Appendix 8.13 Predictive capacity of the Vitrigel-EIT for 114 chemicals
considering DRD

Predictive capacity and relevance
Total 114 test chemicals were tested at the lead laboratory (Appendix 8.14 and Appendix 8.15,
law data).

Hence, the predictive capacity was evaluated by the 114 results comprise the data for

27of the 36 chemicals during Phase III shown in Table 21 and for 87 chemicals obtained at thelead laboratory shown in Appendix 8.14. The test chemicals were selected to ensure that a diverse
range of substances were represented, and aspects such as eye-irritant level per UN GHS
categories, physical state, chemical class. Also, quality and reliability of in vivo data for thechemicals were carefully considered by reference to the Draize eye test Reference Database(Adriaens et al., 2014; Barroso et al, 2017). The 114 test chemicals are composed of 89 liquids
and 25 solids.

Also, their contents are 27 Category 1 chemicals, 1 Category 2 or higher, 1Category 2, 5 Category 2A or higher, 15 Category 2A, 2 Category 2B or higher and 11 Category
2B chemicals, and 52 No Category chemicals by UN GHS classification. There were 27 codedchemicals tested for Phase III and 87 non-coded chemicals were tested at the lead laboratory.
These 114 test chemicals were examined by the Vitrigel-EIT method in accordance with theprotocol versions described in Chapter 3.1.3.4 and Appendix 8.8. However, the temperature atwhich all measurements were made during the chemical exposure experiments was strictly
controlled at 28±2°C (Table 19 and Appendix 8.8). Thus we consider this data sufficient forassessing the suitability of the Vitrigel-EIT method for use in a bottom-up approach for identifying
ocular non-irritants and in a top-down approach for identifying UN GHS Category 1 ocularirritants. In a bottom-up approach, 75 of the test chemicals were classified as irritant and the other39 as non-irritant, with results for 85 of the 114 test chemicals matching their UN GHS categories.

In contrast, 8 of the 62 test chemicals classified as irritants by in vivo data were identified as nonirritants, a false-negative rate of 13%. Additionally, 21 of the 52 test chemicals classified as nonirritants under UN GHS were identified as irritants, a false-positive rate of 40%. Thus, theVitrigel-EIT method achieved a sensitivity of 87% (54/62), a specificity of 60% (31/52), and anaccuracy of 75% (85/114), as shown in Table A8.13-1. Data from the lead laboratory also
demonstrated that predictive capacity could be improved by expanding the sample size. Forexample, the specificity achieved in Phase III of this validation study was lower than that obtained
from the data of 52 non-irritants resulted in a higher specificity. The list of test chemicals thatwere either false negative or false positives is shown in Table A8.13-3.
On the other hand, analysis per a top-down approach for identifying UN GHS Category 1 ocularirritants was also performed as a part of this validation study, as shown in Tables A8.13-2.
Regarding identifying test chemicals classified as UN GHS Category 1 in a top-down approach,the Vitrigel-EIT method demonstrated a sensitivity of 89% (24/27), a specificity of 41% (36/87),
and an accuracy of 53% (60/114). Specificity is an important criterion in a top-down approach,which means that Vitrigel-EIT method is not well suited for use in a top-down approach toidentifying UN GHS Category 1 ocular irritants.

Table A8.13-1. Contingency table used for 114 test chemicals in a bottom-up approach
Vitrigel-EIT

UN GHS
Total
Sensitivity: 87% (54/62)
Specificity: 60% (31/52)
Accuracy: 75% (85/114)

Total

INI

Cat.1, 2A, 2B

54862

No Category

21

3152

7539114

Table A8.13-2. Contingency table used for 114 test chemicals in a top-down approach
Vitrigel-EITUN GHS

TotalINI

Cat.1

24327Cat.2A, 2B, No Category

51

3687

Total

75

39114

Sensitivity: 89% (24/27)
Specificity: 41% (36/87)
Accuracy: 53% (60/114)
Table A8.13-3. False test chemicals in a bottom-up approach for 114 test chemicals
No.*

Test chemicals

Rank

Applicability limitation
pH of 2.5% solution ≤ 5.0

11Lactic acid14

Captan22

Acetic acid (10%)

pH of 2.5% solution ≤ 5.0

41

3,3'-Dithiodipropionic acid

pH of 2.5% solution ≤ 5.0

46

2,6-Dichlorobenzoyl chloride

54

Camphene

55
6266

Insoluble after 5 m

False negative

pH of 2.5% solution ≤ 5.0
Protocol revised

Ethyl 2,6-dichloro-5-fluoro-beta-

pH of 2.5% solution ≤ 5.0

oxo-3-pyridinepropanoate
Glycolic acid (10%)

pH of 2.5% solution ≤ 5.0

Gluconolacton

pH of 2.5% solution < 5.0
after 10 m

67

Methyl amyl ketone

68

Methyl isobutyl ketone

69

N,N-Dimethylguanidine sulfate

70

Glycerol75

2-Ethoxyethyl acetate

76

Ethyl acetate

77

2,4-Pentanediol

78

Triethanolamine

85

Cyclohexanone

89

2,4-Pentanedione90

Butyl acetate

91

XyleneFalse positive

93

EDTA,di-potassium

94

3-Glycidoxypropyltrimethoxysilane

96

Ethyl trimethyl acetate

97

2,2-Dimethyl-3-pentanol

98

Betaine monohydrate

104

1,2,3-trichloropropane

108

2,4-Difuroronitrobenzene

109

Potassium tetrafluoroborate*

Each number corresponds to the number in Appendix 8.14.

Applicability domain
Analysis of the false-negative reactions shows that six of the eight false-negative chemicals wereacidic, and the 2.5% solutions used for exposure had a pH level lower than 5, as shown in Table
25. The TEER values of the HCE models after exposures to each of these six acidic test chemicals
that yielded false-negatives increased from their initial values. Interestingly, it was reported thatisolated rabbit esophageal mucosal epithelium and normal human bronchial epithelial cell layers
in culture displayed increased TEER values when exposed to weak acidic solutions (Farré et al.,2008; Oshima et al., 2012). On the other hand, one of the two non-acidic false-negative chemicals
were water-insoluble solids that were easily separated from the culture medium at room
temperature, as shown in Table A8.13-3. Therefore, the lead laboratory added two restrictions tothe applicability domain in consideration of above scientific rationales:Exclude all test chemicals that have a pH level of 5 or less in solution (affected 9 testedchemicals).

Exclude all solids that have both a logP value of 2.5 or more and a density of either lessthan 0.95 g/cm3 or over 1.10 g/cm3 (affected 3 test chemicals).

Under this applicability domain, 12 of the original 114 test chemicals were excluded, as shown inTables A8.13-4, which improve sensitivity from 87 to 98%, specificity from 60 to 61%, andaccuracy from 75 to 79%, as shown in Table A8.13-5.

Eight of the 36 test chemicals in Phase III are excluded under the new applicability domain:
No. 3-2

2-Benzyl-4-chlorophenol (insoluble)

No. 3-3

2,2-Dimethyl butanoic acid (pH ≤ 5)

No. 3-4

Captan (insoluble)

No. 3-5

tetra-n-Octylammonium bromide (insoluble)

No. 3-13

Myristyl alcohol (insoluble)

No. 3-14

2,6-Dichlorobenzoyl chloride (pH ≤ 5)

No. 3-15

Dibenzyl phosphate (pH ≤ 5)

No. 3-25

tetra-aminopyrimidine sulfate (pH ≤ 5)

After excluding these eight test chemicals, sensitivity improved from between 75 and 83% tobetween 88 and 94% (15 to 16/17), specificity changed from 42% to 36% (4/11), and accuracy
improved from between 64 and 69% to between 68 and 71% (19 to 20/28).
Of the 17 irritants, two others that yielded false-negatives were No. 3-18, camphene, and No. 333, alpha-hexylcinnamaldehyde. Camphene is a waxy, water-insoluble solid, and the falsenegative was due to the technique used for dissolving, as described in section 4.1.4 Phase III.
Alpha-hexylcinnamaldehyde is a water-immiscible liquid and was identified as an irritant by thelead laboratory (Yamaguchi, 2016). The reason for the discordance of the judgment is currently
under investigation although the classification of alpha-hexylcinnamaldehyde in several studies
in vivo was reported as NC and 2A or higher (Barroso et al, 2017).

Table A8.13-4-1. Limitations on applicability (pH level 5 or less in 2.5% solution) in a bottomup approachNo.*

Test chemical

Vitrigel-EIT

GHS categorypHresults

11

Lactic acid1

False negative

322

Acetic acid (10%)

1False negative441

3,3'-Dithiodipropionic acid2A

False negative446

2,6-Dichlorobenzoyl chloride

2A

False negative347

Dibenzyl phosphate

2A55

Ethyl

2,6-dichloro-5-fluoro-beta-

32B

oxo-3-pyridinepropanoate

False negative556

3-Chloropropionitrile

2B

562

Glycolic acid (10%)

2B

False negative

493

EDTA,di-potassium

NC

False positive5*

Each number corresponds to the number in Appendix 8.14.

Table A8.13-4-2. Limitations on applicability (solid chemicals with a logP value of 2.5 or more
and a density under 0.95 g/cm3 or over 1.10 g/cm3 in a bottom-up approach.
No.*

Test chemicalGHS

Vitrigel-EIT

categoryresults

LogP

Density(g/cm3)

10Acid red 92

27.13

2.16

132-Benzyl-4-chlorophenol

1

3.60

1.19

14

Captan

1

2.80

1.74

False negative*

Each number corresponds to the number in Appendix 8.14

Table A8.13-5. Contingency tables used for 102 test chemicals within the applicability domain inbottom-up approach.
Vitrigel-EIT
Cat.1, 2A, 2B
Not Classified

UN GHS
Total

Sensitivity: 98% (50/51)

I
50
2070

NI
1
3132

Total
5151102

Specificity: 61% (31/51)
Accuracy: 79% (81/102)