Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcular^TM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays

Figures & data

Figure 1. (a) Cross-section of healthy human cornea (corneal thickness ca. 550 µm), provided courtesy of Hans E. Grossniklaus, MD. (b) Full thickness bovine cornea (corneal thickness ca. 850–1000 µm) from the bovine corneal opacity and permeability (BCOP) assay (sterile, deionised water, 10-min exposure, 2-h post exposure). Image provided courtesy of Institute for In Vitro Sciences. (c) Summary of depth of injury prediction model, demonstrated using a cross section of a rabbit cornea (corneal thickness ca. 400 µm), modified from Scott et al [4]. Note that minimal irritation comprises ‘non’ and ‘slight’ irritation.

Table 1. Table showing the number of formulations tested, organised according to their EPA eye hazard classification based on historical in vivo rabbit eye data.

EPA Category	Criteria	Personal Protective Equipment (PPE), Signal Word, and Precautionary Statement	Number of Formulations
I	Corrosive (irreversible destruction of ocular tissue) or corneal involvement or irritation persisting for more than 21 days	Goggles face shield, or safety glasses. DANGER. Corrosive. Causes irreversible eye damage	7
II	Corneal involvement or irritation clearing in 8–21 days	Goggles face shield, or safety glasses. WARNING. Causes substantial but temporary eye injury	7
III	Corneal involvement or irritation clearing in 7 days or less	Protective eyewear if appropriate. CAUTION. Causes moderate irritation	8
IV	Minimal effects clearing in less than 24 hours	No signal word or statement required	7
Total	–	–	29

Note that while the signal words and precautionary language differ for each category, importantly, the personal protective equipment requirements only differ across the Category I/II and Category III/IV border.

Figure 2. Schematics showing (a) the classification flowchart for DA-EO + BCOP for EPA hazard classification of eye corrosion/irritation of agrochemical formulations using the EpiOcular^TM EIT and BCOP assays, for formulations predicted to be non- or minimally irritating, and (b) the classification flowchart for DA-BCOP for EPA hazard classification of eye corrosion/irritation of agrochemical formulations using the BCOP assay, for formulations predicted to be irritating. Acronyms: IVIS: in vitro irritancy score; DOI: Depth of Injury.

Table 2. Table showing the compatibility of each of the test systems, EpiOcular^TM EIT or BCOP, with different properties of a test material.

Property	EpiOcular^TMEIT	BCOP
Water soluble	✓	✓
Water insoluble	✓	✓
High vapour pressure	✓	✓
Low vapour pressure	✓	✓
Oxidizing components	✓	If histopathology is included
DNA reactive components	Incompatible	If histopathology is included

Table 3. Data from 29 agrochemical formulations that were tested as well as the formulation type.

Formulation information		Historical in vivo rabbit eye information		EpiOcular^TM EIT	BCOP
Formulation code	Formulation type	Animals tested (Driving classifi-cation)	Study description	Mean cell viability (%)	Opacity scores	OD490	IVIS	Histopathology findings
D	EC	1 (1)	CO (score 1) on D21	13.2	10.3	0.624	19.7	Severe
G	EC	3 (1)	CO (score 1) in a single animal on D21	8.7	71.9	0.684	81.9	Severe
J	EC	1 (1)	CO (score 2) on D21	27.8	11	0.267	15	Severe
F	SL	6 (NR)	Study report not provided; submitting company provided mean scores for tested animals and notation that positive response noted on D21 in at least 1 animal	4.8	12.3	1.775	39	Severe
H	SL	3 (1)	CO (score 1) in a single animal on D21	5.2	8	1.239	26.6	Severe
I	SL	6 (1)	CO (score 2) in a single animal on D21	5.3	75.7	0.12	77.5	Severe
V	SL	3 (2)	CR in 2 animals; cleared by D7	3.7	21	2.571	59.6	Moderate
U	EC	3 (1)	CO and CR effects; cleared by D14	9.8	22.3	0.046	23	Moderate
X	EC	3 (1)	CO in a single animal, cleared by D14	4	5	0.352	10.3	Moderate
R	SL	3 (1)	CO in a single animal on D7, cleared by D14	2.2	5.7	1.562	29.1	Moderate
AB	EC	3 (1)	CO and CR effects; cleared by D7	3.5	11.7	0.456	18.5	Moderate
K	SL	3 (1)	CO in a single animal on D10, cleared by D14; no CO effects in other 2 animals, conjunctival effects in other 2 animals cleared by D4	15.35	0	0.001	0	Minimal
Q	SL	3 (1)	CO in a single animal on D14, cleared by D21	29.1	1.3	0.016	1.6	Moderate
AC	EC	3 (NR)	Study report not available; classification of EPA Cat III provided by submitting company	33	3	0.003	3.1	Mild
W	SL	3 (1)	CO in a single animal on D1, cleared by D7	33.4	4	0.134	6	Mild
E	EC	1 (1)	CO (score 2) on D21	28.5	11	−0.002	11	Mild
L	EC	3 (NR)	Study report not provided; classification of EPA Cat III provided by submitting company	22.75	0.7	−0.001	0.6	Minimal
S	SL	3 (1)	CO, CR, CC on D4, cleared by D7	26.3	1.7	0.011	1.8	Mild
O	SL	3 (3)	No effects in animals tested	36.6	1.7	0.013	1.9	Minimal
Y	EC	3 (1)	CO in a single animal on D14, cleared by D21	19.8	2.3	0.028	2.7	Mild
AA	EC	3 (1)	CO in a single animal on D14; cleared by D21	34.6	2	0.003	2.1	Minimal
A	EC/ME	3 (3)	Minimal effects; no positive scores up to 7 days	93.7	1.3	−0.01	1.2	Minimal
B	SC	3 (3)	No effects in animals tested up to 72 hours	104.6	2	−0.005	1.9	Minimal
C	SC	9 (9)	No effects in animals tested	102.7	0	−0.003	−0.1	Minimal
N	SC	3 (3)	Minimal effects; cleared by D3	85.2	−0.3	0.009	−0.2	Minimal
P	SC	3 (3)	Study report not provided; classification of EPA Cat IV provided by submitting company	101.4	1	0.001	1	Minimal
M	SL	3 (3)	No effects after 24 hours	71.1	0.7	0.014	0.9	Minimal
T	SC	3 (1)	CR in a single animal on D2, cleared by D4	78	1.7	0.014	1.9	Mild
Z	EC	3 (1)	CR response cleared by D3	91.2	4	−0.005	3.9	Minimal

Included are data from the EpiOcular^TM EIT (mean cell viability [%]), the BCOP (opacity score, optical density at 490 nm (OD₄₉₀), IVIS, and histopathology findings), and historical in vivo rabbit eye information (number of animals tested, number of animals driving classification, and the study description).

Abbreviations: Cat: Category; CC: conjunctival chemosis; CO: corneal opacity; CR: conjunctival redness; DX: experimental day number X; EC: emulsifiable concentrate; EC/ME: microencapsulated emulsifiable concentrate; NR: not reported; SC: suspension concentrate; SL: soluble liquid.

Table 4. Table showing the EPA categories predicted with DA-EO + BCOP, DA-BCOP, and historical in vivo rabbit eye data for each of the 29 formulations.

Formulation code	Formulation type	Predicted EPA classification using DA-EO + BCOP	Predicted EPA classification using DA-BCOP	Predicted EPA classification based on historical in vivo rabbit eye data
D	EC	I	I	I
G	EC	I	I	I
J	EC	I	I	I
F	SL	I	I	I
H	SL	I	I	I
I	SL	I	I	I
V	SL	I	I	III
U	EC	II	II	II
X	EC	II^a	II^a	II
R	SL	II	II	II
AB	EC	II	II	III
K	SL	III	IV	II
Q	SL	II^a	IV	II
AC	EC	III	III	III
W	SL	III	III	III
E	EC	III	III	I
L	EC	III	IV	III
S	SL	III	IV	III
O	SL	III	IV	IV
Y	EC	III	IV	II
AA	EC	III	IV	II
A	EC/ME	IV	IV	IV
B	SC	IV	IV	IV
C	SC	IV	IV	IV
N	SC	IV	IV	IV
P	SC	IV	IV	IV
M	SL	IV	IV	IV
T	SC	IV	IV	III
Z	EC	IV	III	III

Colors indicate the level of alignment across category predictions. Green indicates alignment between two or more of the predicted categories. Orange indicates a misalignment of one category prediction, and resulting in no change to risk management requirements for personal protective equipment. Red indicates a misalignment of one or more category prediction, resulting in a change to risk management requirements for personal protective equipment. Specifically, eye goggles are required when using agrochemical formulations labelled EPA Categories I (corrosion) and II (moderate irritation), but are not required when using those labelled EPA Categories III (mild irritation) and IV (minimal or no irritation), though for EPA Category III, the EPA may require eye googles, if they are deemed appropriate.

^a Indicates that histopathology conducted for the BCOP method suggests a mild to moderate eye irritation response, corresponding to either an EPA Category II or III. When this occurs, EPA Category II (the more conservative option) is included in the table below.

Scott L, Eskes C, Hoffmann S, et al. A proposed eye irritation testing strategy to reduce and replace in vivo studies using bottom-up and top-down approaches. Toxicol in Vitro. 2010;24(1):1–9. doi:10.1016/j.tiv.2009.05.019.

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