USE OF THE PORCINE CORNEAL OPACITY AND REVERSIBILITY ASSAY (PORCORA) FOR TESTING DETERGENT AND CLEANING PRODUCTS IDENTIFIED AS IN VIVO

CATEGORY 1 (CAT. 1) DUE TO PERSISTENCE OF OCULAR TISSUE DAMAGE ACCORDING TO THE UN GLOBALLY HARMONIZED SYSTEM (GHS) OF CLASSIFICATION

Chantra Eskes1, Caroline Bertein2*, Micheal Carathers3*, George DeGeorge3, Martina Hermann4, Penny Jones5, Pauline McNamee6, Bennett Varsho3

1SeCAM, Magliaso, Switzerland; 2International Association for Soaps, Detergents and Maintenance Products, Brussels, Belgium; 3MB Research Labs, Spinnerstown (PA), U.S.A.; 4Henkel AG & Co. KGaA, Duesseldorf, Germany; 5Unilever Safety and Environmental Assurance Centre, Bedford, U.K.; 6The Procter & Gamble Company, Egham, U.K.

PURPOSEPURPOSE

The UN GHS for classification was adopted by the European Union by means of the Regulation on Classification, Labelling and Packaging of

substances and mixtures (EC CLP, 2008). The EU CLP established amongst others, the use of lower concentration limits for the additivityapproach for the classification of mixtures in the absence of data, as implemented since June 2015. This leads to the over-labelling of many

detergents and cleaning products that did not require up to then classification according to the previous EU Dangerous Preparation Directive(DPD) classification system (EU, 1999). This over-labelling is corroborated by animal, in vitro and human data. As a consequence, daily usedetergents such as hand dishwashing liquids may display a “corrosive” label similar to truly corrosive products such as alkaline drain cleaners. The

resulting over-labelling can confuse end-users by over-estimation of the real risk. Or in contrary, it can lead to end-users’ trivialization of labelling incases where it is truly merited.

The International Association for Soaps, Detergents and Maintenance Products (A.I.S.E.) believes it is critical to accurately predict the hazardpotential to humans of detergents and cleaning products to ensure correct classification, and that no animal testing should be conducted on

finished products for that purpose. The EU CLP encourages in particular the use of tiered weight-of-evidence strategies and of in vitro assays toavoid unnecessary use of animal testing while ensuring accurate classification. For this reason, A.I.S.E. initiated in 2010 an in vitro program to

ABSTRACT IX - 1019 ABSTRACT IX - 1019

Currently adopted OECD test methods for eye hazard do not allow identification of UN GHS Cat. 2 chemicals. A key reason is that these assays

may not cover all relevant mechanisms of ocular damage. In particular, test methods adopted to identify UN GHS Cat. 1 were not designed topredict chemicals classified in vivo due to tissue effects persisting 21 days after exposure. In contrast, PorCORA was designed to specifically

address (ir)reversibility of corneal damage in ex vivo porcine corneas cultured for 21 days. Detergent and cleaning products having a balanceddistribution of Cat. 1/Cat. 2/No Cat. based on existing, historical in vivo data were tested using PorCORA with an extended washing procedure. All5 in vivo Cat.1 products based on persistence of effects, as well as 9 of 12 in vivo non-Cat. 1 products were correctly identified by the assay.

PorCORA appears therefore as a promising assay for further investigation e.g. within Defined Approaches for eye hazard identification.

RESULTSRESULTS

FormulationPhysical

stateIn vivo

PorCORA

(original)

PorCORA

(modified

rinsing)

Additivity

approachReasons for Cat 1 classification

Laundry powder #1 S LVET – Cat. 1 Reversible n.t. Cat. 1 CO = 4 in 1/3 animals, otherwise Cat 2 (CO)

Laundry liquid #1 L LVET – Cat. 1 Cat. 1 Cat. 1 Cat. 1 Persistence (CO+CR) in 2/6 animals, otherwise Cat 2 (CO+CR+CC)

Laundry liquid #2 L LVET – Cat. 1 Cat. 1 Cat. 1 Cat. 1 Persistence (CR) in 2/3 animals, otherwise Cat 2 (CR)

HDWL #2 L LVET – Cat 1 n.t. Cat. 1 Cat. 1 Persistence (CO) in 1/3 animals, otherwise Cat 2 (CO+CR)

HDWL #4 L LVET – Cat 1 Cat. 1 Cat. 1 Cat. 1 Persistence (CR) in 2/3 animals, otherwise Cat 2 (CO+CR+CC)

HDWL #5 L LVET – Cat 1 Cat. 1 Cat. 1 Cat. 1 Persistence (CO, CR) in 1/3 animals, otherwise Cat 2 (CR+CC)

Laundry powder #2 S LVET – Cat. 2A Reversible n.t. Cat. 1 not applicable

Laundry powder #3 S LVET – Cat. 2B Reversible n.t. Cat. 1 not applicable

Laundry liquid #3 L LVET – Cat. 2A Cat. 1Cat. 1

borderlineCat. 1 not applicable

HDWL #9 L LVET – Cat. 2A Cat. 1 Cat. 1 Cat. 1 not applicable

APC #1 L LVET – Cat. 2A Reversible Reversible Cat. 1 not applicable

APC #2 L LVET – Cat. 2A Cat. 1 Reversible Cat. 1 not applicable

Laundry powder #4 S LVET – No Cat. Reversible n.t. Cat. 1 not applicable

Laundry powder #5 S LVET – No Cat. Reversible n.t. Cat. 1 not applicable

Laundry liquid #4 L LVET – No Cat. Cat. 1 Reversible Cat. 1 not applicable

HDWL #16 L LVET – No Cat. Cat. 1 Cat. 1 Cat. 1 not applicable

APC #3 L LVET – No Cat. Cat. 1 Reversible Cat. 1 not applicable

APC #4 L LVET – No Cat. Cat. 1 Reversible Cat. 1 not applicable

APC: All Purposes Cleaner; Cat.: Category; CC= conjunctival chemosis; CO= Corneal opacity; CR= Conjunctival redness; HDWL: Hand dish wash

liquid; LVET: Low Volume Eye Test.; n.t.: not tested.

International Association for Soaps,Detergents and Maintenance Products

In vivo drivers for Cat. 1 classification (historical data from A.I.S.E. member companies)

PorCORA (original and modified rinsing) predicted classifications as compared to

in vivo and additivity approach classifications according to the UN GHS / EU CLP criteria

Only: only driver of classification; Comb.: combined with other

driver(s) of classification

0

10

20

30

40

50

60

70

80

90

100

CO >= 3 Iris >= 1.5 CO=4 Persistence

Draize

A granular laundry

B liquid laundry

C dishwashing L

D all purpose

Only Comb. Only Comb. Only Comb. Only Comb.

CO ≥ 3 Iritis > 1.5 CO=4 Persistence

Granular laundry (n=5)

Liquid laundry (n=41)

Dishwashing liquid (n=19)

All purpose cleaner (n=22)

Draize (n=87)

0

10

20

30

40

50

60

70

80

90

100

CO >= 3 Iris >= 1.5 CO=4 Persistence

LVETA granular laundry

B liquid laundry

C dishwashing L

D all purpose

Only Comb. Only Comb. Only Comb. Only Comb.

CO ≥ 3 Iritis > 1.5 CO=4 Persistence

Granular laundry (n=20)

Liquid laundry (n=15)

Dishwashing liquid (n=23)

All purpose cleaner (n=16)

LVET (n=74)

●

Example 3: Laundry liquid #4 (No Cat.)

*Corresponding authors:

For A.I.S.E.: caroline.bertein@aise.eu

For PorCORA: mcarathers@mbresearch.com

avoid unnecessary use of animal testing while ensuring accurate classification. For this reason, A.I.S.E. initiated in 2010 an in vitro program toinvestigate the applicability of in vitro test methods for eye irritation and serious eye damage to reliably classify detergent and cleaning products

mixtures.

In particular, persistence (irreversibility) rather than severity of effects was found to be present in the majority of detergents and cleaning

formulations classified as Cat. 1 based on historical in vivo data and according to the UN GHS / EU CLP criteria (see results). For that reason, thePorCORA assay was selected among the assays investigated, due to the fact that it was shown to correctly identify test chemicals causing both

reversible and irreversible serious eye damage as seen in the in vivo rabbit eye test (Donahue et al., 2011; Piehl et al., 2011).

MATERIAL & METHODSMATERIAL & METHODS

The PorCORA makes use of an air-interface culture system to sustain ex vivo porcine corneas in culture for 21 days (similar to the in vivo

observation period described in TG 405), and determines whether corneal injury once inflicted will reverse, i.e., heal (Piehl et al., 2010).

Corneal injury reversibility is measured using sodium fluorescein stain retention which can detect compromised epithelial barrier function.

Treatment is conducted 24 hours after the initiation of cultures. The culture media is removed from the dishes, and corneal surfaces are treatedtopically with 10 mL / 20 mg of test material, with Phosphate Buffered Solution (PBS) for the negative control and with 10% NaOH or 100% EtOH

for the positive controls.

Due to the viscous nature of the liquid formulations tested, in addition to the original rinsing, a modified rinsing step was used to ensure

appropriate removal of the formulations (increased volume of PBS used for washing from 2x1ml to 2x5ml). Once the corneal surface appearedfree of test substance, corneas were transferred to a new sterile dish and media was added to the plate. Culture media was replaced daily.

A total of 18 formulations were tested representing 6 Cat. 1, 6 Cat. 2 and 6 No Cat. materials based on historical invivo data (from both the classical Draize Test and the Low Volume Eye Test (LVET)) according to the UN GHS / EU CLP

criteria. These formulations covered the four categories of detergent and cleaning products of interest, representing 5laundry powders, 4 laundry liquids, 5 hand dish wash liquids and 4 all purpose cleaners.

CONCLUSIONSCONCLUSIONS

�All 5 in vivo UN GHS Cat.1 products based on persistence of effects, as well as 9 of 12 in vivo non-

Cat. 1 products were correctly identified by the PorCORA assay. Furthermore, PorCORA betterpredicted the in vivo eye hazards when compared to the additivity approach.

�The only false negative material observed was the laundry powder #1, which is a UN GHS / EU CLPCat. 1 based on corneal scores of 4 that fully recovered by day 21 based on the LVET data, and would

be non-classified if the former EU DSD / DPD classification criteria was applied. This formulation wasalso under-predicted by the other in vitro assays investigated by A.I.S.E.

� The PorCORA assay, with the modified rinsing conditions, appears therefore to be a promisingassay for the identification of UN GHS / EU CLP Eye Cat. 1 Detergent and Cleaning Productsclassified in vivo based on persistence of effects.

� Further work would be useful to:

• Enlarge the applicability domain of the PorCORA assay based on materials having Draize in

vivo data (e.g., surfactants) and assess its within- and between- laboratory reproducibility,

• Investigate the use of PorCORA in combination with other assays within Defined Approachesto support identification of UN GHS / EU CLP Eye Cat. 2.

REFERENCESREFERENCESEU (1999). Directive 1999/45/EC of the European Parliament and of the Council of 31 May 1999 concerning the approximation of laws, regulations and administrative

provisions of the Member States relating to the classification, packaging and labelling of dangerous preparations. Official Journal of the European Communities L 200, 1-68.

EU (2008). Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on Classification, Labelling and Packaging of substances

and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. Official Journal of the European Union L353,

1-1355.

Donahue D.A., Avalos J., Kaufman L.E., Simion F.A., Cerven D.R. (2011). Ocular irritation reversibility assessment for personal care products using a porcine corneal culture

assay. Toxicology In Vitro 25, 708-714.

Piehl M., Gilotti A., Donovan A., DeGeorge G., Cerven D. (2010). Novel cultured porcine corneal irritancy assay with reversibility endpoint. Toxicology In Vitro 24, 231-239.

Piehl M., Carathers M., Soda R., Cerven D., DeGeorge G. (2011). Porcine Corneal Ocular Reversibility Assay (PorCORA) predicts ocular damage and recovery for global

regulatory agency hazard categories. Toxicology In Vitro 25, 1912-1918.

UN (2015). United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS). ST/SG/AC.10/30/Rev.6, Sixth Revised Edition, New York and

Geneva: United Nations. Available at: http://www.unece.org/trans/danger/publi/ghs/ghs_rev06/06files_e.html.

0%

20%

40%

60%

80%

100%

PorCORA original rinsing (n=17)

PorCORA original +

mod. rinsing (n=18)

UN GHS / EU CLP additivity approach

(n=18)

53%

78%

33%

20% 17% 0%

58%

25%

100%

Concordance

False negatives

False positives

liquid; LVET: Low Volume Eye Test.; n.t.: not tested.

Example 2: Laundry liquid #3 (Cat. 2)Example 1: HDWL #4 (Cat. 1)

driver(s) of classification

Cornea Scores

Mean Score

Day No.

Clear

Corneas

4 3 4 4 3.8 1 0

4 4 4 4 4 2 0

4 4 4 4 4 3 0

4 1 4 4 3.3 7 0

4 4 1 4 3.3 10 0

4 4 4 1 3.3 14 0

1 4 4 4 3.3 21 0

Note: Corneas are not individually identified.

Cornea Scores

Mean Score

Day No.

Clear

Corneas

4 3 2 3 3 1 0

4 3 4 4 3.8 2 0

4 4 3 4 3.8 3 0

2 4 4 4 3.5 7 0

3 3 2 1 2.3 10 0

1 0 0 1 0.5 14 2

0 0 0 1 0.3 21 3

Note: Corneas are not individually identified.

Cornea Scores Mean Score

Day

No. Clear

Corneas

3 3 4 4 3.5 1 0

4 4 2 3 3.3 2 0

2 2 4 4 3 3 0

1 1 0 3 1.3 7 1

1 0 0 1 0.5 10 2

0 0 0 0 0 14 4

ND ND ND ND ND 21 ND

Note: Corneas are not individually identified.

ND = No Data; corneas were preserved because

all corneas no longer retained SF stain.