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Panel discussion—Afternoon session
FdChem. Toxic. Vol. 32, No. 2, pp. 155-157, 1994
Elsevier Science Ltd. Printed in Great Britain 02711-6915/94 56.00 + 0.00
Pergamon
PANEL D I S C U S S I O N J A F T E R N O O N SESSION
Dr Norbert Page: We are pleased that Dr Shelanski has agreed to moderate this afternoon's panel discussion. Dr Shelanski, do you have any opening comments?
than we had five years ago and I presume we will have better information five years from now than we have now. We believe that the Langerhans cell is a necessary instrument in the process of skin sensitization. In its other form as the Kupffer cells or as the pulmonary villus cell it has played a role in development of sensitization in other organs but since we are concerned with skin we have to emphasize the Langerhans cell. [ assume that we have come to some decision that the Langerhans cell is not neurogenie, it is a milder cell by their omission.
RESPONSE: Dr Shelsnski
I would like to see in the future a bank of lymphocytes derived from a thousand or more human donors that are available for the evaluation of sensitizers in an/n vitro system. I realize that much of the technology that would make this possible is still in the future, for example how to keep a lymphocyte culture going, how to make it responsive to the various sensitizers. Would you need thymosin, would you need some of the adrenal hormones, what do you need? But all of these things could be put into one bank so that an in vitro method would be possible that would give human cells from different donors and allow us to evaluate materials with sensitizing propensities on a much more scientific basis. If anybody has that capability I wish he would speak up because it would save the clinician a lot of problems in traumatizing humans unnecessarily in order to get answers to make sure products are safe. it would circumvent one important consideration that has troubled me ever since 1 began clinical work on humans. What would be the long-range effect of the short-term exposure to which we subject volunteers? What would be the effect 20-30 years from now in so far as carcinogenesis? Was that exposure that we subjected these people to, the trigger? Did it have any part in the development of that state? I can speak briefly from my own experience because I do have subjects that I have followed for at least 30 years and I can say in only one case have I found a localized lymphoma that I could relate back to an exposure to an epicutaneously applied material, otherwise I have no information to offer you in that respect. I will now open discussion by the panel. RESPONSE: Dr Rmlemary Osborne
I would like to make one additional note in expanding on what you were saying. Perhaps in terms of skin sensitization we might want a panel of Langerhans cells available for testing. RESPONSE: Dr Shelsnskl
I would assume they would be in the culture, because the cascade in the development of the sensitized state is one that we have better knowledge now
QUESTION: Someone from FDA
Are there in EPA specific requirements for any kind of skin or methodology being used? RESPONSE: Mr Seabaugh
Dr Osterburg compared intact and abraded skin and found there was no difference in responses for the Consumer Product Safety Act. Dr Nixon at Proctor & Gamble has done similar work and on that basis Federal Regulatory Agencies see dermal irritation testing only with intact skin. QUESTION: Someone from FDA
You do not feel there is a difference between the two, and there shouldn't be a requirement for using both kinds? RESPONSES: Mr Seabsugh
For years there was, and the literature had shown there is a difference but for regulatory purposes of pass or fail, as a primary skin irritant, it does not make any difference. RESPONSE:
Dr Osborne
Basically what Nixon and Cohen found when they published a paper in 1975, I believe it was in Journal of Toxicology and Applied Pharmacology. They compared abraded and intact skin in their response to household product chemicals, what they found was that abrasion, or scarifying the skin in a 'tic-tac-toe' pattern, produced increased variability between test animals in the same group. A lot of irradiation localized around the scratch, but that needed to be averaged out over the entire patch area, so its really difficult, and it really does not add anything to the interpretation of the irradiation potential.
155
156
Panel Discussion--Afternoon Session
RESPONSE: Dr S/mlamkl
RESPONSE: Dr I~roumgk
The fact that you get localized inflammation around the scarification stria rather than interstitial skin between the striae that would indicate that so far as irritation is concerned a scarified skin by virtue of the fact that the barrier is no longer in place allows the irritant to get into the skin much deeper to produce an inflammatory response. It does not have to penetrate the stratum corneum. The fact that the inflammation is localized on the striae and not spread throughout is a way of quantifying the irritant effect by virtue of determining how much the inflammatory response spread from the striae to the non-comprised skin. If you want to test the most stringent conditions I would have no reason to object to using both scarified and intact skin.
Along these same lines it may depend upon the method they are using as to whether or not abrading the skin will have an effect. For example in the Buehler test, when you are doing a study under occlusion you're already enhancing absorption if you have an ethanol or acetone vehicle in contact with the skin for six hours. You are already damaging the barrier. It may be that abrading the skin may not have any further effect so you may see no effect of abrasion in certain test protocols but in others you might see an effect of abrasion because of the vehicle effect and the effect of occlusion.
RESPONSE: Mr S¢-baugh
One paper presented at the Society of Toxicology meeting several years ago by Jerry Sullivan that I do not think appeared in the literature concluded for regulatory purposes that abraded skin for dermal irritation testing was not justified. QUESTION: Dr Shelanaki
Did he do the abrasion and intact on the same animal? RESPONSE:
Yes, but I would have to go back and read it.
RESPONSE: Dr S k c l l l k i
Scarification is made with a small sharp needle, so as to produced a division in the squamous and in the stratum corneum without eliciting any frank bleeding and if you have a skilled technician, you can get a uniform site. So far as occlusion, when we do scarification we use stainless-steel chambers as an occlusive device, and we see differences in material and their effect on the striae and on the intact skin in between. It is not just a question of permeation or penetration--it is a question of how quickly the inflammatory response occurs on the skin of the striae as compared to how quickly it will occur on the intact skin. It is a matter of timing rather than a matter of penetration, If you provide in your test protocol for evaluating these things at times when you can differentiate between the effects, then you can see differences.
RESPONSE: Dr Shelauki
Once you sensitize an animal it is going to show up on the intact skin. The question is whether the scarified site is the primary site and whether or not the intact site is secondarily sensitized. I take issue with Dr Osborne. Inflammation is a vascular response: redness, swelling, heat, pain. These classical signs of inflammation are all vascular responses. You can get cytotoxicity, cell necrosis, the release of every kind of intermediary you want, but if you want, but if you do not have a vascular bed to react to the release of those intermediary materials, you do not get inflammation. RESPONSE: Dr Osborne
I understand your point. I was trying to say we can see inflammation in vivo, but what are the important mechanistic endpoints that we can look at in both cell cultures and in vivo? Getting back to scarification versus intact skin I think it would be valuable for you to look at the Nixon paper. It is not just a matter of increased penetration going into the skin, but truly a mixed bag when you have scarification because it is difficult to get that done uniformly, so its various levels of erythema across the entire scratch and it is difficult to get good quantitative data out of that.
RESPONSE: Dr Osbocn¢
We need to understand more about percutaneous absorption and irritation. That is a field that Dr Maibach mentioned this morning, it is a field in which not much has been done including biochemical steps, and whatever I described this morning. In terms of penetration enhancers, I think the point is fairly well taken that there should be some concern. QUESTION:
I would like to ask Dr Shelanski if he is aware of work in which UVB has been used to stimulate inflammation without breaking the stratum corneum. Would that have the same effect of enhancing the rate at which something develops without getting the drying effects from oxygen toxicity and the immediate releases that you get with scarification. RESPONSE: Dr Shclamkl
You are asking if predisposing the skin by using ultraviolet radiation also speeds up the response. I have no data. 1 know that we predispose the skin for certain types of evaluations and after exposing skin to UV radiation or thermal radiation we make it more
Panel Discussion--Afternoon Session susceptible for the evaluation of topical anaesthetics. Since it may be part of the inflammatory response and because the neurological endpoints for pain in the skin are, to some extent, influenced by the cell breakdown products, if you predispose the skin by either UV radiation or thermal radiation you will find that the threshold for pain sensation is much shortened. Thus that applying an analgesic, which is
157
one of the methods used to test topical analgesia, we can determine whether or not we can prolong the threshold before pain is perceived or whether there is any change at all. So I would say that there is from that sort of devious clinical experience that I have, information to support the fact that the UV radiation of skin will enhance the inflammatory response.
Elsevier Science Ltd. Printed in Great Britain 02711-6915/94 56.00 + 0.00
Pergamon
PANEL D I S C U S S I O N J A F T E R N O O N SESSION
Dr Norbert Page: We are pleased that Dr Shelanski has agreed to moderate this afternoon's panel discussion. Dr Shelanski, do you have any opening comments?
than we had five years ago and I presume we will have better information five years from now than we have now. We believe that the Langerhans cell is a necessary instrument in the process of skin sensitization. In its other form as the Kupffer cells or as the pulmonary villus cell it has played a role in development of sensitization in other organs but since we are concerned with skin we have to emphasize the Langerhans cell. [ assume that we have come to some decision that the Langerhans cell is not neurogenie, it is a milder cell by their omission.
RESPONSE: Dr Shelsnski
I would like to see in the future a bank of lymphocytes derived from a thousand or more human donors that are available for the evaluation of sensitizers in an/n vitro system. I realize that much of the technology that would make this possible is still in the future, for example how to keep a lymphocyte culture going, how to make it responsive to the various sensitizers. Would you need thymosin, would you need some of the adrenal hormones, what do you need? But all of these things could be put into one bank so that an in vitro method would be possible that would give human cells from different donors and allow us to evaluate materials with sensitizing propensities on a much more scientific basis. If anybody has that capability I wish he would speak up because it would save the clinician a lot of problems in traumatizing humans unnecessarily in order to get answers to make sure products are safe. it would circumvent one important consideration that has troubled me ever since 1 began clinical work on humans. What would be the long-range effect of the short-term exposure to which we subject volunteers? What would be the effect 20-30 years from now in so far as carcinogenesis? Was that exposure that we subjected these people to, the trigger? Did it have any part in the development of that state? I can speak briefly from my own experience because I do have subjects that I have followed for at least 30 years and I can say in only one case have I found a localized lymphoma that I could relate back to an exposure to an epicutaneously applied material, otherwise I have no information to offer you in that respect. I will now open discussion by the panel. RESPONSE: Dr Rmlemary Osborne
I would like to make one additional note in expanding on what you were saying. Perhaps in terms of skin sensitization we might want a panel of Langerhans cells available for testing. RESPONSE: Dr Shelsnskl
I would assume they would be in the culture, because the cascade in the development of the sensitized state is one that we have better knowledge now
QUESTION: Someone from FDA
Are there in EPA specific requirements for any kind of skin or methodology being used? RESPONSE: Mr Seabaugh
Dr Osterburg compared intact and abraded skin and found there was no difference in responses for the Consumer Product Safety Act. Dr Nixon at Proctor & Gamble has done similar work and on that basis Federal Regulatory Agencies see dermal irritation testing only with intact skin. QUESTION: Someone from FDA
You do not feel there is a difference between the two, and there shouldn't be a requirement for using both kinds? RESPONSES: Mr Seabsugh
For years there was, and the literature had shown there is a difference but for regulatory purposes of pass or fail, as a primary skin irritant, it does not make any difference. RESPONSE:
Dr Osborne
Basically what Nixon and Cohen found when they published a paper in 1975, I believe it was in Journal of Toxicology and Applied Pharmacology. They compared abraded and intact skin in their response to household product chemicals, what they found was that abrasion, or scarifying the skin in a 'tic-tac-toe' pattern, produced increased variability between test animals in the same group. A lot of irradiation localized around the scratch, but that needed to be averaged out over the entire patch area, so its really difficult, and it really does not add anything to the interpretation of the irradiation potential.
155
156
Panel Discussion--Afternoon Session
RESPONSE: Dr S/mlamkl
RESPONSE: Dr I~roumgk
The fact that you get localized inflammation around the scarification stria rather than interstitial skin between the striae that would indicate that so far as irritation is concerned a scarified skin by virtue of the fact that the barrier is no longer in place allows the irritant to get into the skin much deeper to produce an inflammatory response. It does not have to penetrate the stratum corneum. The fact that the inflammation is localized on the striae and not spread throughout is a way of quantifying the irritant effect by virtue of determining how much the inflammatory response spread from the striae to the non-comprised skin. If you want to test the most stringent conditions I would have no reason to object to using both scarified and intact skin.
Along these same lines it may depend upon the method they are using as to whether or not abrading the skin will have an effect. For example in the Buehler test, when you are doing a study under occlusion you're already enhancing absorption if you have an ethanol or acetone vehicle in contact with the skin for six hours. You are already damaging the barrier. It may be that abrading the skin may not have any further effect so you may see no effect of abrasion in certain test protocols but in others you might see an effect of abrasion because of the vehicle effect and the effect of occlusion.
RESPONSE: Mr S¢-baugh
One paper presented at the Society of Toxicology meeting several years ago by Jerry Sullivan that I do not think appeared in the literature concluded for regulatory purposes that abraded skin for dermal irritation testing was not justified. QUESTION: Dr Shelanaki
Did he do the abrasion and intact on the same animal? RESPONSE:
Yes, but I would have to go back and read it.
RESPONSE: Dr S k c l l l k i
Scarification is made with a small sharp needle, so as to produced a division in the squamous and in the stratum corneum without eliciting any frank bleeding and if you have a skilled technician, you can get a uniform site. So far as occlusion, when we do scarification we use stainless-steel chambers as an occlusive device, and we see differences in material and their effect on the striae and on the intact skin in between. It is not just a question of permeation or penetration--it is a question of how quickly the inflammatory response occurs on the skin of the striae as compared to how quickly it will occur on the intact skin. It is a matter of timing rather than a matter of penetration, If you provide in your test protocol for evaluating these things at times when you can differentiate between the effects, then you can see differences.
RESPONSE: Dr Shelauki
Once you sensitize an animal it is going to show up on the intact skin. The question is whether the scarified site is the primary site and whether or not the intact site is secondarily sensitized. I take issue with Dr Osborne. Inflammation is a vascular response: redness, swelling, heat, pain. These classical signs of inflammation are all vascular responses. You can get cytotoxicity, cell necrosis, the release of every kind of intermediary you want, but if you want, but if you do not have a vascular bed to react to the release of those intermediary materials, you do not get inflammation. RESPONSE: Dr Osborne
I understand your point. I was trying to say we can see inflammation in vivo, but what are the important mechanistic endpoints that we can look at in both cell cultures and in vivo? Getting back to scarification versus intact skin I think it would be valuable for you to look at the Nixon paper. It is not just a matter of increased penetration going into the skin, but truly a mixed bag when you have scarification because it is difficult to get that done uniformly, so its various levels of erythema across the entire scratch and it is difficult to get good quantitative data out of that.
RESPONSE: Dr Osbocn¢
We need to understand more about percutaneous absorption and irritation. That is a field that Dr Maibach mentioned this morning, it is a field in which not much has been done including biochemical steps, and whatever I described this morning. In terms of penetration enhancers, I think the point is fairly well taken that there should be some concern. QUESTION:
I would like to ask Dr Shelanski if he is aware of work in which UVB has been used to stimulate inflammation without breaking the stratum corneum. Would that have the same effect of enhancing the rate at which something develops without getting the drying effects from oxygen toxicity and the immediate releases that you get with scarification. RESPONSE: Dr Shclamkl
You are asking if predisposing the skin by using ultraviolet radiation also speeds up the response. I have no data. 1 know that we predispose the skin for certain types of evaluations and after exposing skin to UV radiation or thermal radiation we make it more
Panel Discussion--Afternoon Session susceptible for the evaluation of topical anaesthetics. Since it may be part of the inflammatory response and because the neurological endpoints for pain in the skin are, to some extent, influenced by the cell breakdown products, if you predispose the skin by either UV radiation or thermal radiation you will find that the threshold for pain sensation is much shortened. Thus that applying an analgesic, which is
157
one of the methods used to test topical analgesia, we can determine whether or not we can prolong the threshold before pain is perceived or whether there is any change at all. So I would say that there is from that sort of devious clinical experience that I have, information to support the fact that the UV radiation of skin will enhance the inflammatory response.