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The identification of corrosive agents for human skin In vitro
Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 513-515, 1986
0278-6915[86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
THE IDENTIFICATION OF CORROSIVE AGENTS FOR H U M A N SKIN IN VITRO G. J. A. OLIVER and M. A. PEMBERTON Central Toxicology Laboratory, Imperial Chemical Industries plc, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England Abstract--An in vitro technique, using ex vivo epidermal slices prepared from young rats, has been developed and validated as a predictive screen for the corrosive potential of chemicals to rabbit skin in vivo (Oliver & Pemberton, Fd Chem. Toxic. 1985, 23, 229). A modification of this technique uses full-thickness cadaver skin in vitro to predict corrosive potential to human skin in vivo. Electrical resistance thresholds to corrosive action have been estimated for young-rat and human skin and the effects of 59 chemicals corrosive or irritant to the skin have been investigated in vitro. Rat skin is more susceptible than human skin to corrosive chemical action in vitro. This finding may have significant implications with respect to the assignment of certain chemicals to the correct hazard category.
Introduction
Experimental
It is necessary to be able to assess the possible cutaneous hazard to man of industrial and other chemicals, in order to meet requirements for classification, labelling, packagingr transportation and an overall safety evaluation. Primary or direct effects of chemicals on the skin are categorized as being either irritant (causing local reversible inflammatory responses) or corrosive (causing visible destruction or irreversible alteration at the site of contact). Toxicity tests in animals are generally used to meet the objective of assessing cutaneous hazard to man. The Draize rabbit skin test is the most commonly recommended and utilized protocol (OECD, 1981). We have developed an in vitro test with a high precision for identifying corrosive potential (Oliver & Pemberton, 1985). The technique, which utilizes rat epidermal slices, is based on a premise that corrosive agents directly lyse stratum corneum and that this will be reflected as a fall in the electrical resistance of the skin slice. This correlation has now been established and the in vitro rat epidermal slice technique has a very high sensitivity for chemicals shown to be corrosive in conventional animal tests (Oliver & Pemberton, 1984; Oliver, Pemberton & Rhodes, 1986). The principle of correlating the lysis of stratum corneum to corrosive action lends itself readily to examination in human skin since the experimentation is in vitro and the stratum corneum is an inert, non-viable layer of compacted cells which retains its permeability characteristics for several months in cold storage. Therefore, following minor practical modifications, we have extended our investigation of corrosive chemical activity to human cadaver skin and have compared the response of human skin in vitro with that of animal skin in vitro and in vivo. This adaptation may facilitate the use of human cadaver skin to predict potential skin-corrosive hazard to man directly.
Chemicals, The 59 chemicals (43 corrosives and 16 irritants) used in these experiments represented a range of chemical types. The skin-corrosive chemicals were from the following groups: primary, secondary and tertiary alkylamines; alkylamine salts, ethoxylates and N-oxides; quaternary ammonium compounds; sodium silicates; formulated organic chemicals. The skin-irritant chemicals comprised various inorganic and organic chemicals, including alkylamine salts, ethoxylates and N-oxides. Within the group of corrosive chemicals were representatives from the three transportation categories, packing groups I, II and III (United Nations, 1977). Rat epidermal-slice technique. Wistar albino rats (weighing approximately 70-80g) were humanely killed and the clipped dorsal pelts were removed. "Epidermal slices (0.4 mm thick) were prepared, using an electrokeratotome. A PTFE tube was press-fitted onto this slice, which was secured and sealed to the end of the tube using a rubber 'O' ring before the excess tissue was trimmed away. Each pelt provided between six and eight skin-slice discs. Human skin technique. Cadaver skin of full thickness (i.e. containing a full depth of dermis) was obtained at autopsy. Subcutaneous fat was removed with a scalpel blade and the full-thickness skin was clamped between two halves of a glass cell (Dugard, Walker, Mawdsley & Scott, 1984). Since tissue viability is not required for resistance measurements, the skin slice was suspended in physiological saline. The test chemical (0.3 ml) was applied to the exposed epidermal surface of the skin and removed with a jet of warm water (40-50°C) after the required contact time. Electrical resistance measurements. Skin-resistance measurements were made in vitro using a simple AC half-bridge apparatus. After removal of the chemical, physiological saline (3 ml) was added and air bubbles were dislodged by tapping. Using electrodes on either
513
514
G . J . A . OLIVERand M. A. PEMaERTON
side of the skin, resistance was determined against the external circuit. Visual assessment o f human skin corrosion in vitro. Following removal of the test chemical, 0.5 ml of Harris's haematoxylin was left in contact with the epidermal surface of the skin for 10 min and was then washed off. Breaks in stratum corneum integrity corresponding to the site of potential corrosive lesions were made visible by stain penetration into the tissue.
Young rot 50
91"/o
40
30
58*/*
' Predicted as j corrosive
20
:orrosives Irritants
Results
A number of experiments were performed with inorganic acids to identify the electrical resistance threshold to corrosive action for human and rat skin in vitro. On this basis the resistance threshold between intact and chemically corroded skin from young rats in vitro was determined to be 3.2 Kfl/cm 2 (Oliver et al. 1986). A threshold to the corrosive action on human skin was estimated in a similar manner. However, as in vivo studies on skin corrosives in man were impractical, visual assessments of corrosive lesions were made in vitro by haematoxylin stain penetration. Contact with 0.125-1 M- and with 2M-sulphuric acid for 10 and 30min, respectively, had no detectable effect on electrical resistance or stain penetration. Contact with 6 and 8 M-sulphuric acid for 10 and 30 min produced visible tissue destruction (i.e. positive stain penetration) and reduced electrical resistance to below 12 Kf~/cm 2. Contact with 2 and 4M-sulphuric acid for 10 and 30 min reduced electrical resistance below 16 KQ/cm2 without producing visible tissue destruction. The detection of visible tissue destruction by stain penetration is regarded as a less sensitive indication of corrosive action in vitro than is electrical resistance. Therefore a resistance value of 16 Kf~/cm 2 has been adopted as a threshold between normal and chemically corroded human skin. Chemicals that reduce the electrical resistance below the adopted threshold in vitro are predicted as being skin corrosive. A comparison of the in vitro classification (based on 24-hr contact) and the in vivo classification (derived from tests in the rabbit) is shown in Fig. 1. The in vitro classification using young-rat skin demonstrates high sensitivity and good specificity for in vivo corrosive chemicals, with only 9% false negatives and 37% false correlates. The in vitro model using human skin predicts that 33% fewer chemicals would be corrosive to human skin than were corrosive to animal skin in vitro and in vivo. Because of the difference in electrical resistance thresholds between rat and human skin, the mean time required to reduce skin resistance below the threshold (the break-through time), for all the chemicals tested, has been estimated from the profiles of electrical resistance following contact for 1, 4 and 24 hr. Animal skin (mean break-through time 7.5 hr) was more susceptible than human skin (mean 13.5 hr) to corrosive chemical action (Student's paired t test, P < 0.0005). The ranking of corrosive chemicals based upon breakthrough times was similar on animal skin and human skin (Spearman's rank correlation, r = 0.82; Z test, P < 0.002).
Human
--
Corrodves Irritants
6 3 "/.
2 0
42*/*
Threshold
Predicted as I non--corrosive
3O
Fig. 1. Hazard distribution following 24-hr in vitro contact of skin from young rats and of human skin with chemicals identified by in vivo tests in rabbits as corrosive or irritant to the skin. Discussion
The rat epidermal-slice resistance technique is a rapid, robust, reliable and reproducible system for the in vitro assessment of corrosive potential to animal skin in vivo. This has been confirmed by extensive validation (Oliver et al. 1986). Therefore it is apparent that the underlying hypothesis of the model--that corrosive agents have a greater capacity than other chemicals for exerting a direct physicochemical lytic action on stratum corneum--is proven and there is a satisfactory correlation between the event measured in vitro and the response detected in vivo. The technique of in vitro resistance measurement has been readily transposed from rat to human tissue and the threshold for corrosive action in vitro has been determined. The higher threshold obtained in full-thickness human skin compared to that for rat skin is probably due to a combination of a more effective stratum corneum barrier and a thicker dermis in human skin. An in vitro contact period of 24 hr has been shown to give the highest sensitivity for corrosion prediction in rat tissue (Oliver et al. 1986). Under similar conditions, only two thirds of the chemicals that were 'corrosive' in in vivo rabbit studies and also in in vitro studies using rat skin were shown to be 'corrosive' in in vitro studies with human skin. The four false negatives (9%) in rat tissue in vitro were also negative in human tissue. Therefore, human skin appears to be less susceptible to chemical corrosive action than does animal skin and this may have significant implications with respect to the assignment of certain chemicals to their correct hazard category. However, to validate fully this in vitro approach to the prediction of corrosive action in human skin, it will be necessary to confirm the in vitro findings by appropriate skinpatch testing in man. This is currently being addressed within our laboratory. REFERENCES
Dugard P., Walker M., Mawdsley S. & Scott R. C. (1984). Absorption of some glycol ethers through human skin in vitro. Envir. Hlth Perspect. 57, 193.
In vitro identification of corrosives
OECD (1981), Acute dermal irritation/corrosion. In OECD Guidelines for Testing o f Chemicals: Section 4, no. 404. OECD, Paris. Oliver G. J. A. & Pemberton M. A. (1984). Prediction of skin corrosive potential /n vitro. Hum. Toxicol. 2, 330. Oliver G. J. A. & Pemberton M. A. 0985). An in vitro epidermal slice technique for identifying chemicals with a
F.C.T. 24/6,-7--F
515
potential for severe cutaneous effects. Fd Chem. Toxic 23, 229. Oliver G. J. A., Pemberton M. A. & Rhodes C. 0986). An in vitro skin corrosivity test--modifications and validation. Fd Chem. Toxic. 24, 507. United Nations (1977). Transport of Dangerous Goods. Orange book, special recommendations relating to class 8, p. 173.
0278-6915[86 $3.00+0.00 Pergamon Journals Ltd
Printed in Great Britain
THE IDENTIFICATION OF CORROSIVE AGENTS FOR H U M A N SKIN IN VITRO G. J. A. OLIVER and M. A. PEMBERTON Central Toxicology Laboratory, Imperial Chemical Industries plc, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England Abstract--An in vitro technique, using ex vivo epidermal slices prepared from young rats, has been developed and validated as a predictive screen for the corrosive potential of chemicals to rabbit skin in vivo (Oliver & Pemberton, Fd Chem. Toxic. 1985, 23, 229). A modification of this technique uses full-thickness cadaver skin in vitro to predict corrosive potential to human skin in vivo. Electrical resistance thresholds to corrosive action have been estimated for young-rat and human skin and the effects of 59 chemicals corrosive or irritant to the skin have been investigated in vitro. Rat skin is more susceptible than human skin to corrosive chemical action in vitro. This finding may have significant implications with respect to the assignment of certain chemicals to the correct hazard category.
Introduction
Experimental
It is necessary to be able to assess the possible cutaneous hazard to man of industrial and other chemicals, in order to meet requirements for classification, labelling, packagingr transportation and an overall safety evaluation. Primary or direct effects of chemicals on the skin are categorized as being either irritant (causing local reversible inflammatory responses) or corrosive (causing visible destruction or irreversible alteration at the site of contact). Toxicity tests in animals are generally used to meet the objective of assessing cutaneous hazard to man. The Draize rabbit skin test is the most commonly recommended and utilized protocol (OECD, 1981). We have developed an in vitro test with a high precision for identifying corrosive potential (Oliver & Pemberton, 1985). The technique, which utilizes rat epidermal slices, is based on a premise that corrosive agents directly lyse stratum corneum and that this will be reflected as a fall in the electrical resistance of the skin slice. This correlation has now been established and the in vitro rat epidermal slice technique has a very high sensitivity for chemicals shown to be corrosive in conventional animal tests (Oliver & Pemberton, 1984; Oliver, Pemberton & Rhodes, 1986). The principle of correlating the lysis of stratum corneum to corrosive action lends itself readily to examination in human skin since the experimentation is in vitro and the stratum corneum is an inert, non-viable layer of compacted cells which retains its permeability characteristics for several months in cold storage. Therefore, following minor practical modifications, we have extended our investigation of corrosive chemical activity to human cadaver skin and have compared the response of human skin in vitro with that of animal skin in vitro and in vivo. This adaptation may facilitate the use of human cadaver skin to predict potential skin-corrosive hazard to man directly.
Chemicals, The 59 chemicals (43 corrosives and 16 irritants) used in these experiments represented a range of chemical types. The skin-corrosive chemicals were from the following groups: primary, secondary and tertiary alkylamines; alkylamine salts, ethoxylates and N-oxides; quaternary ammonium compounds; sodium silicates; formulated organic chemicals. The skin-irritant chemicals comprised various inorganic and organic chemicals, including alkylamine salts, ethoxylates and N-oxides. Within the group of corrosive chemicals were representatives from the three transportation categories, packing groups I, II and III (United Nations, 1977). Rat epidermal-slice technique. Wistar albino rats (weighing approximately 70-80g) were humanely killed and the clipped dorsal pelts were removed. "Epidermal slices (0.4 mm thick) were prepared, using an electrokeratotome. A PTFE tube was press-fitted onto this slice, which was secured and sealed to the end of the tube using a rubber 'O' ring before the excess tissue was trimmed away. Each pelt provided between six and eight skin-slice discs. Human skin technique. Cadaver skin of full thickness (i.e. containing a full depth of dermis) was obtained at autopsy. Subcutaneous fat was removed with a scalpel blade and the full-thickness skin was clamped between two halves of a glass cell (Dugard, Walker, Mawdsley & Scott, 1984). Since tissue viability is not required for resistance measurements, the skin slice was suspended in physiological saline. The test chemical (0.3 ml) was applied to the exposed epidermal surface of the skin and removed with a jet of warm water (40-50°C) after the required contact time. Electrical resistance measurements. Skin-resistance measurements were made in vitro using a simple AC half-bridge apparatus. After removal of the chemical, physiological saline (3 ml) was added and air bubbles were dislodged by tapping. Using electrodes on either
513
514
G . J . A . OLIVERand M. A. PEMaERTON
side of the skin, resistance was determined against the external circuit. Visual assessment o f human skin corrosion in vitro. Following removal of the test chemical, 0.5 ml of Harris's haematoxylin was left in contact with the epidermal surface of the skin for 10 min and was then washed off. Breaks in stratum corneum integrity corresponding to the site of potential corrosive lesions were made visible by stain penetration into the tissue.
Young rot 50
91"/o
40
30
58*/*
' Predicted as j corrosive
20
:orrosives Irritants
Results
A number of experiments were performed with inorganic acids to identify the electrical resistance threshold to corrosive action for human and rat skin in vitro. On this basis the resistance threshold between intact and chemically corroded skin from young rats in vitro was determined to be 3.2 Kfl/cm 2 (Oliver et al. 1986). A threshold to the corrosive action on human skin was estimated in a similar manner. However, as in vivo studies on skin corrosives in man were impractical, visual assessments of corrosive lesions were made in vitro by haematoxylin stain penetration. Contact with 0.125-1 M- and with 2M-sulphuric acid for 10 and 30min, respectively, had no detectable effect on electrical resistance or stain penetration. Contact with 6 and 8 M-sulphuric acid for 10 and 30 min produced visible tissue destruction (i.e. positive stain penetration) and reduced electrical resistance to below 12 Kf~/cm 2. Contact with 2 and 4M-sulphuric acid for 10 and 30 min reduced electrical resistance below 16 KQ/cm2 without producing visible tissue destruction. The detection of visible tissue destruction by stain penetration is regarded as a less sensitive indication of corrosive action in vitro than is electrical resistance. Therefore a resistance value of 16 Kf~/cm 2 has been adopted as a threshold between normal and chemically corroded human skin. Chemicals that reduce the electrical resistance below the adopted threshold in vitro are predicted as being skin corrosive. A comparison of the in vitro classification (based on 24-hr contact) and the in vivo classification (derived from tests in the rabbit) is shown in Fig. 1. The in vitro classification using young-rat skin demonstrates high sensitivity and good specificity for in vivo corrosive chemicals, with only 9% false negatives and 37% false correlates. The in vitro model using human skin predicts that 33% fewer chemicals would be corrosive to human skin than were corrosive to animal skin in vitro and in vivo. Because of the difference in electrical resistance thresholds between rat and human skin, the mean time required to reduce skin resistance below the threshold (the break-through time), for all the chemicals tested, has been estimated from the profiles of electrical resistance following contact for 1, 4 and 24 hr. Animal skin (mean break-through time 7.5 hr) was more susceptible than human skin (mean 13.5 hr) to corrosive chemical action (Student's paired t test, P < 0.0005). The ranking of corrosive chemicals based upon breakthrough times was similar on animal skin and human skin (Spearman's rank correlation, r = 0.82; Z test, P < 0.002).
Human
--
Corrodves Irritants
6 3 "/.
2 0
42*/*
Threshold
Predicted as I non--corrosive
3O
Fig. 1. Hazard distribution following 24-hr in vitro contact of skin from young rats and of human skin with chemicals identified by in vivo tests in rabbits as corrosive or irritant to the skin. Discussion
The rat epidermal-slice resistance technique is a rapid, robust, reliable and reproducible system for the in vitro assessment of corrosive potential to animal skin in vivo. This has been confirmed by extensive validation (Oliver et al. 1986). Therefore it is apparent that the underlying hypothesis of the model--that corrosive agents have a greater capacity than other chemicals for exerting a direct physicochemical lytic action on stratum corneum--is proven and there is a satisfactory correlation between the event measured in vitro and the response detected in vivo. The technique of in vitro resistance measurement has been readily transposed from rat to human tissue and the threshold for corrosive action in vitro has been determined. The higher threshold obtained in full-thickness human skin compared to that for rat skin is probably due to a combination of a more effective stratum corneum barrier and a thicker dermis in human skin. An in vitro contact period of 24 hr has been shown to give the highest sensitivity for corrosion prediction in rat tissue (Oliver et al. 1986). Under similar conditions, only two thirds of the chemicals that were 'corrosive' in in vivo rabbit studies and also in in vitro studies using rat skin were shown to be 'corrosive' in in vitro studies with human skin. The four false negatives (9%) in rat tissue in vitro were also negative in human tissue. Therefore, human skin appears to be less susceptible to chemical corrosive action than does animal skin and this may have significant implications with respect to the assignment of certain chemicals to their correct hazard category. However, to validate fully this in vitro approach to the prediction of corrosive action in human skin, it will be necessary to confirm the in vitro findings by appropriate skinpatch testing in man. This is currently being addressed within our laboratory. REFERENCES
Dugard P., Walker M., Mawdsley S. & Scott R. C. (1984). Absorption of some glycol ethers through human skin in vitro. Envir. Hlth Perspect. 57, 193.
In vitro identification of corrosives
OECD (1981), Acute dermal irritation/corrosion. In OECD Guidelines for Testing o f Chemicals: Section 4, no. 404. OECD, Paris. Oliver G. J. A. & Pemberton M. A. (1984). Prediction of skin corrosive potential /n vitro. Hum. Toxicol. 2, 330. Oliver G. J. A. & Pemberton M. A. 0985). An in vitro epidermal slice technique for identifying chemicals with a
F.C.T. 24/6,-7--F
515
potential for severe cutaneous effects. Fd Chem. Toxic 23, 229. Oliver G. J. A., Pemberton M. A. & Rhodes C. 0986). An in vitro skin corrosivity test--modifications and validation. Fd Chem. Toxic. 24, 507. United Nations (1977). Transport of Dangerous Goods. Orange book, special recommendations relating to class 8, p. 173.