Toxic. in VitroVol.6,No. 6,pp. 487-491, 1992 0887-2333/92$5.00+0.00

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## EYE IRRITATION: REFERENCE CHEMICALS

DATA BANK

D. M. BAGLEY*,P. A. BOTHAMt', J. R. GARDNER,,G. HOLLAND,,R. KREILINGII, R. W. LEWISI', D. A. STRINGER¶**and A. P. WALKERI'i" *Colgate-PalmoliveCo., 909 RiverRoad, PO Box 1343,Piscataway,NJ 08855-1343,USA, l'ICICentral ToxicologyLaboratory, AlderleyPark, Macclesfield,Cheshire SKI04TJ, :~ShellResearch Ltd., Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, §UnileverEnvironmental Safety Laboratory, Colworth House, Sharnbrook, BedfordMK44 1LQ, HHoechstAG, Postfach 80 03 20, D-6230Frankfurt

am Main 80, Germany, ¶**ECETOC, Av. E. vanNieuwenhuyse4, (Bte6), B-1160Brussels,Belgiumand ttrepresentingProcter &GambleLtd.,NewcastleTechnicalCentre,POBoxForest HallNo. 2,Newcastle upon Tyne NEI29TS, UK

(Received 28 July 1992)

Abstraet--Alist of55chemicalshas beendevelopedfor whichcomprehensivein vivorabbiteyeirritation data are available. No new in vivotestinghas beencarried out to qualify a chemicalfor inclusionin the list. The 55 chemicalsselectedare available at highand consistent purity and are expectedto be stable

on storage. They have been tested undiluted in in vivo studies, except those chemicals where high concentrations of the substance could be expectedto cause severeeffects.The in vivo data have been generated since 1981 in studies carried out according to OECD Test Guideline 405 and followingthe principlesofGood Laboratory Practice.The data wereobtained from testsnormally usingat leastthree rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the conjunctival sac, and in whichobservations weremade at least at 24, 48 and 72hr after instillation.The chemicalsrepresenta rangeofchemicalclasses(acetates,acids,alcohols,alkalis,aromatics,hydrocarbons, inorganicsand surfactants) and differentdegreesofirritancy.They are ranked for eyeirritation potential

onthebasisofa 'modifiedmaximumaveragescore'.Thereferencedata bankshouldbeofuseinvalidation tests for promising alternatives to the in vivorabbit eye irritation test.This is an essential stepin the progression to regulatory acceptance of alternative procedures.

INTRODUCTION
Historically, toxicological data used in assessing the safety of chemicals for humans have been generated by testing the materials on animals. In recent years a range of regulations has been introduced requiring

more extensive testing of new chemicals. In the foreseeable future it can be expected that further regulationswillrequire the testing of manychemicals that may have beenin use for a numberof years. The
net result of these developmentsis that the numberof animalsused intoxicologicaltests may be expected to increase. This occurs at a time when there is widespread criticism of such use of animalswhen making safety assessments. Many companies and regulatory authorities share the published concern of the animal welfare organizations. For several years industryhas been modifying the existing in vivo techniques in order to minimize distress and the numberof animalsused as an interim

measure until alternative non-sentient methods, acceptable to regulatory authorities, have been developed. The in vivo rabbit eye irritation test, in particular, has frequently been criticized on animal welfare

tTo whomreprint requests should be addressed. Abbreviation: MAS = maximum average score. grounds. In recent years many laboratories have been working to develop in vitro alternatives to this test. Although numerous publications have appeared in the scientific literature a recent review of published alternativetechniques stated: "At the present time all of the alternativesmay have a role as adjuncts to the Draize rabbit eye test but none are sufficiently well validated to replace the test completely" (ECETOC, 1988). A number of validation exercises has been carried
out. Progress towards acceptance of alternativetechniqueshas beenhampered byusing: (a) chemicalsnot freely available or lacking a purity specification; and (b) non-standardized in vivo test protocols. If investigators of potential in vitro alternatives
to the in vivo rabbit eye test had been able to draw
test substances from an internationally accepted sample bank and for which in vivo data were available, then assessmentofthe merits oftheir alternative technique would have been simplified. Proposals have been made that a data bank of reference chemicals should be established in order to assist in the resolution of this problem (Balls et al., 1990; EEC, 1990; Purchase, 1990; US Joint Government/Industry Workshop, 1989).The availability of

a test sample bank would, in effect, assist investigators to conduct 'micro-validation' of their own potential technique.

487

### 488 D.M. BAGLEY et al.



### Validation is a vital step in the acceptance of any
new toxicological test method, in vivo 

### or in vitro. 

### Validation studies establish the reproducibilityof the technique in the hands of different operators in different laboratories (and possibly in the same laboratory) as well as the accuracy of the test system used in predicting the particular toxicological endpoint. Three features are common to any validation exercise. A sample of a test material is evaluated in a standard in vivo 

### test in order to generate 'baseline' toxicological data. A sample of the same material is then tested by the proposed alternative technique. The material is further assessed in other laboratories using the same in vitro 

### technique. These considerations led ECETOC to convene a Task Force with the objective of developing a list of chemicals that would serve as a reference chemicals data bank to be used in assessing the potential of alternative techniques as a replacement for the in vivo 

### eyeirritationtest (often referred to as the Draize test). The initial criterion in the selection of candidates for the list of reference chemicals was that comprehensive in vivo 

### rabbit eye irritation test data should be available. Application of this criterion would ideally ensure that no further in vivo 

### tests would be necessary to qualify a chemical for inclusion in the list. Based on the criterion, potential sources of in vivo 

### eye irritation data were identified from the literature. From these (and other) sources, information relating
to eyeresponses ofindividualrabbits, and the specification of the chemicals selected, was assessed against selection criteria.

Process for the selection of chemicals and selection 

### criteria
A review of the literature revealed a number of publications containing references to in vivo 

### eye irritation tests of identified chemicals. In virtually all

cases data were presented in summary form (and interpreted against different standards or indices) and consequently were inappropriate for the ECETOC exercise. Due to these limitations, the authors and other sources were contacted to establish the availability of individual rabbit eye data, the in vivo 

### test method and the specifications of the chemicals used. The chemicals and the eye irritation data included in the reference chemicals data bank meet the following criteria: Reference chemicals are single chemical entities available at known high consistency and purity and

are expected to be stable in storage. The in vivo 

### rabbit eye irritation data have been generated since 1981 in studies carried out according

*OECD Test Guideline 405 was adopted on 12 May 1981. A revised Test Guideline 405 was adopted on 24 February 1987but the revisions did not amend the essentials of the procedure for the in vivo 

### test. to OECD Test Guideline 405 (OECD, 1987)* and followingthe principlesof Good Laboratory Practice and have been obtained in tests: normally using at least three New Zealand white rabbits tested at the same time; involvinginstillationof0.1 ml or the equivalent weight into the conjunctival sac;
in which anaesthesia was not 

### used;

in which observations were made (at least) 24, 48 and 72 hr following instillation; including individual scores (using the Draize scale) for each tissue assessed in each rabbit;

that enable reversibility/irreversibility to be assessed;

in which chemicals were tested undiluted (except that data from studies using dilutions of the chemical were acceptable when higher concentrations of the chemical could be expected
to cause severe effects).

Chemicals data bank

Chemicals selected 

### Using the criteria defined above, 55 chemicals were selected for inclusion in the samples bank. The in vivo 

### data available precluded the need for any additional animal testing.
Grading of ocular effects


### The eye irritation effects of the chemicals selected have been graded following the weighted scoring scheme of Draize (1944) shown in Table 1. The classification schemes of the EC and US regulatory agencies, and OECD Test Guideline 405, do not include weighting of scores, nor assessment of area of cornea affected (B) or conjunctival discharge (F).

RESULTS

The chemicals selected, together with the purity of the chemicals tested and summarized results of the in vivo 

### tests, are given in Table 2. The eye irritation potential of chemicals is often summarized as the 'maximum average score' (MAS). The MAS is obtained by averaging the individual animal weighted scores at each time of observation (e.g. 4, 24 and 48 hr, etc.) and then selecting the highest (maximum) of these averages. Not all the in vivo 

### assays that qualified chemicals for inclusion in the samples data bank included readings made less than 24 hr after instillation. Consequently, for consistency, the scores given in Table 2

are 'modified MAS' in that they represent maxima calculated at 24 hr (or more) following instillation. All scores, including (where available)observations made within 24 hr of instillation,are given in individual rabbit data sheets in an ECETOC TechnicalEye irritation: reference chemicals data bank

Table 1. Scheme for assessingeye irritation (Draize, 1944) 489

Ocular effects Grade
I. Cornea A. Opacity----degree of density (area most dense taken for reading): No opacity Scattered or diffuse area, details of iris deafly visible Easily discernible translucent areas, details of iris slightlyobscured Opalescent areas, no details of iris visible, size of pupil barely discernible Opaque, iris invisible B. Area of cornea involved: One-quarter (or less) but not zero Greater than one-quarter but less than half Greater than half but less than three-quarters Greater than three-quarters, up to whole area Score (A x B) x 5 Total maximum= 80 II. Iris C. Values: Normal Folds above normal, congestion, swelling,circumcorneal injection (any or all of these or combinations of any thereof), iris stillreacting to light (sluggish reaction is positive) No reaction to light, haemorrhage, gross destruction (any

or all of these) Score C x 5 Total maximum = 10 IlL Conjunctivae D. Redness (refers to palpebral conjuetivue only): Vessels normal Vessels definitelyinjected above normal More diffuse, deeper crimson red, individual vesselsnot easily discernible Diffuse beefy red E. Chemosis (oedema): No swelling Any swellingabove normal (includes nictitatingmembrane) Obvious swellingwith partial eversion of lids Swellingwith lids about half closed Swellingwith lids about half closed to completely closed F. Discharge: No discharge Any amount of difference from normal (does not include small amounts observed in inner canthus of normal animals) Discharge with moistening of the lids and hairsjust adjacent to lids Discharge with moistening of the lids and hairs, and considerable area around the eye Score (D + E + F) x 2 Total maximumffi20 Maximum possible scoreffi80 + 10 + 20 = 110

Table 2. Eye irritation data for individualrabbits
Number of Purity animals Chemical (%) tested Modified MAS
Acetates (7 chemicals) Methyl trimethyl acetate 99 6 2.67 Ethyl trimethyl acetate 99 6 3.83 n-Butyl acetate 99 4 7.50 Ethyl acetate 99 4 15.00 Cellosolve acetate* 99 4 15.00 Ethyl-2-met

hylacetoacetate c97 3 18.00 Methyl acetate 98 4 39.50 *Ethyleneglycolmonoethyl ether acetate
Acids (2 chemicals, 3 tests) Trichloroacetic acid (3%) reagent grade 6 6.67 2,2-Dimethylbutanoic acid 96 6 44.67 Trichloroacetic acid (30%) reagent grade I 106
Alcohols (9 chemicals) Propylene glycol 6 1.33 Glycerol > 99.5 6 1.67 Isoprnpanol 99.9 4 30.50 2-Ethyl-l-hexanol 99 4 51.25 Isobutanol 99.9 4 60.25 n-Butanol 99.8 4 60.75 n-Hexanol 98 4 64.75 [contd]490 D. M. BAGLEYet al.

Table 2---contd
Number of Purity animals Chemical (%) tested Modified MAS Butyl cellosolve* 99 3 68.67 Cyclohexanol 97 4 79.75 *Ethyleneglycolmonobutyl ether
Alkalis (1 chemical, 2 tests) Sodium hydroxide (1%) reagent grade 4 25.75 Sodium hydroxide (10%) reagent grade 1 108
Aromatics (13 chemicals, 14 tests) 4,4-Methylene his (2,6-di-tert-butylphenol) 98 6 0.00 4-Bromopbenetole 99 3 1.33 Xylene [1] 99 4 1.50 3-Chloro-4-fluoronitrobenzene 98 6 1.67 1,3-Di-isopropylbenzene 96 6 2.00 l-Methylpropylbenzene >99.5 6 2.00 3-Ethyltoluene 99 6 2.33 2,4-Difluoronitrobenzene 99 6 4.67 Styrene 99 4 6.75 Toluene 99 4 9.00 Xylene [2] 99 4 9.00 2,6-Dichlorobenzoyl chloride 99 6 23.83 4-Carboxybenzaldehyde > 95 3 50.33 Parafluoroaniline 99 6 69.83 Hydrocarbons (8 chemicals) 3-Methylhexane 99 6 0.67 2-Methylpentane >99.5 6 2.00 1,9-Decadiene 98 6 2.00 Dodecane > 99.5 6 2.00 1,5-Dimethylcyclooctadiene 96 6 2.83 c/s-Cyclooctene 95 6 3.33 Methylcyclopentane >99 6 3.67 1,5-Hexadiene 97 6 4.67 Inorganic chemicals (2 chemicals) Ammonium nitrate (100mg) >99.9 3 18.33 Sodium perborate tetrahydrate (60mg) 98.6 6 30.50
Ketones (4 chemicals, 5 tests) Methyl isobutyl ketone 98 4 4.75 Methyl amyl ketone [I] 4 10.50 Methyl amyl ketone [2] 4 16.25 Methyl ethyl ketone 99 4 50.00 Acetone 99 4 65.75
Miscellaneous (1 chemical) N,N-dimethyl guanidine sulphate(100mg) >95 3 6.67
Surfactants (8 chemicals, 21 tests) Anionics Sodium lauryl sulphate (3%) 98 6 16.00 Sodium lauryl sulphate (15%) 98 6 59.17 Sodum lauryl sulphate (30%) 98 6 60.50 Cationics Benzalkonium chloride [1] (1%) 98 4 34.25 Benzalkonium chloride [2] (1%) 98 6 56.33 Benzalkonium chloride (5%) 98 4 83.75 Benzalkonium chloride (10%) 98 3 108.00 Cetylpyridinium bromide (0.1%) 99 6 2.67 Cetylpyridinium bromide (1%) 99 6 36.00 Cetylpyridinium bromide (6%) 99 4 85.75 Cetylpyridinium bromide (10%) 99 6 89.67 Nonionics Polyethylene glycol400 6 0.00 Polyoxyethyleneoctylphenolt (1%) 98 6 1.67 Polyethylene glycol 600 6 2.00Sorbitanpolyoxyethylenemonooleate~ 98 4 4.00 Polyoxyethyleneoctylphenolt [1] (5%) 98 6 32.33 Polyoxyethyleneoctylphenolt [2] 5%) 98 6 33.83 Polyoxyethyleneoctylphenolt (10%) 98 6 68.67 Zwitterionics Igepon AC-78§ (0.5%) 6 3.67 Igepon AC-78§ (10%) 6 36.83 lgepon AC-78§ (5.0%) 6 51.67
+Tween 80. ~:Triton X-100. §CN oil acid ester of NA isethionate. Numerical suffixes([1], [2])denote instances where the sample of the chemical was retested in the same laboratory.Eye irritation: referencechemicalsdata bank 491

Report (1992). The availability of these data will enable a reader to classify the irritation potential of any of the chemicals selected from the samples bank for use in a micro-validationstudy according to any existing classification scheme.

REFERENCES
Balls M., Blaauboer B., Brusick D., Frazier J., Lamb D., Pemberton M., Reinhardt C., Roberfroid M., Rozenkranz H., Schmid B., Spielmann H., Stammati A-L. andWalum E. (1990)Report and recommendations of the CAAT/ERGATTworkshop on the validation of toxicity test procedures 

### ATLA 18, 313. Draize J. H., Woodard G. and Calvery H. O. (1944) Methods for the study of irritation and toxicity of substances applied to the skin and mucous membranes 

### Journal ofPharmacology 82, 377. ECETOC (1988)

### EyeIrritation Testing.Monograph No. 11. European ChemicalIndustry Ecologyand Toxicology Centre, Brussels. ECETOC (1992)

### Eye Irritation: Reference ChemicalsData Bank. Technical Report No. 48. European Chemical Industry Ecology and ToxicologyCentre, Brussels. EEC (1990) Collaborative study on the evaluation of alternativemethodsto the eyeirritation test. DirectorateGeneral, Employment, Industrial Relations and Social Affairs,Health and SafetyDirectorate. DGV and DGXI, Brussels. OECD (1987) 

### OECD Guidelines for Testing of Chemicals; Test Guideline 405, Acute Eye Irritation~Corrosion. Organisation for Economic Co-operation and Development. Adopted 24 February 1987. Proceedingsofjoint (US)Government/Industry workshop

on progress towards non-animal alternatives to the Draizetest(1989)

### Journal ofToxicology--Cutaneous and Ocular Toxicology 8(I), 69. Purchase I.F.H. (1990)Aninternational referencechemical data bank would acceleratethe development,validation and regulatory acceptance of alternative toxicologytests 

### ATLA 18, 345.