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An in vitro model for identifying skin-corrosive chemicals. I. Initial validation
Toxic. in Vitro Vol. 2, No. I, pp. 7-17, 1988 Printed in Great Britain
0887-2333/88 $3.00+0.00 Pergamon Journals Ltd
A N I N VITRO M O D E L F O R I D E N T I F Y I N G S K I N - C O R R O S I V E C H E M I C A L S . I. I N I T I A L V A L I D A T I O N * G. J. A. OLIVER, M. A. PEMBERTON and C. RHODES Central Toxicology Laboratory, Imperial Chemical Industries plc, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England (Received 10 April 1987; revisions received 6 August 1987)
epidermal slice technique has been developed for identifying chemicals with the potential to cause a corrosive lesion in animal skin in vivo. Skin-corrosive potential has been correlated with the ability to reduce the skin's penetration barrier by lysis of the stratum corneum. This effect was measured as a lowering of the electrical resistance of an epidermal slice following chemical contact in vitro. An initial validation with 68 chemicals showed the technique to have a high sensitivity for corrosive chemicals. The model has potential as a pre-screen for conventional animal tests and, in contrast to in vivo screening methods, has the advantage of providing quantitative and objective data. A b s t r a c t - - A n in vitro
INTRODUCTION Exposure of skin tissue to chemicals may result in a range of responses from no effect to a severe lesion. The assessment of such potential is necessary for the general control and safe handling of chemicals, and to meet existing regulatory requirements for classification, labelling, packaging and transportation. Chemically induced skin lesions can be broadly divided into two distinct categories according to severity. Skin irritation--local, reversible, inflammatory reactions (National Academy of Sciences, 1964) and skin corrosion--visible destruction or irreversible alteration (FDA, 1972). Currently the skin toxicity of materials is assessed in a dermal tolerance test in vivo, usually in the rabbit, which is based on the method of Draize et al. (1944). A major modification to the original method, which has gained regulatory approval, has been a reduction in the exposure or contact time from 24 to 4 hr (OECD, 1981). F o r regulatory purposes, chemicals that have been shown to be corrosive in such tests are categorized according to the contact time necessary to produce the specific skin effect, e.g. a corrosive lesion produced after 3 min or l or 4 hr signifies transport packing groups I, II or III, respectively (United Nations Economic and Social Council, 1977); the risk phrases R35 and R34 are invoked after 3 rain and 4 hr contact, respectively (European Economic Council, 1983). In the context of reducing the numbers of animals used in toxicity experiments, the need has been highlighted for an in vitro screen that would identify particularly those agents with a marked toxic potential to the skin (Balls et al. 1983). This recommendation is relevant to severe skin irritants and corrosive materials and in this paper we describe the development of an in vitro model for predicting chemicals that are corrosive to the skin. *Previously presented in part at the International Conference on Practical in vitro Toxicology, University of Reading, Berkshire, 18-20 September 1985.
A variety of in vitro and e x vivo systems have been used to examine the irritant potential of chemicals (Bettley, 1972; Gibson, 1980; Gibson & Teall, 1983; Kao et al. 1983; Middleton, 1981; Prottey & Ferguson, 1975 & 1976). So far none of these models has been sufficiently well validated with a number of chemicals of diverse structure to be judged a suitable pre-screen, adjunct or replacement for conventional animal tests. Morphologically, the skin is a complex organ and in developing in vitro techniques it is important to emulate the various structural layers of the skin as far as is practicable. Epidermal slices retain two morphological characteristics of the skin, i.e. the stratum corneum which provides a permeability barrier and the underlying viable differentiating epidermis (Middleton, 1981; Oliver & Pemberton, 1983 & 1984). We have used epidermal slices as the basis for an in vitro model for identifying the corrosive potential of chemicals. Retrospective re-analysis of in vivo data indicated that corrosive chemicals exert a physicochemical as well as a biological effect on skin tissue. We postulated that this physico-chemical effect would result in a direct lysis or degradation of normal stratum corneum which in turn could be monitored as a reduction in the electrical resistance of skin slices. MATERIALS AND METHODS
Chemicals. The 68 chemicals used in these experiments were chosen to represent a variety of different structures and physico-chemical properties and comprise: primary, secondary and tertiary alkyl amines, alkyi amine salts, ethoxylates and N-oxides; quaternary ammonium compounds; sodium silicates; organic chemicals; formulated organic chemicals; organic solvents; inorganic acids (Table 1). We categorized these chemicals as skin irritant or skin corrosive (Table 1) on the basis of our evaluation of data from rabbit dermal tolerance tests (OECD, 1981). The corrosive chemicals were further categorized into transport packing groups I, II and III (United
8
G.J.A.
OLIVER et aL
Table I. The identity, physico-chemical properties and in vivo categorization of the 68 chemicals used to validate the rat epidermal slice model for in vitro identification of corrosive potential Chemical no.
Chemical name
In vivo
Physical state
Formulation? solvent
Reactive pH
S* W* W* L W* L
1 I 1
11.4 11.7 11.8 11.5 11.4 11.6
I II III I I I
W* G L
1
8.5 7.5 8.3
II Xi Xi
category:~
Primary alkylamines 1
1-Dodecanamine
2 3 4 5 6
2-Hexadecanamine I-Octadecanamine Amines, coco alkylAmines, tallow alkylAmines, soya alkyl-
1
Secondary alkylamines 7 8 9
Amines, dicoco alkylButanoic acid, 3-amino-, N-coco alkylChemical 8, 48% in water/isopropranol
Tertiary alkylamines 10
Amines, coco alkyldimethyl-
L
10.0
I
Alkylamine salts 11 12 13
Amines, coco alkyl acetate Amines, hydrogenated and tallow alkyl acetate Amines, tallow alkyl acetate
14 15 16 17
Amines, N-coco alkyltrimethylenediAmines, N-tallow alkyltrimethylenedi1,3-Propanediamine, N-9-octadecenylAmines, N-tallow alkyltrimethylenedi-, ethoxylated 10 moles EO
18 19 20 21
Amines, coco alkylbis(2-hydroxyethyl) Amines, coco alkyl, ethoxylated > 10moles EO Amines, tallow alkyl, ethoxylated > 10 moles EO 9-Octadecenylamine, ethoxylated > 10 moles EO
W* W* W*
1 I 2
7.6 6.3 6.4
II II II
W* W* L L
1 I
11.9 12.0 11.5 10.5
1 I I Xi
10.5 13.5 9.4 11.2
II Xi Xi Xi
8.8 8.9 7.6
III Xi Xi
8.3 7.5 7.5 6.5 2.7 7.4 7.2
II III II II1 II II I
L
7.8
I
L
7.6
II
L L L L
11.3 8.1 6.3 7.6
I II II 111
L L
13.1 13.7
III III
L L L
8.8 12.3 10.5
Ill llI lI
E E
6.7 6.2
IIl Xi
G V V L
13.4 13.6 12.3 11.6
I1 III Xi Xi
L L L L
<0.5 <0.5 <0.5 <0.5
III III II III
L L
5.4 5.0
Xi III
Alkyl diamines and ethoxylates
Alkylamine ethoxylates L L V V
Alkylamine N-oxides 22 23 24 25 26 27 28 29 30 31 32 33
1-Tetradecanamine, N,N-dimethyl-N-oxide Amines, coco alkyldimethyl, N-oxide Amines, (hydrogenated tallow alkyl)dimethyl, N-oxide
Quaternary ammonium compounds 1-Dodecanaminium, N,N,N-trimethyl chloride l-Hexadecanaminium, N,N,N-trimethyl chloride QAC, dicoco alkyldimethyl, chloride QAC, bis(hydrogenated tallow alkyl)dimethyl, chlorides QAC, benzyl(hydrogenated tallow alkyl)dimethyl, chlorides QAC, benzyl coco alkyldimethyl, chloride Benzamethanaminium, N,N-dimethyl-N-tetradecyl, chloride QAC, coco alkylbis(hydroxyetbyl)methyl, ethoxylated, chlorides, < 10moles EO QAC, N,N,N, NI,Nt-pentamethyl-NI-tallow alkyltrimethylenedi-, dichlorides
L L W* L L L W* W* L S*
I
1 I 1
Primary alkylamines (CI3-CIs) 34 35 36 37
Amines Ci3~Ci5 Amines, benzylchloride quaternary salt Amines, dimethyl acetate Amines, dimethyl, N-oxide
38 39 40
44
9% BIT/60% propylene glycol 18% BIT/65% dipropylene glycol 44% BIT/12% diethananolamine, 14% triethanolamine, 8% morpholine 3% BIT/Glokill 77 33% BIT/27% ethylenediamine 33% BIT/15% propylene glycol, 1% lignosulphate, 0.25% kettle gum 33% BIT/40% 2.2 dispersing agent% Bentonite
45 46 47 48
Sodium metasilicate Silicate AI40 (32% SIO2/20% Na20) Silicate H100 (31% SIO2/13% Na20 ) Silicate Q79 (29% SIO2/9%, Na:O)
1,2-Benzisothiazolin-3-one formulations
41 42 43
Silicates of sodium
Inorganic acids 49 50 51 52
Hydrochloric acid (20%) Hydrochloric acid (10%) Sulphuric acid (40%) Sulphuric acid (10%)
Miscellaneous 53 54
Block copolymer 5-Nonylsalicylaldoxine
[contd]
In vitro m o d e l f o r testing s k i n c o r r o s i v e s , Table l--contd Chemical no. 55 56 57 58 59 60 61 62 63 64 65 66 67 68
Chemical name
Physical state
Formulation~" solvent
Reactive pH
S* S* L S* S* S* L* L* L L L V L L
3 3
3.5 0.7 5.0 2.0 ND 2.8 ND ND ND 13.3 6.9 8.3 8.2 6.7
2-Bromo-5-methylpyridine 5-Cyano-2,4,6-trichloropyridine Glycerol acetal laurie ester 2-Bromo-2-nitropropane- 1,3-diol. 2 Amino-4,6-dichlorophenol, HCI Trichlorosalieyclic acid n-Tributyltin (0.03%) n-Dibutyltin (0.01%) Aromatic solvent Cleaning agent Washing-up liquid Shampoo Dimethylacetamide Phenol (40%)
2 1 1 1 1
In vivo category:[ Xi III Xi Xi Xi Xi III XI II1 III III III Xi II
BIT = 1,2-Benzisothiazolin-3-one EO = Ethoxylate QAC = Quarternary ammonium compounds S=Solid W=Wax L=Liquid E=Emulsion V=Viscousliquid G=Gel *Formulated chemicals. tFormulation solvent: 1 = ethanol, 2 = acetone, 3 = dimethylformamide. ~In vivo category based on rabbit O E C D protocol (see Materials and Methods); I, II, III = respective corrosive packing group (see Materials and Methods); Xi = irritant. Chemicals 49-52 and 68 were categorized on the basis of historical in-house data on the rat.
Nations Economic and Social Council, 1977). Chemicals 1 to 33 were supplied by Akzo Chemie (UK) Ltd (Littleborough, Greater Manchester). Chemicals 34 to 48, 53 to 63 and 67 were obtained from various Divisions of Imperial Chemical Industries plc. Chemicals 64, 65 and 66 are proprietary materials and were purchased from local retail outlets. Chemicals 49 to 52 and chemical 68 were obtained from BDH Chemicals. p H Measurements. Chemicals were diluted with distilled water (1:9, w/v or v/v) to produce an approximately 10% solution. The reactive pH of this solution was measured using a Beckman pH meter (Model 3550) and a combination electrode (Model 39505). Animals. Alderley Park (Wistar) male albino rats were used at 28 days of age and weighing 60-80 g. The rats were allowed food and water ad lib. Preparation of rat epidermal slices. Animals were anaesthetized (3% Fluothane, ICI plc, Manchester) using a Fluotec apparatus (Cyprane, Keighley, Yorkshire) and the dorsal and flank hair carefully removed using fine clippers (Oster clippers, No. 80, size 40 head, Brookwickward & Co., London W6). The hair cycle is dormant in animals of this age and no further hair growth occurs for approximately 5 days. Epidermal slices were not prepared from animals until at least 48 hr after hair clipping. Animals were killed humanely and the dorsal skin was removed as a single pelt; excess fat was cut away and the remaining skin placed over a cork saddle. Two epidermal slices (18 mm x 80 mm) were cut against the direction of hair growth using a Castroviejo Electrokeratome, 0.3 mm shim (Stortz, St Louis, USA). Each epidermal slice was placed, stratum corneum uppermost, over a rubber 'O' ring. A purpose designed PTFE tube was press-fitted onto the slice and excess tissue was trimmed away. The epidermal slice attached to the PTFE tube was then suspended in physiological saline and maintained at ambient temperature (approximately 20°C; Fig. 1). Each pelt provided six to eight skin slice discs. A minimum of three skin slices was used for the assessment of each chemical.
Physical modification of rat epidermal slice. Rat epidermal slices were modified ex vivo by three different physical procedures of increasing severity. Before cutting individual slices the excised skin pelt was subjected to one of the following procedures: (i) Adhesive tape stripping ( x 5) to remove the superficial cell layers of the stratum corneum. (ii) Slight ( x 1) or severe ( x 5 ) abrasion with medium grade sandpaper. (iii) Immersion in water (50--60°C) for 5 min to remove the epidermis and stratum corneum from the dermis. In vitro application of test chemicals. Each test chemical (0.3ml) was placed onto the stratum
PTFEtube
iiiiii,,
~
ChemicaLs
CLip
DisposabLe
tube~ ,
Rubber'0' ring
Incubation media
T ___..
"--- ~ -
Dermis
.-- .--~.
1 2 3 I I I Scare (cm) Fig. 1, A p p a r a t u s f o r the in vitro e p i d e r m a l slice m o d e l .
10
G. J. A. OLIVERet al.
I
Tritiated water permeability measurements. Tritiated water (1 ml, 106 dpm: Amersham International, plc, Amersham, Bucks.) was added to the epidermal surface of a skin slice mounted in a PTFE tube suspended in isotonic saline (10 ml). Samples (25 #1) of the isotonic saline solution were taken during the steady-state phase of tritiated water penetration (between 3 and 6 hr). A sample (25/zl) of tritiated water was also taken at the beginning and end of the experiment. All aliquots were added to Fisofluor single "LPC" liquid scintillant (Fisons, plc, Loughborough) before counting for tritium in an Intertechnique SL30 scintillation counter. The penetration of tritiated water was calculated as a permeability constant (Kp) as described by Dugard (1983).
RESULTS p H Values o f corrosive and irritant chemicals
Fig. 2. Diagram of the circuit for measuring the electrical resistance of skin slices in vitro, showing capacitance decade (C), resistance decade (R), oscilloscope (O), oscillator (S) and stainless-steel electrodes (A and B).
corneum of the skin slice. After the required contact period the chemical was removed with a jet of warm water (40--45°C) immediately prior (unless otherwise stated) to measuring the electrical resistance across the skin slice. When electrical resistance was measured 20 hr after removal of the test chemical, the epidermal slice was kept in isotonic saline at ambient temperature as described in the previous section. Chemicals were applied as supplied except for (a) solids and waxes when a 50% (w/v) formulation was prepared in either ethanol, acetone or dimethyl formamide (Analar grade solvents, BDH Chemicals) and (b) chemicals 61 and 62 (see Table 1). Electrical resistance measurements. Electrical resistance of skin slices was measured using an a.c. half-bridge apparatus (Fig. 2) which consisted of the following components: a mains wave generator (Levell TG200 DMP, Levell Electronics Ltd, Barnet, Herts.); an oscilloscope with matched X and Y amplifiers (Model MR501, Tektronics, Oregon, USA); a resistance decade box (Model 8000, Time Electronics Ltd, Tonbridge); two capacitance decade boxes (Model Type C100, range 0-100 uf x 1 uf, and Models PC4, range 0-1 uf x 1 x 10-4 uf, J. J. Lloyd Instruments Ltd, Southampton); a colour-coded junction box and two stainless-steel electrodes (8 cm x 1 mm diameter). The wave generator was set to produce a sine wave output of 10 Hz. Amplitude was set at 200 mV and adjusted to give a screen-filling trace on the oscilloscope (100 mV setting). At these settings the potential difference across the epidermal slice was less than 1 V rms. Immediately before measurement of electrical resistance isotonic saline (3 ml) was added to the stratum corneum surface of the skin. Air bubbles were dislodged by slight tapping and the stainless-steel electrodes were placed to complete a circuit across the epidermal slice. Balance in the resistance and capacitance of the external circuit and the epidermal slice was indicated by a 45 ° trace on the oscilloscope.
The reactive pH value for each corrosive and irritant chemical is given in Table 1. The chemicals have been divided into three broad categories, depending on pH, and the distribution of irritant and corrosive chemicals within these categories is shown in Fig. 3. Both irritant and corrosive chemicals were evident at high and low pH (pH ~<2 or >/11.5) but the majority of chemicals were within the pH range 2.1-11.4. The electrical resistance o f intact rat epidermal skin slices
Change in electrical resistance was selected as a rapid and simple measure of stratum corneum integrity. The electrical resistance of untreated rat epidermal skin slices or skin discs ranged from 6-20 kD.skin disc (Fig. 4). Resistance values were compared to 3H20 permeability rates (Kp values) which have been used to assess skin barrier integrity (Dugard, 1983). Resistance values of > 6 k ~ . d i s c corresponded to a Kp 3H20 of 2-4 x 10-3cm/hr which has been reported for Alderley Park strain Wistar rats with an intact stratum corneum (Walker et al. 1983). In all subsequent experiments, skin discs with electrical resistance values greater than 10 kf]. skin disc were used. Electrical resistance values above 10 k ~ corresponded to Kp 3H20 values of less than 3 x 10 -3 cm/hr and in the majority of cases less than 2 x 10 -3 cm/hr (Fig. 4). 100 r-
pH 2.1 - 11.4
pH<~2
pH ~ 1 1 . 5
80
60 o
40 O
20-
0 I
Tr
m
xi
I
II
rrr X i
I
Tr
I£1
Xi
Fig. 3. The distribution of irritant (Xi) and corrosive (packing groups I, II or III) validation chemicals according to their pH values.
In vitro model for testing skin corrosives 10 -1
g
O
.I0 o E
10-2
\
0
\\ •:
"0
\
0
\,'.;" /
/
•e l • /
L. 0 .J
•e•
\\X /
•
10-3
I 0 2
xl I • 6
ELectricat
10
~o
-~ 2 • TM -... ;.b • ,-
I | IPI• 14 • 18
resistance
20
(kg,.disc)
Fig. 4. Relationship between measurements of transcutaneous electrical resistance and tritiated water permeability of young-rat skin in vitro. Measurements were made on intact skin ( 0 ) and on skin physically modified in vitro by slight abrasion (O), tape-stripping (1), severe abrasion (A) and heat separation (V').
The selection o f an electrical resistance value as a threshold indicating impaired stratum corneum
An electrical resistance threshold value that would indicate skin with a reduced barrier function was determined in experiments in which skin discs were modified by either physical or chemical treatment. First, skin discs were modified e x vivo by a number of physical procedures designed to remove the stratum corneum to varying degrees (Fig. 4). The milder forms of treatment (tape-stripping, single abrasion) reduced electrical resistance values to between 1 and 5-6kf~.skin disc whereas more severe treatment (repeated abrasion and heat-treatment) resulted in values below 1.5 k ~ . s k i n disc. The reduction in electrical resistance with progressive removal of stratum corneum correlated with an increase in Kp 3H20 (Fig. 4). Secondly, inorganic acids were applied in vitro to skin discs at varying concentrations for specified contact times. The results (Fig. 5) indicate that electrical resistance is lowered in vitro to approximately 4 kfl. skin disc and below, only under circumstances that produce a corrosive lesion (point or overt necrosis) in vivo. On the basis of the above results, an electrical resistance value of 4 kfl.skin disc (3.2 k ~ . c m ~) was selected as a threshold value for a positive effect in this in vitro model, i.e. chemicals reducing electrical resistance below 4 kf2.skin disc in vitro would be predicted to induce a corrosive lesion in vivo using conventional animal protocols. The effect o f corrosive and irritant chemicals on the electrical resistance o f skin tissue
The electrical resistance values of skin discs after in vitro exposure to 49 corrosive and 19 irritant chemi-
11
cals for contact periods of 1, 4 and 24 hr are shown in Table 2. The total number of corrosive or irritant chemicals that produced a mean resistance value greater or less than 4 kfl. skin disc at each contact time are shown in Fig. 6. Corrosive chemicals are further identified as group I, II or III. In describing the validation of the in vitro corrosivity test, the approach of Cooper et al. (1979) has been adopted. Thus the proportion of corrosive chemicals that are positive is termed "sensitivity"; the proportion of irritant chemicals that are negative is termed "specificity". After a 1-hr exposure in vitro 29% of corrosive chemicals, but no irritant chemicals, were positive. These positive chemicals included corrosive agents from all three packing groups. Following a 4-hr exposure in vitro, two irritants (chemicals 47 and 48) were positive, thus reducing specificity to 90%. However, sensitivity was increased to 65% and with the exception of chemical 32, with a mean resistance value of 4.0 k ~ . disc, all group I corrosive chemicals were positive after this contact period. After 24 hr exposure in vitro, sensitivity was increased to 92%. All group I and II corrosive chemicals were positive with four group III chemicals remaining negative (chemicals 40, 43, 54 and 61). Five irritant chemicals were positive (chemicals 19, 23, 24, 47 and 48) resulting in a specificity of 74%. In vitro exposure conditions were further modified in an attempt to maximize sensitivity without loss of specificity. Following a contact period of 4 hr chemicals were washed off the skin disc but electrical resistance was measured 20 hr later, 24 hr after the initial application. Excluded from this investigation were those chemicals that markedly reduced resistance (to below 1.5 k~.disc) by 4 hr or those that failed to reduce resistance even after 24 hr continuous contact. Results are shown in Table 2 and Fig. 6. Compared with the previous 4-hr exposure results, under these test conditions, where resistance measurement was delayed, specificity was maintained at 90% (chemicals 19 and 24 [Table 2] remaining
0.15
/
._e
.
1.oo
'~
1.5o
2
U.I
lO
In 0.25
In 0.5 MoLarity
I 1
I 2
I 4
of acid
Fig. 5. Electrical resistance of rat epidermal slices in vitro following topical application of sulphuric acid for i hr (O) or 4 hr (A) or of hydrochloric acid for 4 hr (C)). The doses in viva at which no abnormality was detected (n) and at which signs of point necrosis ( 1 ) or necrosis (*) were observed are indicated.
G. J. A. OLIVER et al.
12
positive) but sensitivity was increased to 80% (chemicals 11, 18, 28, 38, 41 and 66 comprising false negatives together with those four corrosive agents that were negative even after 24-hr continuous contact with skin slices).
Specificity and sensitivity and the threshold for a positive effect The in vitro corrosivity test was validated initially on the basis of an electrical resistance threshold for a positive effect of 4 kfl. skin disc. The reasons for the selection of this particular value are given above. In addition, the validation data on the 68 chemicals for each of the four chemical exposure and electrical resistance measurement conditions has been exam-
ined to determine whether the selected threshold value was optimal in relation to both the specificity and sensitivity of the test. The variation in test sensitivity and test specificity with respect to electrical resistance between the values 0 and 10 kl). disc and at intervals of 0.2 kll.disc is shown in Fig. 7. The data demonstrate that to achieve a combination of the highest sensitivity and specificity than 4 kl). disc is the optimal, if empirical, resistance threshold value. DISCUSSION
The principal objective of this work was to develop an in vitro model to identify chemicals that would be classified as corrosive to skin in conventional in vivo
Table 2. The skin electrical resistance values after various periods of contact with the 68 chemicals used to validate the rat epidermal slice model for in vitro identification of corrosive potential Electrical resistance value (kfl.skin disc) after chemical contact in vitro for Chemical no.
1 hr*
l 4 5 6 l0 14 15 16 31 32 34
2.5-+0.7(+) 1.4_+ 0.1 (+) 7.0_+2.7(-) 1.5-+0.3(+) 4.0+_0.4(-) 13.3 -+ 4.6 (--) 4.8_+1.9(-) 1.6_+ 0.3 (+) 6.3_+2.8(-) 19.8_+2.1(-) 9.2_+0.1(-)
Packing group I 1.0_+ 0.1 (+) 0.9_+0.1(+) 1.4-+ 0.1 (+) 0.9-+ 0.1 (+) 1.8 -+ 0.4 (+) 1.3 -+ 0.3 (+) 1.6_+0.3(+) 1.5 -+ 0.1 (+) 1.8-+0.8(+) 0.8_+0.1(+) 2.2-+0.5(+) 1.9 -+ 0.3 (+) 3.5_+0.9(+) 2.0_+0.3(+) 1.5 -+ 0.3 (+) 1.5 -+ 0.3 (+) 1.1 -+ 0.1 (+) 0.8_+0.1(+) 4.0_+0.4(-) 1.0_+ 0.1 (+) 1.4_+0.3(+) 0.9_+0.6(+)
ND(+) ND(+) 2.7-+0.3(+) 2.4_+ 1.3 (+) 2.1_+0.3(+) 1.0_+0.4(+) 0.8 -+ 0.1 (+) ND(+) ND(+) 2.6_+0.1(+) ND(+)
2 7 I1 12 13 18 25 27 29 30 35 36 42 45 51 68
2.5_+1.1(+) 15.1 _+4.1 ( - ) 6.7_+2.2(-) 11.5_+9.2(-) 5.7_+2.2(-) 6.7_+0.6(-) 3.2_+0.6(+) 16.0+_2.7(-) 9.7_+ 1.5(-) 4.3_+1.1(-) 7.0_+3.5(-) 1.0 -+ 0.0 (+) 5.1_+1.6(-) 0.4 _+0.1 (+) 2.7_+0.2(+) 1.1 _+0.3(+)
Packing group II 1.0_+0.3(+) 0.9+0.1(+) 3.5_+0.3(+) 3.9_+0.7(+) 5.1 -+ 1.1 ( - ) 1.1 -+ 0.1 (+) 4.4_+0.6(-) 1.9_+0.4(+) 3.0_+0.6(+) 2.4+0.4(+) 4.1+0.1(-) 1.8_+0.3(+) 1.8_+0.1(+) 1.1-+0.3(+) 3.2-+0.3(+) 1.3_+1.1(+) 2.9_+ 1.8(+) 1.5_+0.3(+) 1.9_+1.4(+) 0.9_+0.3(+) 2.4_+ 1.1 (+) 1.0_+0.3(+) 1.0 -+ 0.0 (+) 0.9_+0.1(+) 3.8_+0.3(+) 1.0_+ 0.1 (+) ND (+) ND (+) 0.7_+0.1(+) ND(+) 0.6_+0.2(+) ND(+)
3.4_+1.3(+) ND(+) 5.0_+2.4(-) 2.3_+0.1(+) 1.4_+0.1(+) 5.4_+1.5(-) 2.3-+0.5(+) 2.8_+2.1(+) 3.6_+2.6(+) 3.4_+ 1.8 (+) 2.2+1.2(+) ND(+) 3.7_+0.8(+) ND (+) ND(+) ND(+)
3 22 26 28 33 37 38 39 41 46 49 50 52 56 63 64 65 66
12.7 _ 6.1 (--) 12.3 -+ 5.0 ( - ) 8.4_+0.6(-) 9.2_+2.2(-) 14.7_+ 3.5 ( - ) 12.7 -+ 4.0 ( - ) 8.0_+3.0(-) 1.7_+ 0.5 (+) 9.0_+4.0(-) 1.1 -+0.3(+) 4.0 -+ 1.0 ( - ) 6.3_+0.6(-) 3.6_+ 1.7(+) 6.3 -+ 1.1 ( - ) 14.0-+5.9(-) 1.6 -+ 0.3 (+) 9.0-+0.0(-) 8.7 -+ 1.5 ( - )
Packing group III 5.9_+ 1.0 ( - ) 3.4_+0.6(+) 6.1 __. 1.3(-) 1.6_ 0.3 (+) 2.4_+0.4(+) 1.4_+ 1.1 (+) 5.4_+0.5(-) 2.0_+0.5(+) 3.4_+ l.l (+) 1.1 -+ 1.1 (+) 9.5_+8.9(-) 1.0_+0.3(+) 8.0_+0.3(-) 2.8_+0.5(+) 1.3 -+ 0.3 (+) 1.6_+0.5(+) 6.2_+2.4(-) 2.2-+0.3(+) 0.9 -+ 0.1 (+) ND(+) 2.3-+0.9(+) 1.1 +_ 1.9(+) 3.8_+0.8(+) 1.0_+ 1.7 (+) l.l _+0.3(+) ND(+) 5.9_+1.0(-) 2.3_+0.8(+) 17.0_+4.1(-) 2.3_+0.3(+) 0.8_+0.1(+) 0.8-+0.3(+) 3.0_+ 1.0 (+) 1.1 -+ 0.0 (+) 5.9_+2.7(-) 1.9-+ l.l (+)
3.9_+0.4(+) 2.2_+ l.l (+) 2.7_+ 1.2 (+) 5.3 _+ 1.2 (--) 2.4_+0.2(+) 3.7_+ 1.1 (+) 10.6 -+ 2.3 ( - ) ND(+) 10.4 -+4.3 ( - ) ND(+) ND(+) ND(+) ND(+) 1.3_+0.1 (+) 3.1_+0.9(+) ND(+) 2.8-+ 1.0 (+) 4.5 -+ 1.2 ( - )
40 43 54 61
13.0_+ 2.0 ( - ) 17.0-+ 5.1 ( - ) ND(-) 20.3 -+ l l . 0 ( - )
4 hr*
10.0_+3.0(-) 16.0 -+ 5.9 ( - ) ND(--) 13.0+_8.5(-)
24 hr*
5.4-+ 1.9 ( - ) 14.2 -+ 3.0 ( - ) 16.9 -+ 3.0 ( - ) 7.7-+2.2(-)
4 hr (resistance measured at 24 hr)*
ND(-) ND(-) ND(-) ND(-) [contd]
13
In vitro model for testing skin corrosives
Table 2--contd Electrical resistance value (k~.skin disc) after chemical contact in vitro for Chemical no.
I hr*
4 hr*
24 hr*
4 hr (resistance measured at 24 hr)*
19 23 24 47 48
8.4_+2.2(-) 13.4_+2.5(-) 11.8-+2.8(-) 4.6-+ 1.3(-) 7.3+2.5(-)
4.9+0.1(-) 15.1+3.5(-) 4.8-+ 1.4(-) 3.5+ 1.5(+) 2.8+0.3(+)
2.7+0.3(+) 2.5+0.1(+) 1.1 +0.1 (+) 1.1 _+0.6(+) 1.6+0.1(+)
3.1+0.5(+) 6.6+0.6(-) 2.9+ 1.8(+) 7.6+ 1.4(-) 7.5+0.2(-)
8 9 17 20
>20(-) 10.8_+2.3(-) 12.7_+6.6(-) 11.0+7.2(-) 10.8+5.3(-) 15.0+2.7(-) ND(-) 6.7_+2.9(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-)
18.0_+3.5(-) 13.7_+9.9(-) >20(-) 13.3_+1.5(-) 8.0+0.8(-) 11.0+1.5(-) ND(-) 8.4+3.2(-) ND(-) 18.9_+5.1(-) 7.2_+3.2(--) 19.0_+2.3(--) 15.7_+2.5(-) ND(-)
12.7_+2.5(-) 5.3+1.8(-) 10.8+3.0(-) 13.3+1.5(-) 8.5+0.4(-) 12.0+0.6(-) 14.1 _+7.0(-) 5.1_+1.8(-) 13.0+4.7(-) 15.1+2.5(--) 5.1 +0.9(--) 14.1 +2.5(--) 17.1 + 3 . 0 ( - ) 4.9+ 1.1 ( - )
ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(--) ND(--) ND(-) ND(-) ND(-) ND(-)
Irritant ehemlenls
21
44 53 55 57 58 59 60 62 67
ND ~ Not determined (but chemicals were categorized as + or - on the basis of their effect on electrical resistance under less stringent or more stringent test conditions respectively) *The (+) or ( - ) refers to the in vitro categorization of the chemical based on an electrical resistance threshold of 4 ki'). skin disc (see Results). Values are means + SD for three measurements.
tests. In this paper we describe the design and use of such an in vitro model and its validation with a large number of chemicals. The chemicals were selected to represent a range of typical industrial materials and formulations of different physico-chemical properties (physical state and pH). All of these chemicals induced skin lesions in animal tests and those categorized as skin corrosive displayed different reactivity, as
indicated by the relative contact period in vivo required to produce a corrosive lesion. In general, for those chemicals for which a pH could be determined, pH did not provide a good indication of corrosive potential. A number of irritant chemicals (4 of 17) were of high (~, 11.5) or low ( 4 2) pH and, conversely, most corrosive chemicals (29 of 46) had a pH of between 2.1 and 11.4. The pH
50-
4 hr (resistance m e a s u r e d a t 2 4 hr) 40
1 hr
4hr
40-
z
24 hr
40
I 5O
30-
20
20-
lo
10-
.3
3O
n
E 2O
"6 "m"
10
< 4 k,O,.disc [-~ )
z
Corrosives
Irritants
Corrosives
i Irritants
'°t I I
lO
20
I
1 Corrosives
~ 2 0
I
I Corrosives
Irritants
'°t 20
I
Irritants
lI
> 4 k~.disc (-)
30
40
Fig. 6. The proportion of the 68 validation chemicals that were positive or negative in the in vitro rat epidermal slice technique based on the electrical resistance threshold of kfl. skin disc after chemical contact in vitro for 1, 4 or 24 hr or after contact for 4 hr with resistance measured at 24 hr. The skin corrosive chemicals that were tested were of groups I, II or III as defined by the United Nations Economic and Social Council (1977).
G. J. A. OUVERet al.
14 loo
9o~
--
7.._..=.._.
,__,-,---?.
80 t-
.,'"
70}6 0 ~-
, ,"
,J
50 I-
.- ....... ......
,'"........ ....'., ~ -'"",,J
..:
Corrosive
Irritant
L-L---5~
r ""
Physico- chemical
,._,-'
1
Keratin tysis
JTissue destruction/irreversibLe change [ < 4 hr
I 0
I -.-~'~"I 1
I 2
I 3
I 4
Resistance
I 5
I 6
I 7
I 8
I 9
I 10
BioLogicaL InfLammatory
irritation
Oedemo/erytherna
threshol.d (k,(),. disc)
T i s s u e death necrosis >4hr
I 100 90
-- - S e v e r i t y
~---
601
~,
\
'..
,~. . . .
40
~ ~o mI~. 20 tO 0
-- -
Fig. 8. Proposed mechanism of action for corrosive and irritant chemicals.
eo I
*~ ~oI
of in vivo r e s p o n s e -
I
I
I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
9
10
Resistance threshold (k,~,. disc)
Fig. 7. The variation in test sensitivity and test specificity with respect to electrical resistance threshold for the 49 corrosive and 19 irritant chemicals. Chemical contact was for 1 ( ), 4 (----) or 24 ( - - - ) or for 4 hr with resistance measured at 24 hr (..... ).
categories selected for this analysis coincide with those quoted in regulatory guidelines as a guide to chemical classification without recourse to further testing (OECD, 1981). Clearly, from these data on this particular selection of chemicals, consideration of pH alone as a guide to skin corrosive potential would fail to identify a high number of corrosive chemicals and falsely indict some irritant chemicals. The choice of in vitro model and index of effect was influenced by the conclusions from a retrospective survey (G. J. A. Oliver & M. A. Pemberton, unpublished data, 1984) of the onset, duration and nature of the macroscopic skin lesions produced in vivo by irritant and corrosive chemicals in animal experiments conducted to current protocols (OECD, 1981). It was apparent that in general for the majority of corrosive chemicals, lesions consistent with a corrosive effect were evident within approximately 4 hr of contact and either accompanied or preceded any inflammatory changes (erythema and/or oedema). In a few instances corrosive lesions were noted after the maximal inflammatory response, at approximately 24 hr or longer after the initial contact. The irritant chemicals induced mainly transient inflammatory reactions. In addition, some irritants caused direct, reversible damage to the outermost skin layers (keratin lysis) which, macroscopically, imparted a "glossy" reflection to the skin surface. It was concluded from this appraisal that, in general, a direct physico-chemical interaction with skin tissue contributes significantly to the development of a corrosive but not an irritant lesion. It was also concluded from the above analysis that there are occasional exceptions to this hypothesis. Thus for a minority of corrosive chemicals the inflammatory
response is predominant and a corrosive lesion would appear to result as the sequel of a biochemically/ pharmacologically mediated tissue necrosis. Conversely, some irritant chemicals directly disrupt and/or remove the outermost layers of skin tissue. The major factors contributing to corrosive and irritant skin responses are summarized in Fig. 8. The apparent property of corrosive chemicals to interact directly with and to lyse the skin in vivo was exploited in designing the in vitro model both in the selection of the tissue and the best measure of effect. It was considered imperative to use skin epidermal slices and thereby include the normal physiological and anatomical structures that would initially be exposed and affected in vivo, particularly the outermost layer, the stratum corneum. The direct cytolytic action of chemicals on skin tissue was examined by measuring their effect on the functional integrity of the stratum corneum. The characteristics of the stratum corneum as a penetration barrier have been studied by various methods, e.g. by determination of rates of chemical absorption (Idson, 1975; Katz & Poulsen, 1971), measurement of transepidermal water loss (Malten et al. 1968; Scott et al. 1982) and measurement of change in electrical status (Ando et al. 1983; Blank & Finesinger, 1964; Dugard & Scheuplein, 1973). We chose to measure the electrical properties of skin slices/discs since this provided the most rapid and simple technique. Water permeability measurements have been used in this laboratory to define rat stratum corneum integrity (Dugard et al. 1984). Water permeability and electrical conductance are proportional and differ only in the rates of permeation of water and ions and in the technical ease of measurement. However, due to the low conductivity of intact skin, the range of resistance values (Fig. 4) and the relative inaccuracy of measurements below 1 kfl/skin disc, we have recorded electrical resistance, rather than conductance. The a.c. half-bridge apparatus measures electrical impedance which has resistive and the relative inaccuracy of measurements below I kf/. skin disc, we have recorded electrical resistance, the resistive component of the skin dominates the negligible capacitive component. In addition to the above major criteria, the epidermal slice technique satisfies a number of further requirements which we consider to be important for
In vitro model for testing skin corrosives
15
the adoption of a practical in vitro toxicity test following 4 hr contact resulted in a higher detection system. The epidermal slice technique (Fig. 1) pro- rate for corrosives (increased sensitivity) without any vides a simple rapid, robust and reproducible means loss of specificity when compared with measurement of screening chemicals on a regular and routine basis. at the end of a. 4-hr contact period. Overall, using a 24-hr contact period or a 4-hr Tissue slices of consistent quality can be quickly prepared. The technique represents an economic use contact period coupled to a 24-hr resistance of animal tissue since the pelt from a single rat measurement, the epidermal slice technique can idenprovides sufficient skin discs to test two or three tify corrosive chemicals in vitro with both high separate chemicals. In addition many factors that specificity and sensitivity. Either set of conditions may influence the final response in vivo may be could be used depending on individual requirements emulated in vitro. In particular, chemicals of different and the relative importance of the likely occurrence physical states (liquids, waxes, gels, solids, powders) of false negative or false positive results. It must be can be applied directly to the normal outer skin remembered that the frequency of occurrence of false surface. Variable contact periods, effective removal of positive and false negative results will depend on the test compound and a degree of occlusion can all be prevalence of corrosive chemicals in a batch of test chemicals as well as on the sensitivity and specificity achieved using this in vitro model. Using water permeability measurement as a refer- of the test model (Cooper et al. 1979). Thus the ence technique, decreased skin resistance was shown validation of an in vitro test model can be described to correlate with a reduction in the skin's barrier in terms of sensitivity and specificity but the practical properties (Fig. 4). From these and additional experi- utility of the model (its predictive value or its accuments (Fig. 5) a resistance value of 4 kf~. skin disc racy) when used as a screen is also influenced by the (3.2 kfl.cm 2) was considered to indicate significantly expected proportion of corrosive chemicals among compromised stratum corneum function. Therefore, those being assessed. In general (a) the higher the a resistance value of 4 k[2. skin disc was adopted as sensitivity of the test then the greater the certainty an arbitrary empirical threshold to corrosive action in that a negative result is true, i.e. the chemical will not vitro. The selection of this value was further vin- be corrosive in vivo; (b) the higher the specificity then dicated by retrospective analysis of the data for the the greater the certainty that a positive result is true, validation chemicals from each of the four i.e. the chemical will be corrosive in vivo; (c) the lower exposure/resistance measurement regimes (see below the prevalence of corrosive chemicals, the higher will be the proportion of false negatives and false and Fig. 7). Using this principle, the validation of the technique positives with decreasing sensitivity and specificity. The detection of false negatives and positives itself with 68 chemicals showed the sensitivity and specificity of the in vitro corrosivity model to be contributes to the definition and understanding of the influenced by both the contact time and the elapsed proposed mode of action of corrosive and irritant time prior to resistance measurement following re- chemicals (Fig. 8). Consideration of the noncorrelates allows a more rationalized appraisal of the moval of the chemical (Fig. 6). Ideally the specificity and sensitivity of an in vitro expected and apparent sensitivity and specificity of screen should be as high as possible with a minimum the model. From the in vivo evidence the four false of false negatives or false positives. This goal can be negatives (24 hr contact) represent chemicals in which related to chemicals of general structure or to specific the inflammatory response is dominant, the corrosive groups or analogues depending on requirements "lesion is relatively slow to develop and probably results from a cascade of biological events leading to (Cooper et al. 1979). When this desired optimal performance for an in overt tissue necrosis (Fig. 7; G. J. A. Oliver & M. A. vitro system is apposed to skin toxicity assessment in Pemberton, unpublished data, 1984). These particular chemicals did not appear to interact directly with vivo, it is pertinent to remember that the evaluation of a chemical's toxic potential in vivo is not absolute the stratum corneum in vivo and therefore would not but is based on subjective assessment and individual be expected to reduce electrical resistance below an opinion of a macroscopic event(s). This consideration adopted threshold value in vitro in the epidermal slice is relevant to the interpretation of the predictive system. The four chemicals are either individual or capacity of this in vitro system since in vivo it may formulated organic chemicals (numbers 40, 43, 54 be difficult to distinguish unequivocally between and 61). Reference to the in vivo response profile of the false "severely irritant" and "corrosive" lesions. Exposure periods of 1 and 4 hr resulted in complete positive chemicals (24 hr contact) also provides a specificity and high specificity respectively but low possible explanation for their in vitro activity. Three of the five chemicals are surfactant materials (chemisensitivity (Fig. 6). Sensitivity was increased to 92% after 24 hr continuous contact in vitro at the expense cals 19, 23 and 24) and two are silicates (chemicals 47 of lowering of specificity to 74%. The procedure in and 48) of high pH (Table 2). For each of these which resistance measurement was delayed following chemicals there was in vivo evidence of keratin lysis chemical removal after a 4-hr exposure was designed (removal of the outer layers of the stratum corneum) to emulate in vivo conditions of contact and thereby in the absence of any indications of a corrosive lesion increase sensitivity without loss of specificity. In vivo (G. J. A. Oliver & M. A. Pemberton, unpublished removal of compound after a specified time may be data 1984). The surface active properties of the incomplete and residual material, within or on the surfactants and/or the high pH of these materials is surface of the skin, may continue to contribute to the consistent with this observation (Spruit, 1970; Spruit final toxic response. This would appear to be upheld & Malten, 1968; Wood & Bettley, 1971) of a reduction in electrical resistance in vitro. in vitro since delaying the resistance measurement
16
G. J. A. OLIVERet al.
For corrosive chemicals the contact time in vitro required to affect the stratum corneum does not appear to correlate with the nominal exposure time in vivo required to induce a corrosive lesion. Thus a proportion of group I and II corrosive agents remain negative in vitro after 1 and 4 hr contact respectively (Fig. 6). The corresponding in vivo exposure times are 3 min and 1 hr for these two classification groups. As discussed earlier this apparent difference may simply reflect a continuing action in vivo from chemical remaining in the tissue after the washing-off process. This view is reinforced by the change in profile of negative/positive results when resistance measurement is delayed after contact in vitro (Fig. 6). Conversely, a proportion of group III corrosive agents are positive in vitro after a l-hr contact period. For these chemicals, their relatively rapid effect on stratum corneum does not lead to a corrosive lesion in vivo unless exposure is extended from 1 to 4 hr. Overall for structurally diverse chemicals, the in vitro contact period required to produce a positive result cannot be used to indicate corrosive potency as illustrated by packing group and as currently defined in in vivo test protocols. In summary, the epidermal slice technique is capable of distinguishing skin corrosive chemicals from other less damaging chemicals. The use of either a 24-hr contact time or a 4-hr contact time coupled with a 24-hr resistance measurement results in both a high sensitivity (92%) and high specificity (74%). On the basis of these data it is proposed that this in vitro model has utility as a prescreen for the identification of skin corrosive potential. Depending on the particular circumstances that will decide the predictive value (see earlier discussion), this prescreen will allow decisions to be made concerning: the need to progress to an animal test, and the design of a confirmatory animal test (contact time, numbers to be used, frequency of observation, etc.) if this is considered necessary to clarify a possible false positive or negative result. Besides the predictive accuracy of the test, other factors, e.g. regulatory considerations, may influence the wider application of results from an in vitro screen. However, a more ready acceptance by regulatory authorities of the results of tests that identify severe responses is logical and feasible. Current guidelines already state that chemicals may be considered corrosive if of high or low pH and without recourse to animal testing "if the result can be predicted" (EEC, 1983; OECD, 1981). In addition to its use as a prescreen, the in vitro corrosivity test provides objective and quantifiable data. This is considered to be a major advantage when compared to subjectively assessed animal tests (Weil & Scala, 1972). The in vitro approach also provides additional qualitative information concerning the action of the chemicals concerned. Finally, the epidermal skin slice model has considerable potential for the more direct assessment of h u m a n hazard since, with suitable modifications, h u m a n skin instead of rat skin can be used. Preliminary electrical resistance experiments have shown that human skin may respond differently to animal skin to some corrosive agents (Oliver & Pemberton, 1986). The results of such experiments may have
considerable significance in the accurate assessment of h u m a n hazard and the appropriate classification of chemicals. Acknowledgements--We would like to thank Dr S. J. Jack-
son, Central Toxicology Laboratory, for organizing the supply of compounds from Akzo Chemic and to thank Akzo Chemic b.v. (Netherlands) and Akzo Chemic (UK) Ltd for supplying chemicals and permitting their use in this work. We are grateful to Dr I. F. H. Purchase for his constructive suggestions and discussion and to Mr I. Pate and Miss M. Boylett for their statistical appraisal of the data. REFERENCES
Ando H. Y., Escobar A., Schnaare R. L. & Sugita E. T. 0983). Skin potential changes in the guinea-pig due to depilation and the repeated application of polyethylene glycol and retinoic acid. J. Soc. cosmet. Chem. 34, 159-169. Balls M., Riddell R. J. & Worden A. N. (Editors) (1983). Animals and Alternatives in Toxicity Testing. Academic Press, London. Bettley F. R. (1972). The irritant effect of detergents. Trans. a. Rep. St. John's Hosp. derm. Soc. 58, 65-74. Blank I. H. & Finesinger J. E. (1964). Electrical resistance of the skin. Archs NeuroL Psychiat., Lond. 56, 544-557. Cooper J. A., Saraachi R. & Cole P. (1979). Describing the validity of carcinogen screening tests. Br. J. Cancer 39, 87-89. Draize J. H., Woodard G. & Calvery H. O. (1944). Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmac. exp. Ther. 82, 377-390. Dugard P. H. (1983). Skin permeability theory in relation to measurements of percutaneous absorption in toxicology. In Dermatotoxicology and Pharmacology. Edited by F. N. Marzulli & H. I. Maibach. 2nd Ed. pp. 525-550. Hemisphere, Washington. Dugard P. H. & Scheuplein R. J. 0973). Effects of ionic surfactants on the permeability of human epidermis: an electrometric study. J. invest. Derm. 60, 263-269. Dugard P. H., Walker M., Mawdsley S. J. & Scott R. C. (1984). Absorption of some glycol ethers through human skin in vitro. Envir. Hlth Perspect. 57, 193-197. European Economic Council (1983). Commission Directive of 29 July 1983 adapting to technical progress for the fifth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, (83-467/EEC). Off. 3. Eur. Commun. 26 (L257), 1-33. FDA (1972). Hazardous substances. Proposed revision of test for primary skin irritants. Fed. Reg. 37, 27635-27636. Gibson W. T. (1980). Lysis of rabbit polymorphonuclear leucocyte granules by surfactants of differing structure and irritancy. Fd Cosmet. Toxicol. 18, 511-515. Gibson W. T. & Teall M. R. (1983). Interactions of C~2 surfactants with the skin: studies on enzyme release and percutaneous absorption in vitro. Fd Chem. Toxic. 21, 581-586. Idson B. (1975). Percutaneous absorption. J. pharm. Sci. 64, 901-924. Kao J., Hall J. & Holland J. M. (1983). Quantitation of cutaneous toxicity: an in vitro approach using skin organ culture. Toxic. appl. Pharmac. 68, 206-217. Katz M. & Poulsen J. B. (1971). Absorption of drugs through the skin. In Concepts in Biochemical Pharmacology. Part 1. Edited by B. B. Brodie & J. R. Gillette; Handbook of Experimental Pharmacology. Vol 28. pp. 103-174. Springer-Verlag, Berlin.
In vitro model for testing skin corrosives
Malten K. E., Spruit D., Boemaars H. G. M. & Keizer M. J. M. (1968). Horny layer injury by solvents. Berufsdermatosen 16, 135-147. Middleton M. C. (1981). New approaches to problems of dermatotoxicity. In Testing for Toxicity. Edited by J. W. Gorrod. pp. 275-296. Taylor & Francis Ltd, London. National Academy of Sciences (1964). Bulletin 118. NAS. Washington, DC. OECD (1981). Acute dermal irritation/corrosion. In OECD Guidelines for Testing of Chemicals. Section 4, no. 404. OECD, Paris. Oliver G. J. A. & Pemberton M. A. (1983). An in vitro model for assessing the corrosive potential of chemicals to the skin (Abstract). Hum. Toxicol. 2, 562-563. Oliver G. J. A. & Pemberton M. A. (1984). Prediction of skin corrosive potential in vitro (Abstract). Hum. Toxicol. 2, 330-331. Oliver G. J. A. & Pemberton M. A. (1986). The identification of corrosive agents for human skin in vitro. Fd Chem. Toxic. 24, 513-515. Prottey C. & Ferguson T. F. M. (1975). Factors which determine the skin irritation potential of soaps and detergents. J. Soc. cosmet. Chem. 26, 29-46.
T.I.V. 2/I--B
17
Prottey C. & Ferguson T. F. M. (1976). The effects of surfactants upon rat peritoneal mast cells in vitro. Fd Cosmet. Toxicol. 14, 425-430. Scott R. C., Oliver G. J. A., Dugard P. H. & Singh H. J. (1982). A comparison of techniques for the measurement of transepidermal water loss. Archs Derm. 274, 57~4. Spruit D. (1970). The evaluation of skin function by the alkali application technique. Curt. Probl. Derm. 3, 148-163. Spruit D. & Malten K. E. (1968). Estimation of the injury of human skin by alkaline liquids. Berufsdermatosen 16, 11-24.
United Nations Economic and Social Council (1977). Transport of dangerous goods. Special recommendations relating to Class 8. p. 173. Walker M., Dugard P. H. & Scott R. C. (1983). Absorption through human and laboratory animals skins; in vitro comparisons. Acta pharm, suec. 20, 52. Weil C. S. & Scala R. A. (1972). Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxic. appl. Pharmac. 19, 276-360. Wood D. C. & Bettley F. R. (1971). The effect of various detergents on human epidermis. Br. J. Derm. 84, 320-325.
0887-2333/88 $3.00+0.00 Pergamon Journals Ltd
A N I N VITRO M O D E L F O R I D E N T I F Y I N G S K I N - C O R R O S I V E C H E M I C A L S . I. I N I T I A L V A L I D A T I O N * G. J. A. OLIVER, M. A. PEMBERTON and C. RHODES Central Toxicology Laboratory, Imperial Chemical Industries plc, Alderley Park, Macclesfield, Cheshire SK10 4TJ, England (Received 10 April 1987; revisions received 6 August 1987)
epidermal slice technique has been developed for identifying chemicals with the potential to cause a corrosive lesion in animal skin in vivo. Skin-corrosive potential has been correlated with the ability to reduce the skin's penetration barrier by lysis of the stratum corneum. This effect was measured as a lowering of the electrical resistance of an epidermal slice following chemical contact in vitro. An initial validation with 68 chemicals showed the technique to have a high sensitivity for corrosive chemicals. The model has potential as a pre-screen for conventional animal tests and, in contrast to in vivo screening methods, has the advantage of providing quantitative and objective data. A b s t r a c t - - A n in vitro
INTRODUCTION Exposure of skin tissue to chemicals may result in a range of responses from no effect to a severe lesion. The assessment of such potential is necessary for the general control and safe handling of chemicals, and to meet existing regulatory requirements for classification, labelling, packaging and transportation. Chemically induced skin lesions can be broadly divided into two distinct categories according to severity. Skin irritation--local, reversible, inflammatory reactions (National Academy of Sciences, 1964) and skin corrosion--visible destruction or irreversible alteration (FDA, 1972). Currently the skin toxicity of materials is assessed in a dermal tolerance test in vivo, usually in the rabbit, which is based on the method of Draize et al. (1944). A major modification to the original method, which has gained regulatory approval, has been a reduction in the exposure or contact time from 24 to 4 hr (OECD, 1981). F o r regulatory purposes, chemicals that have been shown to be corrosive in such tests are categorized according to the contact time necessary to produce the specific skin effect, e.g. a corrosive lesion produced after 3 min or l or 4 hr signifies transport packing groups I, II or III, respectively (United Nations Economic and Social Council, 1977); the risk phrases R35 and R34 are invoked after 3 rain and 4 hr contact, respectively (European Economic Council, 1983). In the context of reducing the numbers of animals used in toxicity experiments, the need has been highlighted for an in vitro screen that would identify particularly those agents with a marked toxic potential to the skin (Balls et al. 1983). This recommendation is relevant to severe skin irritants and corrosive materials and in this paper we describe the development of an in vitro model for predicting chemicals that are corrosive to the skin. *Previously presented in part at the International Conference on Practical in vitro Toxicology, University of Reading, Berkshire, 18-20 September 1985.
A variety of in vitro and e x vivo systems have been used to examine the irritant potential of chemicals (Bettley, 1972; Gibson, 1980; Gibson & Teall, 1983; Kao et al. 1983; Middleton, 1981; Prottey & Ferguson, 1975 & 1976). So far none of these models has been sufficiently well validated with a number of chemicals of diverse structure to be judged a suitable pre-screen, adjunct or replacement for conventional animal tests. Morphologically, the skin is a complex organ and in developing in vitro techniques it is important to emulate the various structural layers of the skin as far as is practicable. Epidermal slices retain two morphological characteristics of the skin, i.e. the stratum corneum which provides a permeability barrier and the underlying viable differentiating epidermis (Middleton, 1981; Oliver & Pemberton, 1983 & 1984). We have used epidermal slices as the basis for an in vitro model for identifying the corrosive potential of chemicals. Retrospective re-analysis of in vivo data indicated that corrosive chemicals exert a physicochemical as well as a biological effect on skin tissue. We postulated that this physico-chemical effect would result in a direct lysis or degradation of normal stratum corneum which in turn could be monitored as a reduction in the electrical resistance of skin slices. MATERIALS AND METHODS
Chemicals. The 68 chemicals used in these experiments were chosen to represent a variety of different structures and physico-chemical properties and comprise: primary, secondary and tertiary alkyl amines, alkyi amine salts, ethoxylates and N-oxides; quaternary ammonium compounds; sodium silicates; organic chemicals; formulated organic chemicals; organic solvents; inorganic acids (Table 1). We categorized these chemicals as skin irritant or skin corrosive (Table 1) on the basis of our evaluation of data from rabbit dermal tolerance tests (OECD, 1981). The corrosive chemicals were further categorized into transport packing groups I, II and III (United
8
G.J.A.
OLIVER et aL
Table I. The identity, physico-chemical properties and in vivo categorization of the 68 chemicals used to validate the rat epidermal slice model for in vitro identification of corrosive potential Chemical no.
Chemical name
In vivo
Physical state
Formulation? solvent
Reactive pH
S* W* W* L W* L
1 I 1
11.4 11.7 11.8 11.5 11.4 11.6
I II III I I I
W* G L
1
8.5 7.5 8.3
II Xi Xi
category:~
Primary alkylamines 1
1-Dodecanamine
2 3 4 5 6
2-Hexadecanamine I-Octadecanamine Amines, coco alkylAmines, tallow alkylAmines, soya alkyl-
1
Secondary alkylamines 7 8 9
Amines, dicoco alkylButanoic acid, 3-amino-, N-coco alkylChemical 8, 48% in water/isopropranol
Tertiary alkylamines 10
Amines, coco alkyldimethyl-
L
10.0
I
Alkylamine salts 11 12 13
Amines, coco alkyl acetate Amines, hydrogenated and tallow alkyl acetate Amines, tallow alkyl acetate
14 15 16 17
Amines, N-coco alkyltrimethylenediAmines, N-tallow alkyltrimethylenedi1,3-Propanediamine, N-9-octadecenylAmines, N-tallow alkyltrimethylenedi-, ethoxylated 10 moles EO
18 19 20 21
Amines, coco alkylbis(2-hydroxyethyl) Amines, coco alkyl, ethoxylated > 10moles EO Amines, tallow alkyl, ethoxylated > 10 moles EO 9-Octadecenylamine, ethoxylated > 10 moles EO
W* W* W*
1 I 2
7.6 6.3 6.4
II II II
W* W* L L
1 I
11.9 12.0 11.5 10.5
1 I I Xi
10.5 13.5 9.4 11.2
II Xi Xi Xi
8.8 8.9 7.6
III Xi Xi
8.3 7.5 7.5 6.5 2.7 7.4 7.2
II III II II1 II II I
L
7.8
I
L
7.6
II
L L L L
11.3 8.1 6.3 7.6
I II II 111
L L
13.1 13.7
III III
L L L
8.8 12.3 10.5
Ill llI lI
E E
6.7 6.2
IIl Xi
G V V L
13.4 13.6 12.3 11.6
I1 III Xi Xi
L L L L
<0.5 <0.5 <0.5 <0.5
III III II III
L L
5.4 5.0
Xi III
Alkyl diamines and ethoxylates
Alkylamine ethoxylates L L V V
Alkylamine N-oxides 22 23 24 25 26 27 28 29 30 31 32 33
1-Tetradecanamine, N,N-dimethyl-N-oxide Amines, coco alkyldimethyl, N-oxide Amines, (hydrogenated tallow alkyl)dimethyl, N-oxide
Quaternary ammonium compounds 1-Dodecanaminium, N,N,N-trimethyl chloride l-Hexadecanaminium, N,N,N-trimethyl chloride QAC, dicoco alkyldimethyl, chloride QAC, bis(hydrogenated tallow alkyl)dimethyl, chlorides QAC, benzyl(hydrogenated tallow alkyl)dimethyl, chlorides QAC, benzyl coco alkyldimethyl, chloride Benzamethanaminium, N,N-dimethyl-N-tetradecyl, chloride QAC, coco alkylbis(hydroxyetbyl)methyl, ethoxylated, chlorides, < 10moles EO QAC, N,N,N, NI,Nt-pentamethyl-NI-tallow alkyltrimethylenedi-, dichlorides
L L W* L L L W* W* L S*
I
1 I 1
Primary alkylamines (CI3-CIs) 34 35 36 37
Amines Ci3~Ci5 Amines, benzylchloride quaternary salt Amines, dimethyl acetate Amines, dimethyl, N-oxide
38 39 40
44
9% BIT/60% propylene glycol 18% BIT/65% dipropylene glycol 44% BIT/12% diethananolamine, 14% triethanolamine, 8% morpholine 3% BIT/Glokill 77 33% BIT/27% ethylenediamine 33% BIT/15% propylene glycol, 1% lignosulphate, 0.25% kettle gum 33% BIT/40% 2.2 dispersing agent% Bentonite
45 46 47 48
Sodium metasilicate Silicate AI40 (32% SIO2/20% Na20) Silicate H100 (31% SIO2/13% Na20 ) Silicate Q79 (29% SIO2/9%, Na:O)
1,2-Benzisothiazolin-3-one formulations
41 42 43
Silicates of sodium
Inorganic acids 49 50 51 52
Hydrochloric acid (20%) Hydrochloric acid (10%) Sulphuric acid (40%) Sulphuric acid (10%)
Miscellaneous 53 54
Block copolymer 5-Nonylsalicylaldoxine
[contd]
In vitro m o d e l f o r testing s k i n c o r r o s i v e s , Table l--contd Chemical no. 55 56 57 58 59 60 61 62 63 64 65 66 67 68
Chemical name
Physical state
Formulation~" solvent
Reactive pH
S* S* L S* S* S* L* L* L L L V L L
3 3
3.5 0.7 5.0 2.0 ND 2.8 ND ND ND 13.3 6.9 8.3 8.2 6.7
2-Bromo-5-methylpyridine 5-Cyano-2,4,6-trichloropyridine Glycerol acetal laurie ester 2-Bromo-2-nitropropane- 1,3-diol. 2 Amino-4,6-dichlorophenol, HCI Trichlorosalieyclic acid n-Tributyltin (0.03%) n-Dibutyltin (0.01%) Aromatic solvent Cleaning agent Washing-up liquid Shampoo Dimethylacetamide Phenol (40%)
2 1 1 1 1
In vivo category:[ Xi III Xi Xi Xi Xi III XI II1 III III III Xi II
BIT = 1,2-Benzisothiazolin-3-one EO = Ethoxylate QAC = Quarternary ammonium compounds S=Solid W=Wax L=Liquid E=Emulsion V=Viscousliquid G=Gel *Formulated chemicals. tFormulation solvent: 1 = ethanol, 2 = acetone, 3 = dimethylformamide. ~In vivo category based on rabbit O E C D protocol (see Materials and Methods); I, II, III = respective corrosive packing group (see Materials and Methods); Xi = irritant. Chemicals 49-52 and 68 were categorized on the basis of historical in-house data on the rat.
Nations Economic and Social Council, 1977). Chemicals 1 to 33 were supplied by Akzo Chemie (UK) Ltd (Littleborough, Greater Manchester). Chemicals 34 to 48, 53 to 63 and 67 were obtained from various Divisions of Imperial Chemical Industries plc. Chemicals 64, 65 and 66 are proprietary materials and were purchased from local retail outlets. Chemicals 49 to 52 and chemical 68 were obtained from BDH Chemicals. p H Measurements. Chemicals were diluted with distilled water (1:9, w/v or v/v) to produce an approximately 10% solution. The reactive pH of this solution was measured using a Beckman pH meter (Model 3550) and a combination electrode (Model 39505). Animals. Alderley Park (Wistar) male albino rats were used at 28 days of age and weighing 60-80 g. The rats were allowed food and water ad lib. Preparation of rat epidermal slices. Animals were anaesthetized (3% Fluothane, ICI plc, Manchester) using a Fluotec apparatus (Cyprane, Keighley, Yorkshire) and the dorsal and flank hair carefully removed using fine clippers (Oster clippers, No. 80, size 40 head, Brookwickward & Co., London W6). The hair cycle is dormant in animals of this age and no further hair growth occurs for approximately 5 days. Epidermal slices were not prepared from animals until at least 48 hr after hair clipping. Animals were killed humanely and the dorsal skin was removed as a single pelt; excess fat was cut away and the remaining skin placed over a cork saddle. Two epidermal slices (18 mm x 80 mm) were cut against the direction of hair growth using a Castroviejo Electrokeratome, 0.3 mm shim (Stortz, St Louis, USA). Each epidermal slice was placed, stratum corneum uppermost, over a rubber 'O' ring. A purpose designed PTFE tube was press-fitted onto the slice and excess tissue was trimmed away. The epidermal slice attached to the PTFE tube was then suspended in physiological saline and maintained at ambient temperature (approximately 20°C; Fig. 1). Each pelt provided six to eight skin slice discs. A minimum of three skin slices was used for the assessment of each chemical.
Physical modification of rat epidermal slice. Rat epidermal slices were modified ex vivo by three different physical procedures of increasing severity. Before cutting individual slices the excised skin pelt was subjected to one of the following procedures: (i) Adhesive tape stripping ( x 5) to remove the superficial cell layers of the stratum corneum. (ii) Slight ( x 1) or severe ( x 5 ) abrasion with medium grade sandpaper. (iii) Immersion in water (50--60°C) for 5 min to remove the epidermis and stratum corneum from the dermis. In vitro application of test chemicals. Each test chemical (0.3ml) was placed onto the stratum
PTFEtube
iiiiii,,
~
ChemicaLs
CLip
DisposabLe
tube~ ,
Rubber'0' ring
Incubation media
T ___..
"--- ~ -
Dermis
.-- .--~.
1 2 3 I I I Scare (cm) Fig. 1, A p p a r a t u s f o r the in vitro e p i d e r m a l slice m o d e l .
10
G. J. A. OLIVERet al.
I
Tritiated water permeability measurements. Tritiated water (1 ml, 106 dpm: Amersham International, plc, Amersham, Bucks.) was added to the epidermal surface of a skin slice mounted in a PTFE tube suspended in isotonic saline (10 ml). Samples (25 #1) of the isotonic saline solution were taken during the steady-state phase of tritiated water penetration (between 3 and 6 hr). A sample (25/zl) of tritiated water was also taken at the beginning and end of the experiment. All aliquots were added to Fisofluor single "LPC" liquid scintillant (Fisons, plc, Loughborough) before counting for tritium in an Intertechnique SL30 scintillation counter. The penetration of tritiated water was calculated as a permeability constant (Kp) as described by Dugard (1983).
RESULTS p H Values o f corrosive and irritant chemicals
Fig. 2. Diagram of the circuit for measuring the electrical resistance of skin slices in vitro, showing capacitance decade (C), resistance decade (R), oscilloscope (O), oscillator (S) and stainless-steel electrodes (A and B).
corneum of the skin slice. After the required contact period the chemical was removed with a jet of warm water (40--45°C) immediately prior (unless otherwise stated) to measuring the electrical resistance across the skin slice. When electrical resistance was measured 20 hr after removal of the test chemical, the epidermal slice was kept in isotonic saline at ambient temperature as described in the previous section. Chemicals were applied as supplied except for (a) solids and waxes when a 50% (w/v) formulation was prepared in either ethanol, acetone or dimethyl formamide (Analar grade solvents, BDH Chemicals) and (b) chemicals 61 and 62 (see Table 1). Electrical resistance measurements. Electrical resistance of skin slices was measured using an a.c. half-bridge apparatus (Fig. 2) which consisted of the following components: a mains wave generator (Levell TG200 DMP, Levell Electronics Ltd, Barnet, Herts.); an oscilloscope with matched X and Y amplifiers (Model MR501, Tektronics, Oregon, USA); a resistance decade box (Model 8000, Time Electronics Ltd, Tonbridge); two capacitance decade boxes (Model Type C100, range 0-100 uf x 1 uf, and Models PC4, range 0-1 uf x 1 x 10-4 uf, J. J. Lloyd Instruments Ltd, Southampton); a colour-coded junction box and two stainless-steel electrodes (8 cm x 1 mm diameter). The wave generator was set to produce a sine wave output of 10 Hz. Amplitude was set at 200 mV and adjusted to give a screen-filling trace on the oscilloscope (100 mV setting). At these settings the potential difference across the epidermal slice was less than 1 V rms. Immediately before measurement of electrical resistance isotonic saline (3 ml) was added to the stratum corneum surface of the skin. Air bubbles were dislodged by slight tapping and the stainless-steel electrodes were placed to complete a circuit across the epidermal slice. Balance in the resistance and capacitance of the external circuit and the epidermal slice was indicated by a 45 ° trace on the oscilloscope.
The reactive pH value for each corrosive and irritant chemical is given in Table 1. The chemicals have been divided into three broad categories, depending on pH, and the distribution of irritant and corrosive chemicals within these categories is shown in Fig. 3. Both irritant and corrosive chemicals were evident at high and low pH (pH ~<2 or >/11.5) but the majority of chemicals were within the pH range 2.1-11.4. The electrical resistance o f intact rat epidermal skin slices
Change in electrical resistance was selected as a rapid and simple measure of stratum corneum integrity. The electrical resistance of untreated rat epidermal skin slices or skin discs ranged from 6-20 kD.skin disc (Fig. 4). Resistance values were compared to 3H20 permeability rates (Kp values) which have been used to assess skin barrier integrity (Dugard, 1983). Resistance values of > 6 k ~ . d i s c corresponded to a Kp 3H20 of 2-4 x 10-3cm/hr which has been reported for Alderley Park strain Wistar rats with an intact stratum corneum (Walker et al. 1983). In all subsequent experiments, skin discs with electrical resistance values greater than 10 kf]. skin disc were used. Electrical resistance values above 10 k ~ corresponded to Kp 3H20 values of less than 3 x 10 -3 cm/hr and in the majority of cases less than 2 x 10 -3 cm/hr (Fig. 4). 100 r-
pH 2.1 - 11.4
pH<~2
pH ~ 1 1 . 5
80
60 o
40 O
20-
0 I
Tr
m
xi
I
II
rrr X i
I
Tr
I£1
Xi
Fig. 3. The distribution of irritant (Xi) and corrosive (packing groups I, II or III) validation chemicals according to their pH values.
In vitro model for testing skin corrosives 10 -1
g
O
.I0 o E
10-2
\
0
\\ •:
"0
\
0
\,'.;" /
/
•e l • /
L. 0 .J
•e•
\\X /
•
10-3
I 0 2
xl I • 6
ELectricat
10
~o
-~ 2 • TM -... ;.b • ,-
I | IPI• 14 • 18
resistance
20
(kg,.disc)
Fig. 4. Relationship between measurements of transcutaneous electrical resistance and tritiated water permeability of young-rat skin in vitro. Measurements were made on intact skin ( 0 ) and on skin physically modified in vitro by slight abrasion (O), tape-stripping (1), severe abrasion (A) and heat separation (V').
The selection o f an electrical resistance value as a threshold indicating impaired stratum corneum
An electrical resistance threshold value that would indicate skin with a reduced barrier function was determined in experiments in which skin discs were modified by either physical or chemical treatment. First, skin discs were modified e x vivo by a number of physical procedures designed to remove the stratum corneum to varying degrees (Fig. 4). The milder forms of treatment (tape-stripping, single abrasion) reduced electrical resistance values to between 1 and 5-6kf~.skin disc whereas more severe treatment (repeated abrasion and heat-treatment) resulted in values below 1.5 k ~ . s k i n disc. The reduction in electrical resistance with progressive removal of stratum corneum correlated with an increase in Kp 3H20 (Fig. 4). Secondly, inorganic acids were applied in vitro to skin discs at varying concentrations for specified contact times. The results (Fig. 5) indicate that electrical resistance is lowered in vitro to approximately 4 kfl. skin disc and below, only under circumstances that produce a corrosive lesion (point or overt necrosis) in vivo. On the basis of the above results, an electrical resistance value of 4 kfl.skin disc (3.2 k ~ . c m ~) was selected as a threshold value for a positive effect in this in vitro model, i.e. chemicals reducing electrical resistance below 4 kf2.skin disc in vitro would be predicted to induce a corrosive lesion in vivo using conventional animal protocols. The effect o f corrosive and irritant chemicals on the electrical resistance o f skin tissue
The electrical resistance values of skin discs after in vitro exposure to 49 corrosive and 19 irritant chemi-
11
cals for contact periods of 1, 4 and 24 hr are shown in Table 2. The total number of corrosive or irritant chemicals that produced a mean resistance value greater or less than 4 kfl. skin disc at each contact time are shown in Fig. 6. Corrosive chemicals are further identified as group I, II or III. In describing the validation of the in vitro corrosivity test, the approach of Cooper et al. (1979) has been adopted. Thus the proportion of corrosive chemicals that are positive is termed "sensitivity"; the proportion of irritant chemicals that are negative is termed "specificity". After a 1-hr exposure in vitro 29% of corrosive chemicals, but no irritant chemicals, were positive. These positive chemicals included corrosive agents from all three packing groups. Following a 4-hr exposure in vitro, two irritants (chemicals 47 and 48) were positive, thus reducing specificity to 90%. However, sensitivity was increased to 65% and with the exception of chemical 32, with a mean resistance value of 4.0 k ~ . disc, all group I corrosive chemicals were positive after this contact period. After 24 hr exposure in vitro, sensitivity was increased to 92%. All group I and II corrosive chemicals were positive with four group III chemicals remaining negative (chemicals 40, 43, 54 and 61). Five irritant chemicals were positive (chemicals 19, 23, 24, 47 and 48) resulting in a specificity of 74%. In vitro exposure conditions were further modified in an attempt to maximize sensitivity without loss of specificity. Following a contact period of 4 hr chemicals were washed off the skin disc but electrical resistance was measured 20 hr later, 24 hr after the initial application. Excluded from this investigation were those chemicals that markedly reduced resistance (to below 1.5 k~.disc) by 4 hr or those that failed to reduce resistance even after 24 hr continuous contact. Results are shown in Table 2 and Fig. 6. Compared with the previous 4-hr exposure results, under these test conditions, where resistance measurement was delayed, specificity was maintained at 90% (chemicals 19 and 24 [Table 2] remaining
0.15
/
._e
.
1.oo
'~
1.5o
2
U.I
lO
In 0.25
In 0.5 MoLarity
I 1
I 2
I 4
of acid
Fig. 5. Electrical resistance of rat epidermal slices in vitro following topical application of sulphuric acid for i hr (O) or 4 hr (A) or of hydrochloric acid for 4 hr (C)). The doses in viva at which no abnormality was detected (n) and at which signs of point necrosis ( 1 ) or necrosis (*) were observed are indicated.
G. J. A. OLIVER et al.
12
positive) but sensitivity was increased to 80% (chemicals 11, 18, 28, 38, 41 and 66 comprising false negatives together with those four corrosive agents that were negative even after 24-hr continuous contact with skin slices).
Specificity and sensitivity and the threshold for a positive effect The in vitro corrosivity test was validated initially on the basis of an electrical resistance threshold for a positive effect of 4 kfl. skin disc. The reasons for the selection of this particular value are given above. In addition, the validation data on the 68 chemicals for each of the four chemical exposure and electrical resistance measurement conditions has been exam-
ined to determine whether the selected threshold value was optimal in relation to both the specificity and sensitivity of the test. The variation in test sensitivity and test specificity with respect to electrical resistance between the values 0 and 10 kl). disc and at intervals of 0.2 kll.disc is shown in Fig. 7. The data demonstrate that to achieve a combination of the highest sensitivity and specificity than 4 kl). disc is the optimal, if empirical, resistance threshold value. DISCUSSION
The principal objective of this work was to develop an in vitro model to identify chemicals that would be classified as corrosive to skin in conventional in vivo
Table 2. The skin electrical resistance values after various periods of contact with the 68 chemicals used to validate the rat epidermal slice model for in vitro identification of corrosive potential Electrical resistance value (kfl.skin disc) after chemical contact in vitro for Chemical no.
1 hr*
l 4 5 6 l0 14 15 16 31 32 34
2.5-+0.7(+) 1.4_+ 0.1 (+) 7.0_+2.7(-) 1.5-+0.3(+) 4.0+_0.4(-) 13.3 -+ 4.6 (--) 4.8_+1.9(-) 1.6_+ 0.3 (+) 6.3_+2.8(-) 19.8_+2.1(-) 9.2_+0.1(-)
Packing group I 1.0_+ 0.1 (+) 0.9_+0.1(+) 1.4-+ 0.1 (+) 0.9-+ 0.1 (+) 1.8 -+ 0.4 (+) 1.3 -+ 0.3 (+) 1.6_+0.3(+) 1.5 -+ 0.1 (+) 1.8-+0.8(+) 0.8_+0.1(+) 2.2-+0.5(+) 1.9 -+ 0.3 (+) 3.5_+0.9(+) 2.0_+0.3(+) 1.5 -+ 0.3 (+) 1.5 -+ 0.3 (+) 1.1 -+ 0.1 (+) 0.8_+0.1(+) 4.0_+0.4(-) 1.0_+ 0.1 (+) 1.4_+0.3(+) 0.9_+0.6(+)
ND(+) ND(+) 2.7-+0.3(+) 2.4_+ 1.3 (+) 2.1_+0.3(+) 1.0_+0.4(+) 0.8 -+ 0.1 (+) ND(+) ND(+) 2.6_+0.1(+) ND(+)
2 7 I1 12 13 18 25 27 29 30 35 36 42 45 51 68
2.5_+1.1(+) 15.1 _+4.1 ( - ) 6.7_+2.2(-) 11.5_+9.2(-) 5.7_+2.2(-) 6.7_+0.6(-) 3.2_+0.6(+) 16.0+_2.7(-) 9.7_+ 1.5(-) 4.3_+1.1(-) 7.0_+3.5(-) 1.0 -+ 0.0 (+) 5.1_+1.6(-) 0.4 _+0.1 (+) 2.7_+0.2(+) 1.1 _+0.3(+)
Packing group II 1.0_+0.3(+) 0.9+0.1(+) 3.5_+0.3(+) 3.9_+0.7(+) 5.1 -+ 1.1 ( - ) 1.1 -+ 0.1 (+) 4.4_+0.6(-) 1.9_+0.4(+) 3.0_+0.6(+) 2.4+0.4(+) 4.1+0.1(-) 1.8_+0.3(+) 1.8_+0.1(+) 1.1-+0.3(+) 3.2-+0.3(+) 1.3_+1.1(+) 2.9_+ 1.8(+) 1.5_+0.3(+) 1.9_+1.4(+) 0.9_+0.3(+) 2.4_+ 1.1 (+) 1.0_+0.3(+) 1.0 -+ 0.0 (+) 0.9_+0.1(+) 3.8_+0.3(+) 1.0_+ 0.1 (+) ND (+) ND (+) 0.7_+0.1(+) ND(+) 0.6_+0.2(+) ND(+)
3.4_+1.3(+) ND(+) 5.0_+2.4(-) 2.3_+0.1(+) 1.4_+0.1(+) 5.4_+1.5(-) 2.3-+0.5(+) 2.8_+2.1(+) 3.6_+2.6(+) 3.4_+ 1.8 (+) 2.2+1.2(+) ND(+) 3.7_+0.8(+) ND (+) ND(+) ND(+)
3 22 26 28 33 37 38 39 41 46 49 50 52 56 63 64 65 66
12.7 _ 6.1 (--) 12.3 -+ 5.0 ( - ) 8.4_+0.6(-) 9.2_+2.2(-) 14.7_+ 3.5 ( - ) 12.7 -+ 4.0 ( - ) 8.0_+3.0(-) 1.7_+ 0.5 (+) 9.0_+4.0(-) 1.1 -+0.3(+) 4.0 -+ 1.0 ( - ) 6.3_+0.6(-) 3.6_+ 1.7(+) 6.3 -+ 1.1 ( - ) 14.0-+5.9(-) 1.6 -+ 0.3 (+) 9.0-+0.0(-) 8.7 -+ 1.5 ( - )
Packing group III 5.9_+ 1.0 ( - ) 3.4_+0.6(+) 6.1 __. 1.3(-) 1.6_ 0.3 (+) 2.4_+0.4(+) 1.4_+ 1.1 (+) 5.4_+0.5(-) 2.0_+0.5(+) 3.4_+ l.l (+) 1.1 -+ 1.1 (+) 9.5_+8.9(-) 1.0_+0.3(+) 8.0_+0.3(-) 2.8_+0.5(+) 1.3 -+ 0.3 (+) 1.6_+0.5(+) 6.2_+2.4(-) 2.2-+0.3(+) 0.9 -+ 0.1 (+) ND(+) 2.3-+0.9(+) 1.1 +_ 1.9(+) 3.8_+0.8(+) 1.0_+ 1.7 (+) l.l _+0.3(+) ND(+) 5.9_+1.0(-) 2.3_+0.8(+) 17.0_+4.1(-) 2.3_+0.3(+) 0.8_+0.1(+) 0.8-+0.3(+) 3.0_+ 1.0 (+) 1.1 -+ 0.0 (+) 5.9_+2.7(-) 1.9-+ l.l (+)
3.9_+0.4(+) 2.2_+ l.l (+) 2.7_+ 1.2 (+) 5.3 _+ 1.2 (--) 2.4_+0.2(+) 3.7_+ 1.1 (+) 10.6 -+ 2.3 ( - ) ND(+) 10.4 -+4.3 ( - ) ND(+) ND(+) ND(+) ND(+) 1.3_+0.1 (+) 3.1_+0.9(+) ND(+) 2.8-+ 1.0 (+) 4.5 -+ 1.2 ( - )
40 43 54 61
13.0_+ 2.0 ( - ) 17.0-+ 5.1 ( - ) ND(-) 20.3 -+ l l . 0 ( - )
4 hr*
10.0_+3.0(-) 16.0 -+ 5.9 ( - ) ND(--) 13.0+_8.5(-)
24 hr*
5.4-+ 1.9 ( - ) 14.2 -+ 3.0 ( - ) 16.9 -+ 3.0 ( - ) 7.7-+2.2(-)
4 hr (resistance measured at 24 hr)*
ND(-) ND(-) ND(-) ND(-) [contd]
13
In vitro model for testing skin corrosives
Table 2--contd Electrical resistance value (k~.skin disc) after chemical contact in vitro for Chemical no.
I hr*
4 hr*
24 hr*
4 hr (resistance measured at 24 hr)*
19 23 24 47 48
8.4_+2.2(-) 13.4_+2.5(-) 11.8-+2.8(-) 4.6-+ 1.3(-) 7.3+2.5(-)
4.9+0.1(-) 15.1+3.5(-) 4.8-+ 1.4(-) 3.5+ 1.5(+) 2.8+0.3(+)
2.7+0.3(+) 2.5+0.1(+) 1.1 +0.1 (+) 1.1 _+0.6(+) 1.6+0.1(+)
3.1+0.5(+) 6.6+0.6(-) 2.9+ 1.8(+) 7.6+ 1.4(-) 7.5+0.2(-)
8 9 17 20
>20(-) 10.8_+2.3(-) 12.7_+6.6(-) 11.0+7.2(-) 10.8+5.3(-) 15.0+2.7(-) ND(-) 6.7_+2.9(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-)
18.0_+3.5(-) 13.7_+9.9(-) >20(-) 13.3_+1.5(-) 8.0+0.8(-) 11.0+1.5(-) ND(-) 8.4+3.2(-) ND(-) 18.9_+5.1(-) 7.2_+3.2(--) 19.0_+2.3(--) 15.7_+2.5(-) ND(-)
12.7_+2.5(-) 5.3+1.8(-) 10.8+3.0(-) 13.3+1.5(-) 8.5+0.4(-) 12.0+0.6(-) 14.1 _+7.0(-) 5.1_+1.8(-) 13.0+4.7(-) 15.1+2.5(--) 5.1 +0.9(--) 14.1 +2.5(--) 17.1 + 3 . 0 ( - ) 4.9+ 1.1 ( - )
ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(-) ND(--) ND(--) ND(-) ND(-) ND(-) ND(-)
Irritant ehemlenls
21
44 53 55 57 58 59 60 62 67
ND ~ Not determined (but chemicals were categorized as + or - on the basis of their effect on electrical resistance under less stringent or more stringent test conditions respectively) *The (+) or ( - ) refers to the in vitro categorization of the chemical based on an electrical resistance threshold of 4 ki'). skin disc (see Results). Values are means + SD for three measurements.
tests. In this paper we describe the design and use of such an in vitro model and its validation with a large number of chemicals. The chemicals were selected to represent a range of typical industrial materials and formulations of different physico-chemical properties (physical state and pH). All of these chemicals induced skin lesions in animal tests and those categorized as skin corrosive displayed different reactivity, as
indicated by the relative contact period in vivo required to produce a corrosive lesion. In general, for those chemicals for which a pH could be determined, pH did not provide a good indication of corrosive potential. A number of irritant chemicals (4 of 17) were of high (~, 11.5) or low ( 4 2) pH and, conversely, most corrosive chemicals (29 of 46) had a pH of between 2.1 and 11.4. The pH
50-
4 hr (resistance m e a s u r e d a t 2 4 hr) 40
1 hr
4hr
40-
z
24 hr
40
I 5O
30-
20
20-
lo
10-
.3
3O
n
E 2O
"6 "m"
10
< 4 k,O,.disc [-~ )
z
Corrosives
Irritants
Corrosives
i Irritants
'°t I I
lO
20
I
1 Corrosives
~ 2 0
I
I Corrosives
Irritants
'°t 20
I
Irritants
lI
> 4 k~.disc (-)
30
40
Fig. 6. The proportion of the 68 validation chemicals that were positive or negative in the in vitro rat epidermal slice technique based on the electrical resistance threshold of kfl. skin disc after chemical contact in vitro for 1, 4 or 24 hr or after contact for 4 hr with resistance measured at 24 hr. The skin corrosive chemicals that were tested were of groups I, II or III as defined by the United Nations Economic and Social Council (1977).
G. J. A. OUVERet al.
14 loo
9o~
--
7.._..=.._.
,__,-,---?.
80 t-
.,'"
70}6 0 ~-
, ,"
,J
50 I-
.- ....... ......
,'"........ ....'., ~ -'"",,J
..:
Corrosive
Irritant
L-L---5~
r ""
Physico- chemical
,._,-'
1
Keratin tysis
JTissue destruction/irreversibLe change [ < 4 hr
I 0
I -.-~'~"I 1
I 2
I 3
I 4
Resistance
I 5
I 6
I 7
I 8
I 9
I 10
BioLogicaL InfLammatory
irritation
Oedemo/erytherna
threshol.d (k,(),. disc)
T i s s u e death necrosis >4hr
I 100 90
-- - S e v e r i t y
~---
601
~,
\
'..
,~. . . .
40
~ ~o mI~. 20 tO 0
-- -
Fig. 8. Proposed mechanism of action for corrosive and irritant chemicals.
eo I
*~ ~oI
of in vivo r e s p o n s e -
I
I
I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
9
10
Resistance threshold (k,~,. disc)
Fig. 7. The variation in test sensitivity and test specificity with respect to electrical resistance threshold for the 49 corrosive and 19 irritant chemicals. Chemical contact was for 1 ( ), 4 (----) or 24 ( - - - ) or for 4 hr with resistance measured at 24 hr (..... ).
categories selected for this analysis coincide with those quoted in regulatory guidelines as a guide to chemical classification without recourse to further testing (OECD, 1981). Clearly, from these data on this particular selection of chemicals, consideration of pH alone as a guide to skin corrosive potential would fail to identify a high number of corrosive chemicals and falsely indict some irritant chemicals. The choice of in vitro model and index of effect was influenced by the conclusions from a retrospective survey (G. J. A. Oliver & M. A. Pemberton, unpublished data, 1984) of the onset, duration and nature of the macroscopic skin lesions produced in vivo by irritant and corrosive chemicals in animal experiments conducted to current protocols (OECD, 1981). It was apparent that in general for the majority of corrosive chemicals, lesions consistent with a corrosive effect were evident within approximately 4 hr of contact and either accompanied or preceded any inflammatory changes (erythema and/or oedema). In a few instances corrosive lesions were noted after the maximal inflammatory response, at approximately 24 hr or longer after the initial contact. The irritant chemicals induced mainly transient inflammatory reactions. In addition, some irritants caused direct, reversible damage to the outermost skin layers (keratin lysis) which, macroscopically, imparted a "glossy" reflection to the skin surface. It was concluded from this appraisal that, in general, a direct physico-chemical interaction with skin tissue contributes significantly to the development of a corrosive but not an irritant lesion. It was also concluded from the above analysis that there are occasional exceptions to this hypothesis. Thus for a minority of corrosive chemicals the inflammatory
response is predominant and a corrosive lesion would appear to result as the sequel of a biochemically/ pharmacologically mediated tissue necrosis. Conversely, some irritant chemicals directly disrupt and/or remove the outermost layers of skin tissue. The major factors contributing to corrosive and irritant skin responses are summarized in Fig. 8. The apparent property of corrosive chemicals to interact directly with and to lyse the skin in vivo was exploited in designing the in vitro model both in the selection of the tissue and the best measure of effect. It was considered imperative to use skin epidermal slices and thereby include the normal physiological and anatomical structures that would initially be exposed and affected in vivo, particularly the outermost layer, the stratum corneum. The direct cytolytic action of chemicals on skin tissue was examined by measuring their effect on the functional integrity of the stratum corneum. The characteristics of the stratum corneum as a penetration barrier have been studied by various methods, e.g. by determination of rates of chemical absorption (Idson, 1975; Katz & Poulsen, 1971), measurement of transepidermal water loss (Malten et al. 1968; Scott et al. 1982) and measurement of change in electrical status (Ando et al. 1983; Blank & Finesinger, 1964; Dugard & Scheuplein, 1973). We chose to measure the electrical properties of skin slices/discs since this provided the most rapid and simple technique. Water permeability measurements have been used in this laboratory to define rat stratum corneum integrity (Dugard et al. 1984). Water permeability and electrical conductance are proportional and differ only in the rates of permeation of water and ions and in the technical ease of measurement. However, due to the low conductivity of intact skin, the range of resistance values (Fig. 4) and the relative inaccuracy of measurements below 1 kfl/skin disc, we have recorded electrical resistance, rather than conductance. The a.c. half-bridge apparatus measures electrical impedance which has resistive and the relative inaccuracy of measurements below I kf/. skin disc, we have recorded electrical resistance, the resistive component of the skin dominates the negligible capacitive component. In addition to the above major criteria, the epidermal slice technique satisfies a number of further requirements which we consider to be important for
In vitro model for testing skin corrosives
15
the adoption of a practical in vitro toxicity test following 4 hr contact resulted in a higher detection system. The epidermal slice technique (Fig. 1) pro- rate for corrosives (increased sensitivity) without any vides a simple rapid, robust and reproducible means loss of specificity when compared with measurement of screening chemicals on a regular and routine basis. at the end of a. 4-hr contact period. Overall, using a 24-hr contact period or a 4-hr Tissue slices of consistent quality can be quickly prepared. The technique represents an economic use contact period coupled to a 24-hr resistance of animal tissue since the pelt from a single rat measurement, the epidermal slice technique can idenprovides sufficient skin discs to test two or three tify corrosive chemicals in vitro with both high separate chemicals. In addition many factors that specificity and sensitivity. Either set of conditions may influence the final response in vivo may be could be used depending on individual requirements emulated in vitro. In particular, chemicals of different and the relative importance of the likely occurrence physical states (liquids, waxes, gels, solids, powders) of false negative or false positive results. It must be can be applied directly to the normal outer skin remembered that the frequency of occurrence of false surface. Variable contact periods, effective removal of positive and false negative results will depend on the test compound and a degree of occlusion can all be prevalence of corrosive chemicals in a batch of test chemicals as well as on the sensitivity and specificity achieved using this in vitro model. Using water permeability measurement as a refer- of the test model (Cooper et al. 1979). Thus the ence technique, decreased skin resistance was shown validation of an in vitro test model can be described to correlate with a reduction in the skin's barrier in terms of sensitivity and specificity but the practical properties (Fig. 4). From these and additional experi- utility of the model (its predictive value or its accuments (Fig. 5) a resistance value of 4 kf~. skin disc racy) when used as a screen is also influenced by the (3.2 kfl.cm 2) was considered to indicate significantly expected proportion of corrosive chemicals among compromised stratum corneum function. Therefore, those being assessed. In general (a) the higher the a resistance value of 4 k[2. skin disc was adopted as sensitivity of the test then the greater the certainty an arbitrary empirical threshold to corrosive action in that a negative result is true, i.e. the chemical will not vitro. The selection of this value was further vin- be corrosive in vivo; (b) the higher the specificity then dicated by retrospective analysis of the data for the the greater the certainty that a positive result is true, validation chemicals from each of the four i.e. the chemical will be corrosive in vivo; (c) the lower exposure/resistance measurement regimes (see below the prevalence of corrosive chemicals, the higher will be the proportion of false negatives and false and Fig. 7). Using this principle, the validation of the technique positives with decreasing sensitivity and specificity. The detection of false negatives and positives itself with 68 chemicals showed the sensitivity and specificity of the in vitro corrosivity model to be contributes to the definition and understanding of the influenced by both the contact time and the elapsed proposed mode of action of corrosive and irritant time prior to resistance measurement following re- chemicals (Fig. 8). Consideration of the noncorrelates allows a more rationalized appraisal of the moval of the chemical (Fig. 6). Ideally the specificity and sensitivity of an in vitro expected and apparent sensitivity and specificity of screen should be as high as possible with a minimum the model. From the in vivo evidence the four false of false negatives or false positives. This goal can be negatives (24 hr contact) represent chemicals in which related to chemicals of general structure or to specific the inflammatory response is dominant, the corrosive groups or analogues depending on requirements "lesion is relatively slow to develop and probably results from a cascade of biological events leading to (Cooper et al. 1979). When this desired optimal performance for an in overt tissue necrosis (Fig. 7; G. J. A. Oliver & M. A. vitro system is apposed to skin toxicity assessment in Pemberton, unpublished data, 1984). These particular chemicals did not appear to interact directly with vivo, it is pertinent to remember that the evaluation of a chemical's toxic potential in vivo is not absolute the stratum corneum in vivo and therefore would not but is based on subjective assessment and individual be expected to reduce electrical resistance below an opinion of a macroscopic event(s). This consideration adopted threshold value in vitro in the epidermal slice is relevant to the interpretation of the predictive system. The four chemicals are either individual or capacity of this in vitro system since in vivo it may formulated organic chemicals (numbers 40, 43, 54 be difficult to distinguish unequivocally between and 61). Reference to the in vivo response profile of the false "severely irritant" and "corrosive" lesions. Exposure periods of 1 and 4 hr resulted in complete positive chemicals (24 hr contact) also provides a specificity and high specificity respectively but low possible explanation for their in vitro activity. Three of the five chemicals are surfactant materials (chemisensitivity (Fig. 6). Sensitivity was increased to 92% after 24 hr continuous contact in vitro at the expense cals 19, 23 and 24) and two are silicates (chemicals 47 of lowering of specificity to 74%. The procedure in and 48) of high pH (Table 2). For each of these which resistance measurement was delayed following chemicals there was in vivo evidence of keratin lysis chemical removal after a 4-hr exposure was designed (removal of the outer layers of the stratum corneum) to emulate in vivo conditions of contact and thereby in the absence of any indications of a corrosive lesion increase sensitivity without loss of specificity. In vivo (G. J. A. Oliver & M. A. Pemberton, unpublished removal of compound after a specified time may be data 1984). The surface active properties of the incomplete and residual material, within or on the surfactants and/or the high pH of these materials is surface of the skin, may continue to contribute to the consistent with this observation (Spruit, 1970; Spruit final toxic response. This would appear to be upheld & Malten, 1968; Wood & Bettley, 1971) of a reduction in electrical resistance in vitro. in vitro since delaying the resistance measurement
16
G. J. A. OLIVERet al.
For corrosive chemicals the contact time in vitro required to affect the stratum corneum does not appear to correlate with the nominal exposure time in vivo required to induce a corrosive lesion. Thus a proportion of group I and II corrosive agents remain negative in vitro after 1 and 4 hr contact respectively (Fig. 6). The corresponding in vivo exposure times are 3 min and 1 hr for these two classification groups. As discussed earlier this apparent difference may simply reflect a continuing action in vivo from chemical remaining in the tissue after the washing-off process. This view is reinforced by the change in profile of negative/positive results when resistance measurement is delayed after contact in vitro (Fig. 6). Conversely, a proportion of group III corrosive agents are positive in vitro after a l-hr contact period. For these chemicals, their relatively rapid effect on stratum corneum does not lead to a corrosive lesion in vivo unless exposure is extended from 1 to 4 hr. Overall for structurally diverse chemicals, the in vitro contact period required to produce a positive result cannot be used to indicate corrosive potency as illustrated by packing group and as currently defined in in vivo test protocols. In summary, the epidermal slice technique is capable of distinguishing skin corrosive chemicals from other less damaging chemicals. The use of either a 24-hr contact time or a 4-hr contact time coupled with a 24-hr resistance measurement results in both a high sensitivity (92%) and high specificity (74%). On the basis of these data it is proposed that this in vitro model has utility as a prescreen for the identification of skin corrosive potential. Depending on the particular circumstances that will decide the predictive value (see earlier discussion), this prescreen will allow decisions to be made concerning: the need to progress to an animal test, and the design of a confirmatory animal test (contact time, numbers to be used, frequency of observation, etc.) if this is considered necessary to clarify a possible false positive or negative result. Besides the predictive accuracy of the test, other factors, e.g. regulatory considerations, may influence the wider application of results from an in vitro screen. However, a more ready acceptance by regulatory authorities of the results of tests that identify severe responses is logical and feasible. Current guidelines already state that chemicals may be considered corrosive if of high or low pH and without recourse to animal testing "if the result can be predicted" (EEC, 1983; OECD, 1981). In addition to its use as a prescreen, the in vitro corrosivity test provides objective and quantifiable data. This is considered to be a major advantage when compared to subjectively assessed animal tests (Weil & Scala, 1972). The in vitro approach also provides additional qualitative information concerning the action of the chemicals concerned. Finally, the epidermal skin slice model has considerable potential for the more direct assessment of h u m a n hazard since, with suitable modifications, h u m a n skin instead of rat skin can be used. Preliminary electrical resistance experiments have shown that human skin may respond differently to animal skin to some corrosive agents (Oliver & Pemberton, 1986). The results of such experiments may have
considerable significance in the accurate assessment of h u m a n hazard and the appropriate classification of chemicals. Acknowledgements--We would like to thank Dr S. J. Jack-
son, Central Toxicology Laboratory, for organizing the supply of compounds from Akzo Chemic and to thank Akzo Chemic b.v. (Netherlands) and Akzo Chemic (UK) Ltd for supplying chemicals and permitting their use in this work. We are grateful to Dr I. F. H. Purchase for his constructive suggestions and discussion and to Mr I. Pate and Miss M. Boylett for their statistical appraisal of the data. REFERENCES
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