FdChem.Toxic.Vol.22, no. 2, pp. 157 161, 1984 0278-6915/84$3.00+0.00

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# Review Section

PREDICTION OF OCULAR IRRITANCY POTENTIAL FROM

DERMAL IRRITATION TEST RESULTS

S. J. 

### WILLIAMS Central Research and Development Department, E.I. du Pont de Nemours & Company, Haskell Laboratory for Toxicology and Industrial Medicine, Elkton Road, P.O. Box 50, Newark, DE 19711, USA (Received 12April 1983) Summary In a surveyofthetestfilesofHaskell Laboratory, itwas foundthat ofthematerialsthathad been tested on rabbit skin for primary irritation or corrosivity, 60 were severeirritants or corrosive on the skin and had also been tested for ocular irritancy. Of these 60 materials only 39 were severe eye irritants, while 15weremildirritantsor non-irritantsand the remainderwereconsideredto bemoderate irritants. Thus, to suggest that materials are eye irritants solely on the basis of their skin-irritation properties may be misleading, even when severe dermal irritants are considered.



### Introduction
The use ofanimals, particularly rabbits, in the evaluation of chemical and cosmetic safety has been criticized in the past few years. Criticisms have been based upon a number of factors: the lack of reproducibility of eye and skin irritation test results between and within laboratories (Weil& Scala, 1971), the numbers of animals used in such tests, and the testing ofmaterials that elicit extreme responses such
as pain or death. The replacement of animals by in vitro or other predictive systems of safety evaluation for possible human exposure is not yet possible. However, it may be possible to reduce the numbers of animals used in such tests and to decrease the likelihood of causing
severe reactions in test animals (Griffith, Nixon, Bruce et al. 1980; Williams, Graepel & Kennedy, 1982). Alterations of test design, predictive screens based on chemical behaviour in other test systems, or evaluation of physico-chemical properties of test materials may prove useful to this end. One suggested method by which the numbers of rabbits used in ocular irritancy evaluation might be reduced is to pre-test material for primary skin irritation (Interagency Regulatory Liaison Group, 1981) or other properties (e.g. pH, partition coefficients), so that further testing might not be required. This recommendation, which seems reasonable, has been restated recently (Murphy, Osterberg, Seabaugh & Bierbower, 1982) but has never been systematically tested. The purpose of this report is to test the hypothesis that materials found to cause severe primary skin irritation can reliably be predicted to cause a similar degree of response in the eye. This was done using

Abbreviations: CFR = Code of Federal Regulations; FHSA = Federal Hazardous Substances Act. historical data for materials shown in our laboratory to be either severe primary irritants or corrosive to the skin.



### Method
Literature search The test files of Haskell Laboratory, dated from 1968 to the present, were searched for all materials that had been tested at the Laboratory for either primary skin irritation or skin corrosion. This listwas condensed to include only those materials that were found to be either severe primary skin irritants or corrosive to the skin and has also been tested for ocular irritation. For each of the final 60 materials that were selected, the test reports or original data were used to evaluate the relative responses of each tissue. In making thesecomparisons, the ocular responses were categorized as follows: Class 1--Severe: severe injury of any ocular tissue at any time, or any degree ofdamage thatpersisted for 21 days or longer; Class2--Moderate: mild to moderate injury ofany ocular tissue that resolved in 5-14 days; Class 3 Mild or non-irritant: mild injury of any ocular tissue that resolved in 3 days or less, or no observable change. Skin-testingprocedures Unless otherwise indicated, the following procedures, which are used internallyat Haskell Laboratory for material evaluation, were used to evaluate primary skin irritation. Six male albino rabbits (2-3 kg) were clipped free of hair on the trunk and lateral areas and placed in stocks. Doses of 0.5ml of undiluted test material (0.5 g of moistened solids)
were applied to intact skin under 1.5 × 1.5in. 12-ply
157158 S. J. WILLIAMS

gauze patches. The patches were secured with adhesive tape and occluded with rubber sheeting wrapped loosely around the trunk. After 24hr, the rabbits
were taken out of the stocks, the patches were removed and the skin reactions were scored for erythema and oedema (16 CFR 1500.41: 1973) immediately and at 48 (sometimes 72) hr after the initial application. A composite score was not calculated. When the test systems described under the Federal Hazardous Substances Act (16 CFR 1500.41: 1973)
or the Department ofTransport Skin Corrosion Test (49 CFR 173.240: 1975) were used, these are indicated (Table 1). Only the results obtained on intact skin have been used for the comparisons. 

### Eye-testing procedures Unless otherwise indicated, the procedures used to
assess ocular irritation were those used routinely at Haskell for material evaluation. A dose of 0.1 ml of liquid or 0.1 g of solid material was instilled into the lower conjunctival sac of the right eye of each of two albino rabbits (2-2.5 kg, free of ocular lesions). Twenty seconds after contact, the treated eye of one rabbit was washed for 1 min with a constant stream oftap-water. Test rabbits were observed both grossly and with a hand slit-lamp at 1 and 4 hr after dosing. On days 1, 2 and 3, gross observations and biomicroscopic examinations were made followed by hand slit-lamp examination of the eye stained with fluorescein. The observations were graded using a modified Draize system (Draize, 1959), but a composite score was not calculated. Assessment of injury
was based largely upon descriptive notation and duration ofeffect. Where noted in Table 1, the FHSA procedure for eye irritation evaluation was used (16 CFR 1500.42: 1973). Only the test results obtained in

Table 1. Ocular responsesto materialsfound to be severe primary skinirritants or to becorrosive on the skin Eye

Material Skin response Response Irritancy class* 1. Triphenylborane DOTt--corrosive

2. Triphenylboron NaOH complex Necrotic erythema; severe oedema 3. Car wash,liquid FHSA:~(+)--severeerythema and oedema 4. Chrome polish andcleaner FHSA$ (+)--severeerythema; moderate oedema 5. Cyanamide,sodiumsalt Necrotic erythema; severe oedema 6. p-Aminodicyclohexylmethane,acetate Necrotic erythema; moderate oedema

7. 1,4-Dimercapto-2,3-butanediol Necrotic erythema;mild oedema 8. Hand cleaner FHSA$ (+) necrotic erythema; moderate to severe oedema 9. 1,2-Diaminocyclohexanol/ 1,6-hexanediamine DOTt~orrosive

10. MixtureA FHSA:~(+)--necrotic erythema; mildoedema 11. 1,l-Dichloroethene polymerwith Necrotic erythema; moderate 1,1-difluoroethene and oedema 1-chloro-l-fluoroethene 12. Mixture B Severeto necrotic erythema; mild oedema(85~ solution) 13. Mixture C Severe, necrotic erythema; severeoedema 14. Mixture D Blanching, necrotic erythema; moderate oedema 15. Mixture E Severeerythemaand oedema

16. MixtureF Severe, necrotic erythema; severeoedema 17. MixtureG Necrotic erytbema; severe oedema

18. Mixture H Severeerythemaand oedema
19. Fixer Moderate necrotic erythema; moderate oedema 20. MixtureI Moderate to severeerythema and oedema Severe,permanentcorneal opacity with vascularization; necrotic Corrosive; permanentcorneal opacity
FHSA§ (+~severe corneal opacity; eversion ofeyelids FHSA§ (+) moderatecorneal opacity at 72hr Severe, generalized, penetrating corneal opacity FHSA§ (+) severe, irreversible corneal and conjunctival effects; corrosive Mild corneal opacity, reversible in 3 days; eyenormalin 14days FHSA§ ( )

Generalized, permanent,penetrating corneal opacity Severe, permanentcorneal opacity
Severe,permanentcorneal opacity

Moderate, permanentcorneal opacity l (6% solution) Permanent,penetrating, moderate 1 corneal opacity Severe,permanentcorneal, iritic and 1 conjunctival effects Minimal,transientconjunctival 3 redness Mild, transientconjunctivitis 3
Mild, generalized corneal opacity; 1 severeconjuntivitis; normalin 14 days Persistent, moderatecorneal opacity; moderate to severeconjunctivitis Severegeneralized corneal opacity; perforation with haemorrhage Slight, reversible after 2 days 1

l


### 3 [contd]Prediction of ocular irritation 159

Table l--continued

Material Skin response Eye Irritancy Response class*

21. Mixture J

22. Mixture K

23. Mixture L

24. N,N'-Bis-(3-aminopropyl)-1,6-hexanediamine with N-(3-aminopropyl)-1,6-hexanediamine (1:1) 25. N-Butyl-1,6-hexanediamine

26. N.N'-Dibutyl-1,6-hexanediamine

27. 2-"Cocoyl"-1

-(sodium carboxymethyl)-l-(2-hydroxyethyl)-2- imidazoliniumhydroxide 28. Sulphonated glyceroltrioleate

29. Paint, antifoulingmarine

30. Paint stripper

31. 2-Methylpentanediamine

32. Mixture M

33. 50% Solution of mono- and diphosphate esters of mixed alcohols in kerosine 34. 6-tert-Butyl-3-ethylphenol

35. Mixture N

36. e-Tridecyl-og-hydroxy-poly(oxy- 1,2- ethanediyl)

37. Mixture O

38. Mixture P

39. 2,2,3-Trinuoro-3-oxopropanoic acid, methyl ester 40. 3-(Methylsulphonyl)acrylonitrile

41. 2-Ethylpyridine

42. 5-Ethenyl-2-methylpyridine

43. Polynactin (84%)

44. Mixture Q

45. Polishingcompound

46. Hindered phenol antioxidant

47. Mixture R 48. Gas booster Necrotic erythema; severe oedema Severe erythema; moderate oedema DOTt~orrosive

Severeerythema and oedema

Necrotic erythema; severe oedema Necrotic erythema; severe oedema Severe erythema with sloughing;moderate oedema

Severe blanchingand necrotic erythema; severe oedema

Necrotic erythema; severe oedema

Necrotic erythema;mild oedema DOTt--corrosive

Moderate increasingto severe erythema at 5 days DOTt--corrosive

DOTt--corrosive

Severe erythema; mild oedema

Necrotic erythema; mild oedema

Necrotic erythema; severe oedema

Necrotic erythema; severe oedema DOTt--vorrosive

Severe to necrotic erythema; moderate oedema Severe erythema; mild oedema

Severe erythema; mild oedema
FHSA$ (+)---necrotic erythema Severe erythema; blanching

FHSA~: (+)--blanching; necrotic erythema DOTt--corrosive

Severe erythema and oedema FHSA:~ (+)--moderate to severe erythema and oedema Mild to moderate conjunctivitis: normal in 3 days Slight, transient conjunctivitis

Permanent corneal opacity with vascularization Persistent, severe corneal opacity and conjunctivitis

Severe corneal opacity; corrosive
Severe, permanent, penetrating corneal opacity Moderate corneal opacity with vascularization;severe conjunctivitis

Severe conjunctival blanching;corneal opacity at 14days with some vascularization Moderate corneal opacity decreased to slightby 7 days; moderate, reversibleconjunctivitis Severecorneal opacity and conjunctivitis Opalescentcornea; conjunctival necrosis Slightcorneal cloudiness; normal in 1 day Severecorneal opacity and conjunctivitis;reversiblein 3 days Mild to moderate corneal opacity; severe iritisand conjunctivitis Slightcorneal clouding; mild conjunctivitis;reversiblein 2-3 days Permanent, general, moderate corneal opacitywith vascularization; severe conjunctivitis Slight, transient corneal opacity; mild to moderate conjunctivitis;reversible in 3 days Mild corneal opacity and conjunctivitis;reversiblein 3 days Corrosive; corneal perforation

FHSA~ (+~irreversible, corrosive

Persistent general corneal opacity and moderate conjunctivitis General, moderate permanent corneal opacity; moderate conjunctivitis Moderate corneal opacity and conjunctivitis;normal in 7 days Slight transient microscopic corneal opacity; moderate conjunctivitis; normal in 7 days Slight but persistent corneal opacity; 1 severe conjunctivitis Moderate, penetrating corneal 2 opacity; moderate conjunctivitis; normal in 5 days Slight conjunctivitis, normal in 2 days 3 FHSA§ (-)~slight, transient 3 conjunctival redness [contd]160 S. J. WILLIAMS

Table 1 continued

Material Skin response Eye lrritancy Response class*
49. Zinc chloride (10% solution)

50. Zinc ammonium chloride galvanizing flux A (10% solution) 51. Zinc ammonium chloride galvanizing flux B (10% solution) 52. Wax, cream

53. Ultraviolet curing lacquer A

54. Ultraviolet curing lacquer B

55. Ultraviolet curing lacquer C

56. Ultraviolet curing lacquer D

57. Ultraviolet curing lacquer E

58. Ultraviolet curing lacquer F

59. Varnish A Necrotic erythema; severe oedema

Necrotic erythema

Necrotic erythema

FHSA+ + (+)--necrotic erythema Moderate erythema and oedema, necrotic at 6 days; localized haemorrhaging Moderate to severe erythema, necrotic and sloughing at 6 days; haemorrhaging Necrotic at 11 days; localized haemorrhaging Necrotic at 11 days; localized haemorrhaging

Moderate erythema with severe oedema, necrotic at 11 days; localized haemorrhaging Moderate erythema, severe oedema, localized haemorrhaging, necrotic at 6 days Moderate erythema and oedema; necrotic at 6 days

60. Varnish B Moderate erythema with moderate to severe oedema Moderate, penetrating corneal 2 opacity; mild conjunctivitis reversiblein 7 14 days Mild, transient conjunctivitis 3
Mild, transient conjunctivitis 3
FHSA§ (-)--transient, mild 3 conjunctivitis Moderate corneal opacity; persistent I conjunctivitis
Severe, permanent corneal l opacification; moderate conjunctivitis Severe, permanent corneal 1 opacification Moderate to severe corneal opacity 1 and conjunctivitis;normal in 21 days Severe, permanent corneal opacity 1

Severe, permanent corneal oedema 1 with vascularization

Severe, permanent corneal opacity 1 with superficial vascularization; severe conjunctivitis Generalized, permanent, moderate to 1 severe corneal opacity; moderate conjunctivitis *See text for description of eye-irritancy class designation. tDepartment of Transportation test for skin corrosion (49 CFR 173.240: 1975). :~Dermal irritation test protocol specified by the Federal Hazardous Substances Act (16 CFR 1500.41: 1973). §Ocular irritation test protocol specifiedby the Federal Hazardous Substances Act (16 CFR 1500.42: 1973).



### unwashed eyes are used for comparison with dermal irritation test results. In all except a few indicated instances, the concentrations of materials tested for ocular irritation were the same as those tested for dermal irritation.

Results and Discussion
The results inTable 1demonstrate that asa general principle, primary skin irritation test results are not predictive of ocular irritancy potential. We started with the hypothesis that severe or corrosive dermal irritants would also be severe ocular irritants. This clearly is not the case. Fifteen of 60 severe primary dermal irritants were classified as mild irritants or non-irritants when tested in the eye; another six materials were classified as moderate irritants. Also, it should be noted that the definition of a severe eye irritant used here was conservative and would be consistent with the definition of a substantial irritant
as defined in the NAS criteria document (National Academy of Sciences---National Research Council, 1977) and by Griffith et al. 

### (1980). If the FHSA criteria were used to classify these materials as eye 

### irritants, prediction of eye irritancy on the basis of skinresponseswould still becorrect foronly45 of60 materials. While a direct relationship between primary dermal irritation and ocular irritancy is not apparent from this retrospective study,itispossiblethat some subclass of the materials examined here could be foundthat wouldyielda positivecorrelationbetween skin and eye irritation. This subclassificationmight be based upon some set ofphysico-chemicalproperties, such as pH, octanol-waterpartitioncoefficients
or others (Williams, 1982),whichmightyielda tiered screeningsystemwithbetter predictability.However, good physico-chemicaldata are very difficultto obtain for complex formulationssuch as some ofthose that have been tested or must be tested at Haskell Laboratories as a part of product safety evaluation. Moreover, it has been demonstrated recently that there is no simple relationship between ocular irritancyandeasilyobtainablechemicalparameters such
as pH (Murphy et al. 

### 1982)even with chemically well-defined samples. Anotherpossiblereasonforthe lack ofcorrelation betweenthe resultsofthetwo tests isthat the current

### Prediction ofocular irritation 161

methods for evaluating primary skin irritation may be too rigorous a model for probable exposure. For example, two materials studied here (Table 1, nos 45 and 52) were also evaluated by the proposed revision test forprimary skin irritants (Federal Register, 1972) and were found to be non-irritants, as defined. In this test, material is applied in a similar manner to that described for the FHSA test, but test patches areheld in place for 4 rather than 24hr. In the eyetests itcan be argued that, while exposure may be exaggerated, the protective responses of the eye remain physiological. This is not the case for current dermal test procedures since occlusion ofthe contact area allows for an extended time of intimate dermal exposure under conditions of abnormal hydration. A better correlation between severe primary skin irritation and eye irritation might be obtained if the skin irritation test were performed using non-occluded conditions. In a review of the acute toxicity of 300 materials (Smyth, Carpenter, Weil et al. 1962) in which a non-occluded method of dermal-irritation evaluation was used, only four out of 63 severe skin irritants (score />6) were classified as less than severe irritants when tested in the eye(score ~< 4). Therefore, the lack of predictability of ocular responses based upon dermal irritancy may be related to overestimation of the latter by current test procedures.

REFERENCES 

### Draize J. H. (1959). Dermal toxicity. In Appraisal ofthe Safety ofChemicals inFoods,Drugs and Cosmetics. p.46. Association of Food and Drug Officials of the United States, Austin, TX. Federal Register (1972). Proposed revisiontest forprimary skin irritants. Fed.Reg. 37, 27635. Griffith J. F., Nixon G. A., Bruce R. D., Reer P. J. & Bannan E. A. (1980). Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye. Toxic. appl.Pharmac. 55, 501. lnteragency Regulatory Liaison Group (1981). Recommended Guideline for Acute Eye Irritation Testing. IRLG Testing Standards and Guidelines Work Group, Washington, DC. Murphy J. C., Osterberg R. E., Seabaugh V. M. & Bierbower G. W. (1982). Ocular irritancy responses to various pH's of acids and bases with and without irrigation. 7bxicology 23, 281, National Academy ofSciences-National Research Council (1977). Principles andProceduresfor Evaluating the Toxicity ofHousehold Substances. p. 41. National Academy ofSciences, Washington, DC. SmythH. F., Jr, Carpenter C. P., Weil C. S.,PozzaniU.C. & Streigel J. A. (1962). Range-findingtoxicitydata: List VI.Am. ind.Hyg. Ass. J. 23, 95. Weil C. S. & Scala R. A. (1971). Study of intra- and interlaboratory variabilityin theresults ofrabbit eyeand skin irritation tests. Toxic. appl.Pharmac. 19, 276. Williams S. J. (1982). Heat precipitation of acid soluble collagen as a screen for severe corneal opacifacients. (Abstract 622) Toxicologist 2 (1), 178. Williams S. J., Graepel G. J. & Kennedy G. L. (1982). Evaluation ofocular irritancypotential: Intralaboratory variabilityand effect ofdosage volume. Toxicology Lett. 12, 235.