Fd Chem. Toxic. Vol. 30, No. 12, pp. 1061-1067, 1992

0278-6915/92 $5.00 + 0.00 Copyright© 1992Pergamon Press Ltd

Printed in Great Britain.All rights reserved

ANALYSIS OF ALTERNATIVE METHODS FOR DETERMINING OCULAR IRRITATION T. M.~RTINS*, J. PAULUHN and L. MACHEMER Bayer AG, Department of Toxicology, PO Box 101709, 5600 Wuppertal 1, Germany (Accepted 10 August 1992)

Abstract--According to classification and labelling requirements, chemicals, dyes, agrochemicals and pharmaceutical formulations have to be evaluated for their potential to induce eye irritancy or corrosion. An attempt was made to analyse the predictive power of the bovine eye-chicken egg chorioallantoic membrane (BE-CAM) assay in comparison with results obtained using the conventional Draize method. In summary, results showed limited correlation between reactions/n vitro and responses of eyes in vivo. In a pilot study, ultrasonic pachymetry showed high sensitivity and fairly good correlation between corneal thickness and clinical observations in eyes.

INTRODUCTION The procedure described by Draize et al. (1944) has formed the basis of eye irritation testing for many years. With some minor modifications it has been adopted worldwide by regulatory bodies for assessment of the ocular irritation potential of chemicals. Since its introduction the Draize rabbit eye test has been criticized for a variety of reasons, and particular efforts have been made to develop alternatives to this test (ECETOC, 1988). However, for classification and labelling purposes, none of these techniques has so far been validated at a level that is accepted by international regulatory authorities (Kalweit et aL, 1990). An attempt was made to analyse the predictability of the alternative BE--CAM assay (van Erp and Weterings, 1990; Weterings and van Erp, 1987) in comparison with results obtained in the Draize test using various chemicals, dyes, agrochemicals and pharmaceutical formulations. In a pilot study, corneal ultrasonic pachymetry was tested as a method of establishing objective and sensitive data on eye irritancy (Jacobs and Martens, 1989; Kennah et al., 1989). MATERIALS

AND METHODS

Test substances. This paper presents the distribution of results obtained in the Draize rabbit eye tests with

671 substances tested continuously during the last years. The substances tested encompass a variety of chemical classes and indication areas: dyes, formulated and non-formulated, mainly various diazo compounds, anthraquinones, and substituted sulphonic acids; basic chemicals and solvents (i.e. various aromatic and aliphatic substituted hydrocarbons); polymers (e.g. antifoams and emulsifiers); pesticides, formulated and non-formulated, encompassing mainly insecticides, herbicides and fungicides; pharmaceutical formulationst. Approximately one in 10 of all products tested on eyes representing the entire spectrum of irritating properties in vivo were selected and examined in vitro. A total of 61 chemicals was tested in the BE-CAM assay, of which 34 were transparent liquids. Substances used in the investigations of corneal thickness are commercially available: saline, 1% sodium dodecyl sulphate (SDS), 0.1 N-NaOH, acetone, citric acid and ethylenediamine. Tests o f irritant/corrosive effects on eyes. The investigations were performed in the albino rabbit (New Zealand White) in accordance with the EEC (1983 and 1984), OECD (1987) and EPA (1984 and 1989) guidelines. The tests were performed for the purpose of classification and labelling of the products. The results were evaluated with respect to the nature, intensity, and reversibility or irreversibility of the findings, taking into consideration internationally recognized evaluation guidelines (EPA, 1981 and 1988; OECD, 1983). Bovine eye-chorioallantoic membrane assay

*To whom correspondence should be addressed. Abbreviations: BE-CAM--bovine eye-chick egg chorioallantoic membrane; SDS = sodium dodecyl sulphate. tMore detailed information on the nature (i.e. structure, physicochemical properties and composition) of the substances tested is far beyond the scope of this publication. However, all data are documented and if there is specific interest in some aspects of the results the authors are certainly willing to furnish more information.

With some minor modifications the procedure for the BE assay was in accordance with the technique described by Weterings and van Erp (1987) and the CAM assay was performed according to the method described by Luepke (1985), modified according to the protocol reported by Kalweit et al. (1990). The experimental details are briefly summarized.

1061

1062

T. M)~l~Xlr~set al. Table 1. Score table for the bovine eye (BE) assay Opacity

Assessment -

< = > Acetone

Score 0

1 2 3

Fluorescein staining

Epithelial detachment

Assessment

Assessment

-

Score 0

< = > Toluene

0.5 I 1.5

Score

--

0

Wrinkling Loosening

2 4

Bovine eye assay. Calf eyes were obtained from a local slaughterhouse. They were enucleated within 10 min following bolt-shot and transported in saline at ambient temperature. The eyes were used for the assay within 4 hr after excision. A plastic egg-tray was used to hold the eyes and the whole was kept in the humid atmosphere of a closed water-bath at 37°C for 10 min. A silicone ring dipped in saline was placed on the central part of the cornea and the eyes were equilibrated in the water-bath for a further 5 min. The test and reference substances were applied to the cornea inside the silicone ring in a volume of 0.1 ml or in the case of solids in an amount entirely covering the cornea within the ring. Nine eyes were used for each experiment--four eyes per test substance, two eyes per reference substance (acetone, toluene) and one untreated control eye. After 45 sec the eyes were rinsed with saline and incubated in the water-bath for 10 rain. The extent of corneal damage was assessed according to Table 1. As a deviation from the test procedure described by Weterings and van Erp (1987), corneal injury resulting from exposure to the test substances was standardized against the results obtained with the reference substances to minimize observer variation. The final score was obtained by adding the scores for opacity, fluorescein staining (as an indicator of epithelial integrity) and epithelial detachment. The mean score was calculated from the final scores for each of the four eyes. The mean scores were evaluated according to Table 2. Chorioallantoic membrane assay. Fertile White Leghorn eggs (weight 60-70 g; Lohmann Tierzucht GmbH, Cuxhaven, Germany) were incubated in an automatically rotating incubator (temperature 37.538.0°C, relative humidity 55-60%). The eggs were candled on day 7 of incubation and the outline of the air chamber was marked on the shell; unfertilized eggs or eggs containing a dead embryo were discarded. On days 9 until 10 of incubation the shell was scratched around the air chamber by means of a rotating polisher. The inner egg membrane was removed. The test and reference substances were Table 2. Evaluation of bovine eye-chicken egg chorioanantoic membrane (BE-CAM) a s s a y CAM score Evaluation

BE score

Rinsed

Non-rinsed

3.5

0-2.9 3.0--4.9 5.0-8.0 > 8.0

0-3.9 4.0-6.9 7.0-11.0 > 11.0

of irritation No Slight Moderate

Severe

applied to the CAM in a volume of 0.3 ml. All substances tested were applied to the CAM undiluted; 20 sec after administration the CAM was carefully rinsed with saline for 10 sec. 10 eggs were used for each experiment--six eggs per test substance, two eggs per reference substance (0.1 N-NaOH, 1% SDS). An additional test was performed with all transparent liquids; in this test the CAM was not rinsed and the reactions were recorded through the transparent substance. The blood vessels, including the capillary system, and the albumen were examined and scored for irritant effects (haemorrhage = H, lysis = L, coagulation = C) over a period of 300 sec. As soon as a reaction was identified the time (in sec) was noted and used to calculate an irritation score for each egg according to the following equation: Score =

5 × [(301 - sec H)/300] + 7 × [(301 - sec L)/300] + 9 × [(301 -- sec C)/300].

The mean score was calculated from the scores for each of the six eggs. The mean scores were evaluated according to Table 2. The results were also standardized against the results obtained with the reference substances to minimize potential observer variation. As it transpired that observer variability was negligible, the evaluation of the CAM assay was based on the scores calculated according to the above equation. The highest score of either BE assay or CAM assay was used for assessment of the irritant potential of the substance. The evaluation system used in this investigation was slightly different from those used by Weterings and van Erp (1987) and Kalweit et al. (1990), mainly as a consequence of the differences in the systems used for evaluation in vivo. Ultrasonic corneal pachymetry

Corneal thickness was measured non-invasively in albino rabbits using an ultrasonic pachymeter (STORZ Omega Scan, Heidelberg, Germany) equipped with a water tip probe (20 MHz) before, and 1, 24, 48, 72 hr and 7 days after, administration of the test substance. The pachymetric data were compared with the macroscopic findings on eyes, recorded simultaneously. Clinical evaluation was in accordance with the guidelines mentioned above. 100 #1 of the test substances were instilled into each eye of two rabbits. In order to prevent discomfort to the animals and to avoid the blinking reflex (because the water cone of the tip probe had to come into contact with the surface of the cornea), the eyes were anaesthetized before substance administration and before each ultrasonic measurement, using xylocaine 4%; 24 hr after instillation the eyes were rinsed with saline. Ultrasonic measurements were performed on five different corneal sections (central, inferior, superior, nasal and temporal pole), three times per section, in order (a) to obtain an integrative value

Alternative methods to the Draize test

No irritation ( 4 4 5~ ) %~ : : s ~ ii3i°:: f / A ~ -i "::~°rr°si°n(32) 4~8~ ~

~ I i ~

~V:srt'on (28)

Slight l irritation(126) - -

Moderate irritation(40)

Fig. I. Relative distribution of the local irritant/corrosive effects on eyes for a total of 671 products tested. The figures in parentheses represent the absolute numbers of substances tested. of the corneal thickness, (b) to detect a potential influence of the initial thickness on the intensity and time-course of corneal oedema, and (c) to identify thickness gradients of different sections. The mean corneal thickness was calculated from the values for all sections. The pre-exposure thicknesses of the different corneal sections were compared by one-way analysis of variance.

RESULTS Tests o f irritant~corrosive effects on eyes The relative distribution of the results of the eye tests is shown in Fig. 1: 85% of the 671 substances tested had little or no effect. A final evaluation of the irritant/corrosive effects on eyes was possible for all products. What was most evident in this survey was that the proportion of products tested in our institute having severe irritant or corrosive effects was less than 10%. Kobel and Gfeller (1985) reported similar results of tests on eyes of industrial chemicals. B E - C A M assay Of 61 substances, seven could be evaluated only in the BE assay and not in the CAM assay; these substances either adhered firmly to the CAM and could

1063

not be removed by the standard rinsing treatment, or intensively stained the CAM, thereby preventing evaluation of the reactions. It was not, therefore, possible to draw any definite conclusions about the irritant potential of these seven products. Good reproducibility was achieved in each experiment; the mean coefficient of variation for all experiments was 5.8%. The results of the BE-CAM assay and the Draize eye irritation studies are summarized in Table 3. Of 54 substances tested in the BE-CAM assay (CAM rinsed), 41 (76%) were either in exact agreement with (33%) or showed only slight (i.e. 1 grade) deviation from (43%) the results in vivo. Of 17 substances that induced severe reactions in the Draize eye test, seven were considerably (i.e. more than 1 grade deviation) underestimated by the results obtained in the BE-CAM assay. This high rate of false negatives caused particular concern; analysis of the structure, physicochemical properties and composition revealed no similarities of the substances missed. Of the 28 substances that induced no or slight irritation in the Draize test, six were considerably overestimated by the BE-CAM assay. Of the 34 transparent liquids tested in the BECAM assay, 23 (67%) were in exact agreement with (38%) or showed only slight deviation from (29%) the results in vivo. Only one of 11 substances that induced a severe response in the rabbit eye was considerably underestimated by the BE-CAM assay. Of the 18 transparent substances inducing no or slight irritation in the Draize test, 10 were considerably overestimated by the BE-CAM assay. Ultrasonic corneal pachymetry The average pre-exposure corneal thickness in rabbits (n = 24 eyes) in this study was 364 #m (SD = 19), which corresponds well with data reported in the literature (Jacobs and Martens, 1989). Figure 2 shows that there were no significant differences in the thicknesses of different corneal sections before administration of test substances. Figure 3 shows the mean corneal thickness for each eye of each rabbit treated with saline, SDS 0.1%, 0.1 N-NaOH, and ethylenediamine. As acetone

,.~ 4 0 0 -

Table3. Comparisonof chemicalstestedin the Draizetestand in the bovineeye-chickeneggchodoallantoicmembrane(BE-CAM)assay Draize BE-CAM* No Slight Moderate Severer No Slight Moderate Severe No Slight

4 14 3 l 2 6 4

I 2 I 2 0 0

0 4 2 3 0 0

0 7 0 I0 0 I

Moderate 0 2 1 Severe 2 4 3 9 *BE-CAM1= CAMrinsed;BE-CAM2 = CAMnot rinsed. ?Severeirritationor corrosionin vivo.

~ 380 ~ 360 ,'~

340

~ 320 0

Central Temporal Nasal Superior Inferior

Corneal section Fig. 2. Mean thickness of rabbit corneas before exposure to test substances. Vertical bars represent SD.

T. Mgg~NS et al.

1064

effect on corneal thickness during the follow-up observation period; 1% SDS and 0 . 1 N - N a O H caused a slight transitory increase of corneal thickness, even though only slight reactions of the mucous membranes but no opacity were observed. As values returned to normal, thickness remained almost constant during the follow-up period, demonstrating good reproducibility. Ethylene-

and citric acid induced distinct thickness gradients, the pachymetric data of the inferior and superior poles o f the cornea were plotted separately for each animal in Fig. 4. The corresponding Draize scores are given in Table 4. Saline caused no visible changes but a mild transitory increase in corneal thickness was recorded after 1 hr. The diagram of saline in Fig. 3 also shows that local anaesthesia had no discernible 380--

Saline 1/Right

370 360 350

------

I/Left

.....

2/Right

. . . . . . . . . 2/Left

340 _

/

330 -oh` 420 --

I

L

I

I

1

I

/\.

SDS I%

3/Right

~ \ \\\ \

400 380

--~-

3/Left

.....

4/Right

. . . . . . . . . 4/Left 360 "4

340 l

[

t

I

I

,I

°~

420

NaOH 0.1N

400 0

5/Right

38O 360 340 --

\'- . . . . . . . . . 222:-~-"~:':'~'

/

320 -¥

I

800 --

I

I

5/Left

.....

6/Right

.........6/Left

I

Ethylenediamine A /.//':,~

700 600 500

I

----

?/Right

;"

/

'

.,'..

~

/

~

?/Left

.....

8/Right

. . . . . . . . . 8/Left

400 300 7o J Before

------

I 1 I t lhr 24hr 48hr 72hr Time following instillation

I ?d

Fig. 3, Mean corneal thickness before and 1 hr to 7 days after instillation of saline, 1% sodium dodecyl sulphate, 0.1 N-NaOH and ethylenediamine into the right/left conjunctival sac of rabbits nos 1--8. The corresponding clinical signs are given in Table 4.

Alternative methods to the Draize test

diamine caused a rapid and steep increase in corneal thickness; even though the cornea remained completely opaque, the thickness decreased during the following hours, obviously owing to ulcerative changes. Acetone and citric acid caused marked increases in corneal

1065

superior pole, which failed to correlate with an opacity gradient, were measured in animal no. 9 and in the right eye of animal no. 12. DISCUSSION

thickness that, in general, corresponded well to opacity grades and other inflammatory signs. Significant differences in thickness between the inferior and the

Of the substances tested on eyes in our institute, 85% had no effect or caused only minor irritation.

700 9/Right inferior pole 600

-

- - - -

5OO

.....

9/Right superior pole 9/Left inferior pole

. . . . . . . . . 9/Left superior pole

400 300 7"

I

0

I

I

I

I

700 10/Right inferior pole 600 500

o

.....

10/Left inferior pole

T

I

0

--ca 900 eJ

10/Right superior pole

. . . . . . . . . 10/Left superior pole

400

300

- - - - - -

I

I

I

I

Citric acid

s00

--

700

1

/ ---....

...'...

/

11/Right inferior pole

O .

"

.

600

" ,

/

",°

.

,"

"£

,

~

1----

ll/Right superior pole

.....

ll/Left inferior pole

500 . . . . . . . . . ll/Left superior pole

400 300

I

800

I

I

B

Citric acid 12/Right inferior pole

700 600 •

-- ....

12/Left inferior pole

500 •:

. . . . . . . . . 12/Left superior pole

400 300 0

7-

I

I

I

I

I

I

Before

lhr

24hr

48hr

72hr

7d

Time following instillation Fig. 4. Corneal thickness o f the inferior/superior pole before and I hr to 7 days after instillation of acetone and citric acid into the right/left conjunctival sac of rabbits nos 9-12. The corresponding clinical signs are given in Table 4.

1066

T.M.~RTINS et al.

Severe irritants and corrosive chemicals accounted for only less than 10% of the substances tested. These substances are the ones that cause the animals most

stances tested was considerably underestimated or o v e r e s t i m a t e d b y t h e r e s u l t s o f t e s t s in vitro. T h e r e was a trend towards overestimation in the 'non-rinsed'

discomfort and that give rise to concern with regard to animal protection; however, assessment of these

assay and a trend towards underestimation in the 'rinsed' assay, indicating a certain influence of the

h a z a r d s is n e c e s s a r y b e c a u s e o f t h e g r e a t i m p o r t a n c e

exposure

of human eye damage with the possibility of subsequent visual impairment or loss of sight (Kobel and

conducted with substances that had certain physicochemical properties, such as intensive dyes and

G f e l l e r , 1985). M o r e o v e r , p a i n r e a c t i o n s o f r a b b i t s such as squealing, hunched back or blepharospasm

adhesives. The effects on the BE and the CAM

were

only

to reflect the effects observed on the cornea and the

immediately after exposure, The results obtained in the BE--CAM assay revealed limited predictability of ocular irritant potential,

mucous membranes, respectively (Weterings and van E r p , 1987). N e v e r t h e l e s s , i t is q u e s t i o n a b l e w h e t h e r t h e r e a c t i o n s e l i c i t e d in vivo a r e c o m p a r a b l e w i t h t h e

Fewer

observed

than

in less than

40%

of the

1%

and

usually

substances

tested

time. The

BE-CAM

assay could

not

be

are considered

in the

r e a c t i o n s in vitro: c o r n e a l o p a c i t y in vivo is c o n t r i -

BE-CAM assay were in exact agreement with the r e s u l t s in vivo. A s i g n i f i c a n t p r o p o r t i o n o f t h e s u b -

buted to by oedema of the stroma and infiltration by i n f l a m m a t o r y c e l l s , w h e r e a s c o r n e a l o p a c i t y in vitro is

Table 4. Draize gradings for saline (animals no. 1, 2), 1% sodium dodecyl sulphate (animals no. 3, 4), I N-NaOH (animals no. 5, 6), ethylenediamine (animals no. 7, 8), acetone (animals no. 9. 10), citric acid (animals no. 11, 12) Gradings for right (r) or left (I) eye of animal no: 1 Assessment time Tissue effect* r

lhr

Cornea O A Iris

2

3

4

5

6

7

8

9

1

r

I

r

1

r

1

r

1

r

I

r

1

r

1

r

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

4 4

4 4

4 4

4 4

0

0

0

0

0

0

0

0

0

0

0

0

x

x

x

x

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 2 2

0 2 2

0 0 1 1 11

n n 2 2 3b 3b

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

4 4

0

0

0

0

0

0

0

0

0

0

0

0

x

0 0 0

0 0 0

0 0 0

0 0 0

1 1 0 0 0 0

1 1 0 0 0 0

I 0 0

1 0 0

1 1 0 0 0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0

0

0

0

0

0

0

0

0

0

0

10 I

r

11

12

1

r

1

r

I

1 I 4 4

1 1 4 4

I 4

3/1 2/2

0

0

0 0

0

0

0

1

n n 2 2 3b 3b

0 0 4 4 3 3

0 0 4 4 3 2

0

0

2 2

2 2

0 4 3

0 3 3

4 4

4 4

4 4

1 1 4 4

1 1 4 4

x

x

x

0

0

0 0

0

1

0

1

n n 2 2 1 1

n n 2 2 11

2 2 3

2 1 1

22 11 23

1 3 3

1 2 3

1 4 3

1 4 3

0 0

4 4

4 4

4 4

4 4

1 1 4 4

11 44

0

x

x

x

x

0

0 0

0

1

1

1 3 3

2 3 3

1 2 4 4 33

1 3/1 4 1/3

Conjunctivae R

C D Cornea O A 24 hr

Iris

2/0 3/I 1/3 2/2

1 3/1 4 1/3

Conjunctivae R

C D Cornea 0 A 48 hr

Iris

0

2/03/I 1/32/2

1 3/1 4 I/3 1

Conjunctivae R

C D Cornea 0 A 72 hr

Iris

0 0 0 0 00

0 0 0 0 00

0 0 0 0 00

0 0 0 0 00

1 1 0 0 00

1 1 0 0 00

n n n n I1

n n n n 11

2 2 1 1 10

22 11 13

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

4u 4u 4 4

4u 4u 4 4

1 0 4 0

2 2 44

0

0

0

0

0

0

0

0

0

0

0

0

x

x

0

0 0

0 I 2 2

x

x

0

2/03/1 1/32/2

I 4

3/1 1/3

1

I

I

2 3 3

11 3 3 33

Conjunctivae 00 0 0 00

00 0 0 00

00 0 0 00

00 0 0 00

11 0 0 00

I1 0 0 00

nn n n 11

nn n n 11

21 0 0 00

22 01 23

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

# #

# #

# #

# #

0 0 0 0

1 I 4 4

0

0

0

0

0

0

0

0

0

0

0

0

#

#

#

#

0

0

0 0

0

1

I

1

R

0

0

0

0

0

0

0

0

0

0

0

0

2

2

1

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

# # #

I

0 0

# # #

1 0

0 0

# # #

0

0 0

# # #

1

C D

0 0

0 0

0 0 1 0

1 1

2 2

2 3

1 1

R

C D Cornea O A 7d

Iris

1/0 3/2 2/2 2/2

1 1/0 4 2/2

Conjunctivae

"O = opacity; A = area of opacity; R = redness; C = chemosis; D = discharge; u = ulceration; # = killed; b = bloody discharge; x = evaluation not possible owing to seve)e corneal opacity; n = evaluation not possible owing to necrotic changes; Opacity n/n = opacity grade lower section/opacity grade upper section; Area n/n = area of opacity lower section/area of opacity upper section.

Alternative methods to the Draize test most probably attributable to denaturation of superficial corneal proteins in the absence of protective mechanisms. Irritation in vivo results in congestion of the conjunctival vascular bed and escape of intravascular fluids, whereas exposure to the C A M results in destruction of the blood vessels with haemorrhage and coagulation. Thus, according to Fraizer (1990), the B E - C A M assay may be considered only as a correlative test and therefore may be adequate for screening purposes, whereas mechanistic tests are required for replacements. A further limitation of the B E - C A M assay is that differentiation between severe irritation and a corrosive effect is not possible. The BE~EAM assay is also unable to monitor the time-course of eye damage, which is essential and required for adequate evaluation of effects. Like other tests in vitro, the B E - C A M assay does not permit evaluation of sensory irritation or discomfort of the animals. It must be emphasized that this is not intended to call into question the value of this assay in general for evaluating substances of a defined class. Nevertheless, the B E - C A M assay was established to test the predictive value, not only for pure chemicals of certain classes but also for dyes and complex formulations, all of which have to be evaluated for their potential to induce irritancy or corrosion. Ultrasonic pachymetry provided a fairly good correlation between corneal thickness and clinical observations, except for the substance inducing corrosive changes. The method demonstrated high sensitivity and good reproducibility. Measuring times were short (approx. 5 min per eye), the instrument was simple to operate and results were easy to interpret. F r o m the data it may also be concluded that it is sufficient to evaluate the thickness of the inferior pole of the cornea, since this is the section most affected when substances are administered into the lower conjunctival sac. In future, when a larger number of substances have been tested, it should be possible to establish a cut-off thickness separating irritants from non-irritants. The accuracy of corneal pachymetry could lessen inter- and intralaboratory discrepancies in scoring (Weil and Scala, 1971), thereby reducing the n u m b e r of animal tests. Studies have shown that lower dose volumes predict human eye irritants more accurately and cause less pain to animals (Freeberg et al., 1984; Griffith et al., 1980; Williams et al., 1982). As mild irritants or slight differences may not be detected at reduced volumes, they may be identifiable by measurement of corneal thickness, owing to the high sensitivity of this method. Thus, use of ultrasonic pachymeters might permit application of small volumes without loss in sensitivity of the animal model, thereby reducing the discomfort of the animals.

REFERENCES

Draize J. H., Woodard G. and Calvery H. O. (1944) Methods for the study of irritation and toxicity of sub-

1067

stances applied topically to the skin and the mucous membranes. Journal of Pharmacology and Experimental Therapeutics 82, 377-390. ECETOC (1988) Eye irritation testing. Monograph no. 11, Brussels. EEC (1983) Criteria for irritation/corrosion. Directive 83/467/EEC. EEC (1984) Eye irritation. Directive 84/449/EEC. EPA (1981) Eye irritation testing--an assessment of methods and guidelines for testing materials for eye irritancy. EPA document 560/11-82-001, NTIS, Springfield, VA. EPA (1984) Pesticide Assessment Guidelines--Subdivision F: Hazard evaluation--humans and domestic animals; $ 81-4. Primary eye irritation study, pp. 55a-59. NTIS, Springfield, VA. EPA (1988) Standard evaluation procedure: eye irritation studies. EPA document 540/09-88-105, NTIS, Springfield, VA. EPA (1989) TSCA Test Guidelines; $ 798.4500 'Primary eye irritation'; Federal Register 40, 480-485. Fraizer J. M. (1990) Validation of in vitro models. Journal of the American College of Toxicology 9, 355359. Freeberg F. E., Griffith J. F., Bruce R. D. and Bay P. H. S. (1984) Correlation of animal test methods with human experience for household products. Journal of Toxicology-Cutaneous & Ocular Toxicology 1, 53. Griffith J. F., Nixon G. A., Bruce R. D., Reer P. J. and 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 hazards to the human eye. Toxicology and Applied Pharmacology 55, 501-513. Jacobs G. A. and Martens M. A. 0989) An objective method for the evaluation of eye irritation in vivo. Food and Chemical Toxicology 27, 255-258. Kalweit S., Besoke R., Gerner I. and Spielmann H. (1990) A national validation project of alternative methods to the Draize rabbit eye test. Toxicology in Vitro 4, 702706. Kennah H. E., Hignet S., Laux P. E., Dorko J. D. and Barrow C. S. (1989) An objective procedure for quantitating eye irritation based upon changes in corneal thickness. Fundamental and Applied Toxicology 12, 258-268. Kobel W. and Gfeller W. (1985) Distribution of eye irritation scores of industrial chemicals. Food and Chemical Toxicology 23, 311-312. Luepke N. P. (1985) Hen's egg chorioallantoic membrane test for irritation potential. Foodand Chemical Toxicology 23, 287-291. OECD 0983) Provisional Data Interpretation Guides for Initial Hazard Assessment of Chemicals; no. 2.3., Acute eye irritation/corrosion. ENV/CHEM/CM/83.3. OECD (1987) Guidelines for Testing of Chemicals, Section 4--Health Effects; no. 405, Acute eye irritation/corrosion. van Erp Y. H. M. and Weterings P. J. J. M. (1990) Eye irritancy screening for classification of chemicals. Toxicology in Vitro 4, 267-269. Weil C. S. and Scala R. A. (1971) Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests. Toxicology and Applied Pharmacology 19, 276-360. Weterings, P. J. J. M. and van Erp Y. H. M. (1987) Validation of the BECAM assay: an eye irritancy screen test. In Alternative Methods in Toxicology. No. 5. Edited by A. M. Goldberg, pp. 515-521. Mary Ann Liebert, New York. Williams S. S., Graepel G. J. and Kennedy G. L. (1982) Evaluation of ocular irritancy potential: intralaboratory variability and effect of dosage volume. Toxicology Letters 12, 235-241.