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

Printed in Great Britain Pergamon Press Ltd 

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-Palmolive Co., 909 River Road, PO Box 1343, Piscataway, NJ 08855-1343, USA, l'ICI Central 

Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SKI0 4TJ, :~Shell Research Ltd., Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, §Unilever Environmental Safety Laboratory, 

Colworth House, Sharnbrook, Bedford MK44 1LQ, HHoechst AG, Postfach 80 03 20, D-6230 Frankfurt 

am Main 80, Germany, ¶**ECETOC, Av. E. van Nieuwenhuyse 4, (Bte 6), B-1160 Brussels, Belgium and 

ttrepresenting Procter & Gamble Ltd., Newcastle Technical Centre, PO Box Forest Hall No. 2, Newcastle 

upon Tyne NEI2 9TS, UK 

(Received 28 July 1992) 

Abstraet--A list of 55 chemicals has been developed for which comprehensive in vivo rabbit eye irritation 

data are available. No new in vivo testing has been carried out to qualify a chemical for inclusion in the 

list. The 55 chemicals selected are available at high and consistent purity and are expected to be stable 

on storage. They have been tested undiluted in in vivo studies, except those chemicals where high 

concentrations of the substance could be expected to cause severe effects. The in vivo data have been 

generated since 1981 in studies carried out according to OECD Test Guideline 405 and following the 

principles of Good Laboratory Practice. The data were obtained from tests normally using at least three 

rabbits evaluated at the same time, involving instillation of 0.1 ml (or equivalent weight) into the 

conjunctival sac, and in which observations were made at least at 24, 48 and 72 hr after instillation. The 

chemicals represent a range of chemical classes (acetates, acids, alcohols, alkalis, aromatics, hydrocarbons, 

inorganics and surfactants) and different degrees of irritancy. They are ranked for eye irritation potential 

on the basis of a 'modified maximum average score'. The reference data bank should be of use in validation 

tests for promising alternatives to the in vivo rabbit eye irritation test. This is an essential step in 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 

regulations will require the testing of many chemicals 

that may have been in use for a number of years. The 

net result of these developments is that the number of 

animals used in toxicological tests may be expected to 

increase. This occurs at a time when there is widespread criticism of such use of animals when making 

safety assessments. 

Many companies and regulatory authorities share 

the published concern of the animal welfare organizations. For several years industry has been modifying 

the existing in vivo techniques in order to minimize 

distress and the number of animals used 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 whom reprint 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 

alternative techniques stated: "At the present time all 

of the alternatives may 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 alternative techniques has been hampered by using: (a) chemicals not 

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 assessment of the merits of their 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 reproducibility of 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 

eye irritation test (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 eye responses of individual rabbits, 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 1987 but the revisions did not amend the essentials 

of the procedure for the in vivo test. 

to OECD Test Guideline 405 (OECD, 1987)* and 

following the principles of Good Laboratory Practice 

and have been obtained in tests: 

normally using at least three New Zealand 

white rabbits tested at the same time; 

involving instillation of 0.1 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 Technical 
Eye irritation: reference chemicals data bank 

Table 1. Scheme for assessing eye 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 

slightly obscured 

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 still reacting 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 definitely injected above normal 

More diffuse, deeper crimson red, individual vessels not 

easily discernible 

Diffuse beefy red 

E. Chemosis (oedema): 

No swelling 

Any swelling above normal (includes nictitating membrane) 

Obvious swelling with partial eversion of lids 

Swelling with lids about half closed 

Swelling with 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 hairs just 

adjacent to lids 

Discharge with moistening of the lids and hairs, and 

considerable area around the eye 

Score (D + E + F) x 2 Total maximum ffi 20 

Maximum possible score ffi 80 + 10 + 20 = 110 

Table 2. Eye irritation data for individual rabbits 

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 

*Ethyleneglycol monoethyl 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. BAGLEY et al. 

Table 2---contd 

Number of 

Purity animals 

Chemical (%) tested Modified MAS 

Butyl cellosolve* 99 3 68.67 

Cyclohexanol 97 4 79.75 

*Ethyleneglycol monobutyl 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-Car boxybenzaldehyde > 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 (100 mg) >99.9 3 18.33 

Sodium perborate tetrahydrate (60 mg) 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(100 mg) >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 glycol 400 6 0.00 

Polyoxyethyleneoctylphenolt (1%) 98 6 1.67 

Polyethylene glycol 600 6 2.00 

Sorbitanpolyoxyethylenemonooleate~ 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: reference chemicals data 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-validation study 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. and Walum E. (1990) Report and recommendations 

of the CAAT/ERGATT workshop 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 of Pharmacology 82, 377. 

ECETOC (1988) Eye Irritation Testing. Monograph No. 11. 

European Chemical Industry Ecology and Toxicology 

Centre, Brussels. 

ECETOC (1992) Eye Irritation: Reference Chemicals Data 

Bank. Technical Report No. 48. European Chemical 

Industry Ecology and Toxicology Centre, Brussels. 

EEC (1990) Collaborative study on the evaluation of 

alternative methods to the eye irritation test. DirectorateGeneral, Employment, Industrial Relations and Social 

Affairs, Health and Safety Directorate. 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. 

Proceedings of joint (US) Government/Industry workshop 

on progress towards non-animal alternatives to the 

Draize test (1989) Journal of Toxicology--Cutaneous and 

Ocular Toxicology 8(I), 69. 

Purchase I.F.H. (1990) An international reference chemical 

data bank would accelerate the development, validation 

and regulatory acceptance of alternative toxicology tests 

ATLA 18, 345.