pyrene

When seeded at a low cell density in Petri dishes not pre-conditioned with feeder layers, fetal hamster lung cells at 2-4 passages grew with greatly varied plating efficiencies (PE). Addition of insulin to the serum-supplemented medium gave relatively constant PE. However, the presence of insulin in the medium enhanced the toxicity of benzo(alpha)pyrene (BP) on these cells. Cytotoxic and cell transforming effects of BP in the presence of insulin were studied.

Epithelial alterations in fetal tracheal explants of syrian golden hamsters exposed to diethylinitrosamine in utero

The fetal tracheae (13th--15th day of gestation) of Syrian golden hamsters exposed in utero to diethylnitrosamine (DEN: 200--400mg/kg body wt.) were divided into cranial and caudal halves. Both sections were then organ-cultured for 4--6 weeks. The cranial explants developed 1.5 times as many epithelial tracheal lining alterations as the caudal explants. These changes were found 25--42 days after the beginning of explant culture and histologically demonstrated hyperplasia, metaplasia and dysplasia. No alterations occurred in the control explants.

Letter to the Editor: A regulatory view on the discussion on the role of alternative methods in the risk assessment of chemicals in the context of REACH

In a paper published recently in Archives of Toxicology, Lilienblum et al. present their view on available and ready to use alternative methods for the risk assessment of chemicals (Lilienblum et al. 2008). As they refer speciWcally to the new European legislation (REACH) which came into force mid 2007 we would like to comment some of the aspects of the paper from the perspective of regulators. First of all there is general agreement with the evaluation of the current status of alternative methods of the authors (Lilienblum et al. 2008). Considering complex endpoints, we consent that emphasis should be given to reWne tests with the aim to reduce the number of animals rather than to try to replace the tests by developing in vitro alternatives to the current testing methods. However, we would more clearly express the potential for the vitro methods for some of the endpoints which are discussed in the publication. For example, the in vitro dermal resorption test and the in vitro test on phototoxicity are both validated test methods and accepted at the regulatory level. Hence, in our view no in vivo tests are necessary to cover these endpoints. In addition, we see a high value in the available prediction tool for skin irritation and corrosion (ECB 2008) which is extremely helpful in a tiered approach. For classiWcation and labelling negative results can be used as they have clearly been identiWed in the validation study (Hulzebos et al. 2005). Hence, even under the GHS rules where two levels for positive results are required (which the prediction tool is not able to distinguish) the tool is nevertheless helpful considering that negative results for skin irritation and corrosion account for more than 70% of the cases in the EU New Chemicals Database. It can be assumed that using this alternative method would reduce the need for in vivo testing under REACH in 70% of 15,500 chemicals with a production volume below 10 tons per year. We also would put more emphasis in the potential the currently available in vitro test strategy can oVer for genotoxicity and mutagenicity testing. We agree with the assessment and the critique presented in the Lilienblum paper (Lilienblum et al. 2008). However, we would like to draw the attention to the fact that the currently used regulatory tiered testing approach reduces the necessity to perform in vivo testing to a small number of cases. Based on the numbers in the New Chemicals Data Base, the current approach to start with in vitro testing and to proceed with in vivo testing only in the positive in vitro cases was required in only around 25% of the cases which reduces the number of required in vivo tests under REACH to a quite remarkable low number of cases. Furthermore, we would like to give some considerations to the topic of toxicokinetic and metabolism which is extensively discussed in the Lilienblum paper. We agree with the assessment that toxicokinetic and metabolism is of importance and is a crucial issue and that metabolism often plays a key role in intraand inter-species diVerences (to document the regulatory need we refer to work of Bernauer et al. 2000, 2002, 2003, 2006; Abraham et al. 2005; Mielke et al. 2005; Bernauer and Gundert-Remy 2008). We agree with other scientists that the interpretation of testing results need additional data for extrapolation of the results to humans (Greim 2007) and toxicokinetic information is regarded as very helpful for the development of integrated testing strategies (ITS) which are propagated in the REACH guidance documents according to REACH legislation. In addition, toxicokinetic data can be used to support waiving arguments (Annex VIII, 8.6.1 90 days study; Annex IX, 8.7 studies on reprotoxiccity; Annex X, 8.4 germ cell mutageU. Gundert-Remy (&) · U. Bernauer · S. Madle · A. Oberemm · A. Schulte · H.-B. Richter-Reichhelm Berlin, Germany e-mail: ursula.gundert-remy@bfr.bund.de

Tubular explant culture of fetal Syrian golden hamster tracheae.

Two types of organ culture technique (stationary and rocking) were compared for their ability to maintain the epithelial structures in the tubular explants of fetal Syrian golden hamster tracheae. A simple new technique has been devised for the organ culture of small tubular explants of fetal tracheae. A large pore-sized membrane filter is introduced into the rocking culture, which improves unfavourable in vitro effects of the stationary technique.

Chromosomal aberrations in tracheal epithelial cells of the fetal Syrian golden hamster after transplacental N-diethylnitrosamine administration

Transplacental effect of N-diethylnitrosamine (DEN) on chromosomal morphology, was investigated in fetal tracheal epithelium of Syrian hamsters. At the 15th day of pregnancy, Syrian golden hamsters were injected subcutaneously with a tumorigenic dose of DEN (50, 100 and 200 mg/kg body wt). Two hours later, the fetal tracheae were isolated, epithelial cells of the respiratory mucosa were separated from mesenchymal tissue and were transferred to cell culture. At 24, 48 and 72 h after cultivation, chromosomal damages were examined. The results showed clearly that a high incidence of chromosomal aberrations, particularly chromatid-type exchanges, were seen in the epithelial cells from DEN-treated groups.

Effects of serum concentrations on the clonal growth of Syrian golden hamster fetal lung cells

In order to gain an insight into the mechanisms of clonal growth of early-passage Syrian golden hamster fetal lung (SGHFL) cells, a study was undertaken, in which the effects of various serum concentrations on the cloning efficiency and on the colony size of these cells were examined. The results suggest that both cloning efficiency and colony size are influenced by at least two factors present in the serum.