Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Avian cell – per se
Reexamination Certificate
1999-03-11
2001-01-09
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Avian cell, per se
C435S350000, C435S351000, C435S352000, C435S325000, C435S004000, C424S581000, C424S582000
Reexamination Certificate
active
06171858
ABSTRACT:
The invention relates to a process for determining the toxicity of electromagnetic radiation, the phototoxicity and/or photosensitivity of substances or substance mixtures as well as use of said process.
Phototoxicity as defined in the present invention refers to an impairment of the local tolerance brought about by electromagnetic radiation within the range from 1 mm to 200 nm as the result of exposure to infrared light, visible light or ultraviolet radiation.
In particular, a phototoxic reaction refers to a reaction in which radiation energy and/or sensitizing substances penetrate the skin in an exogenic or endogenic manner and then cause photodermatosis. In this process, such phototoxic substances not only absorb UV radiation, but they can also be chemically altered by it. They are capable of absorbing in various radiation ranges, as a function of their chemical structure. It is assumed that there is a corresponding correlation between the quantity of radiation energy and the substance struck by the radiation. A small amount of energy and a large amount of substance have the same effect as a small amount of substance and a large amount of radiation energy. At a maximum congruence of the two reaction parameters, the phototoxic reaction will be particularly intense. As a rule, a phototoxic reaction appears approximately 8 to 12 hours after the initial exposure. Now and then, this can also take place earlier. Common symptoms are a very pronounced reddening associated with blistering and subsequent post-inflammatory pigmentation. Even a sunburn can be seen as a phototoxic reaction.
In contrast to a phototoxic reaction, a photoallergic reaction is defined as a reaction which occurs even in response to smaller quantities of radiation and just a small amount of activatable substance. Moreover, this is not dose dependent. The mechanism here is the same as that of an allergy, whereby the foreign substance altered by the radiation can turn into the hapten, which then combines with the protein to form an allergen. A photo-allergic reaction is usually manifested in the form of eczema. It appears between 48 and 72 hours after the initial exposure to the sun. As we know from the developmental mechanism of allergies, they never occur after the first exposure, since this is the instance that leads to the formation of antibodies. Photoallergic reactions are not restricted to the areas of skin exposed to the radiation. As a spreading phenomenon, they also occur on other parts of the body which have not been irradiated. As compared to the absorption spectrum, the action spectrum is shifted more towards the long-wave range. Particularly problematic photoallergic reactions are reactions to chemically related substances such as, for example, para substances. The amino-, hydroxy- and nitro-group, substituted in the para position on the phenyl ring, is the typical technical-structural feature.
It is a known practice to employ chicken embryos in order to study the toxicological properties of test substances. For this purpose, the substance specimens are normally applied to the embryo via the extraembryonal blood vessel system.
Two recent scientific publications from the literature can be mentioned as representative of the common use of chicken embryos as a model system for pharmacological (Roberts, W. G and Hasan, T. in CANCER RESEARCH, volume 52, pages 924 through 930, 1992) or toxicological test series (Goldberg, S. J.; Dawson, B. V.; Johnson, P. D.; Hoyme, H. E. and Ulreich, J. B., PEDIATRIC RESEARCH, volume 32, pages 23 through 26, 1992).
The publication by Roberts and Hasan describes the pharmacological/pharmacotopological properties of photodynamic agents in proliferating vascular and non-vascular tissues in chicken embryos. The chicken embryogenesis serves as a model system for the angiogenesis in solid tumors.
The publication by Goldberg et al., in contrast, describes the use of chicken embryos as a method to examine the teratogenicity of substances, particularly dichloroethylene. The above-mentioned publications, however, neither suggest nor anticipate the approach of studying phototoxic and/or photosensitive substances in chicken embryos.
Up until now, only an animal model or cell systems have been available for studying potentially phototoxic or photosensitive substances.
The animal model is the “Draize test” which detects the irritation caused by photo-toxic substances in the eye of rabbits. Its use is limited, not only due to cost considerations but also particularly from an animal-rights standpoint.
The cell systems available, that is to say, bacteria, yeast and other eucaryotic cells, are likewise inadequate as models for phototoxicity since the physiological marginal conditions of the phototoxic or photosensitive substance behavior cannot be adequately duplicated.
The present invention has the objective of creating a technically simple method for testing potentially phototoxic substances. On the one hand, this test should take the physiological conditions on the skin into account and, on the other hand, also meet future legislative plans and ethical considerations. This objective is achieved by the features of claim
1
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Thus, the present invention relates to a process for determining the phototoxicity and/or photosensitivity of substances and/or substance mixtures, involving putting the chemical substance or substance mixture into contact with non-human vertebrate embryos or tissues or tissue components of a vertebrate embryo, except for human skin cell cultures, whereby, following the contact, there is an additional treatment with radiation ranging from 1 mm to 200 nm, followed by evaluation of the pathology of the embryo, tissue or cell.
According to a preferred embodiment of the present process, prior to contact, eggs of the Aves class, that is to say, birds, are incubated as the form containing vertebrate embryos and, at a later point in time after the embryonal gastrulation, some of the egg white is removed from the egg via at least one opening and, if applicable, further incubation takes place and another opening is provided in the upper section of the egg shell for purposes of the radiation.
Even though it is fundamentally possible to employ any non-human vertebrate embryo or a tissue or a tissue component of a vertebrate embryo, except for human skin cell cultures, practical considerations have proven it to be advantageous to give preference to the use of incubated eggs of the Aves class, in other words, birds. Examples of these are the eggs of ostriches, rheas, emus, kiwis, tataupas, gallinaceous birds, hoatzins, quail, pigeons, sand grouse, rails, jacanas, cranes, bustards, gulls, wading birds, auks, divers, grebes, penguins, petrels, duck-like anseres, steganopods, glossy ibises, flamingos, diurnal raptors, cuckoos, turacos, parrots, doves, kingfishers, bee-eaters, hoopoes, hornbills, owls, nightjars, swifts, hummingbirds, mousebirds, trogons, toucans, barbets, honey guides, woodpeckers and passerine birds. As incubated eggs, preference is given to eggs of the Galliformes order, that is to say, gallinaceous birds, whereby special preference is given to eggs of chickens, or else turkeys.
The period of incubation normally depends on the development time of the embryo in question.
According to a preferred embodiment of the present invention, the egg is provided with at least one opening after the first incubation of the eggs. This opening should preferably be at least of such a size that it allows part of the egg white to be removed through the opening. This is preferably done in such a way that part of the egg white, preferably 5 to 10 ml of the egg white present, is removed by means of a suction device, preferably a pipette such as, for instance, an Eppendorf pipette.
Following this aspiration procedure, another opening is made in the upper section of the eggshell so that later the radiation procedure can be carried out. This is normally done in such a way that a mechanical device is used to create another, this time larger, opening in the eggshell which can be accomplis
Hölzle Erhard
Lehmann Percy
Neumann Norbert J.
Plewig Gerd
Rosenbruch Martin
Coe Susan D.
Connolly Bove & Lodge & Hutz LLP
Lankford , Jr. Leon B.
Neumann Norbert J.
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