Toxicology in Vitro - COnnecting REpositories · 2016. 12. 9. · Classiﬁcation and labeling of industrial products with extreme pH by making use of in vitro methods for the assessment

Toxicology in Vitro 25 (2011) 1435–1447

Contents lists available at ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Classification and labeling of industrial products with extreme pH by making useof in vitro methods for the assessment of skin and eye irritation and corrosionin a weight of evidence approach

Julia Scheel a,⇑, Andreas Heppenheimer b, Elke Lehringer a, Juergen Kreutz a, Albrecht Poth b,Holger Ammann a, Kerstin Reisinger a, Norbert Banduhn a

a Henkel AG & Co. KGaA, Düsseldorf, Germanyb Harlan Cytotest Cell Research GmbH, Rossdorf, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 January 2011Accepted 18 April 2011Available online 27 April 2011

Keywords:Extreme pHCorrosionIrritationIn vitroClassificationLabeling

0887-2333 � 2011 Elsevier Ltd.doi:10.1016/j.tiv.2011.04.017

Abbreviations: AR, alkali/acid reserve; CCM, convCLP, classification, labeling and packaging regulatiomonobutyl ether; DPD, dangerous preparationssubstances directive; EPI-200-SIT, EpiDerm™ skin iharmonized system; GLP, good laboratory practice; Hisolated chicken eye; IRE, isolated rabbit eye; MEA, mopretreatment; MTT, 3-(4,5-dimethylthiazol-2-yl)-2mide; NHEK, normal human keratinocytes; NTA, n(quantitative) structure activity relationship; RhE, recoHSM, human skin model; WoE, weight of evidence.⇑ Corresponding author. Tel.: +49 211 797 2413; fa

E-mail address: [email protected] (J. Scheel

Open access under CC BY

Classification and labeling of products with extreme pH values (62 or P11.5) is addressed in chemicalslegislation. Following determination of pH and alkaline/acid reserve, additional in vitro tests are needed,especially to substantiate results less than corrosive. However, only limited experience with the practicalapplication of in vitro methods to determine appropriate classifications for pH extreme products is avail-able so far. Expert judgment and weight of evidence are given major roles under the globally harmonizedsystem of classification and labeling of chemicals (GHS) and should be performed on a sound data basis.We have used a tiered testing strategy to assess 20 industrial products (cleaning and metal pretreatment)regarding their corrosive and irritating properties towards human skin models in vitro in the EpiDerm™skin corrosion and/or skin irritation test. Nine dilutions of individual compounds were additionallytested. Non-corrosive samples were tested in the Hen’s egg test chorioallantoic membrane (HET-CAM).We demonstrate how data is combined in a weight of evidence expert judgment, and give examples ofclassification decisions. To our knowledge this is the first comprehensive analysis of industrial productswith extreme pH values to determine irritating and corrosive properties by making use of in vitro meth-ods in a weight of evidence approach.

� 2011 Elsevier Ltd. Open access under CC BY-NC-ND license.

1. Introduction and packaging regulation (CLP or EU GHS) (EU, 2008). Weight of

Appropriate classification and labeling with regard to the corro-sive and irritating potential of products to skin and eyes representsa fundamental requirement in chemicals legislation. Tiered weightof evidence (WoE) strategies are generally suggested for testingand assessment in accordance with international chemicals legisla-tion, specifically under the globally harmonized system of classifi-cation and labeling of chemicals (GHS) (UN, 2003, 2009) and itsregional implementation like the European classification, labeling

entional calculation method;n; DEGBE, diethylene glycoldirective; DSD, dangerousrritation test; GHS, globallySM, human skin model; ICE,noethanolamine; MPT, metal,5-diphenyltetrazolium bro-itrilotriacetic acid; (Q)SAR,

nstructed human epidermis;

x: +49 211 798 12413.).

-NC-ND license.

evidence means that all available information relevant for the pur-pose is considered together through expert judgment, like physico-chemical data, results of suitable in vitro tests, relevant animal dataand human experience, (Q)SAR, results from grouping and read-across approaches as well as human data, if available.

A generic approach to assess the dangerous/hazardous proper-ties of preparations in the EU consists in the application of calcula-tion methods which are routinely used and especially consideredsuitable in cases where no specific, possibly non-additive effectsare expected. With regard to mixtures or products with pH valuesin the extremely low acidic or high alkaline range, the CLP states –similar to previous EU legislation (DSD and DPD, (EU, 1976, 1999))– that the application of such generic calculation methods is insuf-ficient. ‘‘A mixture is considered corrosive to skin (skin corrosiveCategory 1) if it has a pH of 2 or less or a pH of 11.5 or greater. Ifconsideration of alkali/acid reserve suggests the substance ormixture may not be corrosive despite the low or high pH value,then further testing shall be carried out to confirm this, preferablyby use of an appropriate validated in vitro test.’’ This readsanalogously for effects on the eye: ‘‘A mixture is considered tocause serious eye damage (Category 1) if it has a pH 62.0 or

http://dx.doi.org/10.1016/j.tiv.2011.04.017mailto:[email protected]://dx.doi.org/10.1016/j.tiv.2011.04.017http://www.sciencedirect.com/science/journal/08872333http://www.elsevier.com/locate/toxinvithttp://creativecommons.org/licenses/by-nc-nd/3.0/http://creativecommons.org/licenses/by-nc-nd/3.0/

1436 J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447

P11.5. If consideration of alkali/acid reserve suggests the mixturemay not have the potential to cause serious eye damage despitethe low or high pH value, then further testing needs to be carriedout to confirm this, preferably by use of an appropriate validatedin vitro test’’ (EU, 2008).

The alkali/acid reserve referred to in the regulation was pro-posed over 20 years ago by Young et al. (1988). It represents a titra-tion method by which substances or preparations may be classifiedas irritating or corrosive to the skin which is in particular usefulwhen the irritating or corrosive properties of a preparation aredue to the acidity/alkalinity.

Quite a number of in vitro methods to assess skin and eye irri-tation/corrosion have been developed as alternatives to thein vivo rabbit tests (OECD, 2002a, 2002b), some of which haveundergone formal validation. Several in vitro methods to assesscorrosive effects of substances and mixtures to the skin have beenofficially adopted by OECD over the past decade including the hu-man skin model test (OECD, 2004a, 2004b, 2006). In contrast toskin corrosion which refers to the production of irreversible tissuedamage of the skin following the application of a test material, skinirritation refers to the production of reversible damage. Only re-cently OECD adopted an in vitro procedure that may be used forthe hazard identification of skin irritants by measuring cell viabil-ity in reconstructed human epidermis (RhE), which in its overalldesign closely mimics the biochemical and physiological proper-ties of the upper parts of the human skin. Currently three validatedtest methods, i.e. EpiDerm™, EpiSkin™ and SkinEthic™, are avail-able that comply with this guideline (OECD, 2010a).

For the assessment of eye irritation, some organotypic modelshave gained partial regulatory acceptance: The Bovine CornealOpacity and Permeability Test Method (BCOP) and the IsolatedChicken Eye (ICE) test method have been recently implementedat OECD level to screen for corrosives and severe eye irritants(OECD, 2009a, 2009b). In Europe, the HET-CAM (Hen’s Egg TestChorioallantoic Membrane) and the Isolated Rabbit Eye (IRE) testhave also been accepted for this purpose (EU, 2009). In addition,the Cytosensor Microphysiometer test method has gained valida-tion status for identification of severe irritants (water solublematerials) and not-classified (water-soluble surfactants and sur-factant-containing mixtures) and for which the OECD guideline iscurrently being drafted (OECD, 2010b). At the current stage,in vitro eye irritation methods may especially be useful as part ofWoE assessments rather than as stand-alone classificationmethods.

In this study, we have used a tiered testing strategy to generatedata for 20 industrial products (cleaners and metal pre-treatmentproducts) and 9 individual compounds to assess their corrosiveand irritating properties with EpiDerm™ human skin models(Epi-200) and in the HET-CAM. The information from the in vitrotests was assessed in the context of all available data, includinghistorical in vivo data for individual components in a weight of evi-dence approach.

2. Materials and methods

2.1. Test samples

Test samples were provided by Henkel AG & Co. KGaA, Düssel-dorf. All samples were liquids. Trade names are not disclosed dueto intellectual property reasons, but a description of the basicchemistry of the product classes is provided in Table 1, as well asthe CAS numbers and test concentrations of individual compounds(Table 2). Dilutions of compounds were prepared with purifiedwater (aqua bidest.). Controls and references are described belowin the context of the individual protocols.

2.2. Conventional calculation method (CCM) according to DPD

The conventional calculation method is a standard method inthe EU to provide an estimate of the hazardous properties of apreparation based on the classification of its ingredients (EU,1999). In the case that specific concentration limits have been as-signed to substances, these must be used for the calculation; in allother instances generic limits are applied. A preparation isconsidered

� corrosive, ifP

(Pcor/Lcor) P 1� irritating, if

P(Pcor/Lirr + Pirr/Lirr) P 1

Pcor/irr are the percentages by weight or volume of each corro-sive substance which is assigned to a corrosive (cor) or irritating(irr) classification in the preparation; Lcor/irr are the correspondingconcentration limits. For eye effects, two separate calculations areperformed to assess severe eye irritation and eye irritation. We re-fer to the calculation method and classification symbols of DPD andDSD which is still valid for the classification of products until June2015. Also, since not for all product constituents GHS classifica-tions were available at the time of the study, a similar exercisewith GHS provisions could not be conducted.

2.3. Determination of pH and the acid or alkali reserve

The procedure was performed as described previously (Younget al., 1988). In brief, for liquids, the pH of the undiluted liquidwas determined where possible. The acid/alkali reserve is usuallydetermined by titration with 2 N sodium hydroxide for acid andwith 2 N sulphuric acid for alkaline solutions. Acid/alkali reserve(AR) is expressed as NaOH/H2SO4 (equivalent) in [g] per 100 g li-quid required to adjust the pH to pH 4 (for acids) or pH 10 (foralkaline substances or products). A sample is classified as

� corrosive, if pH + 1/12 alkali reserve P 14.5 or pH � 1/12 acidreserve 6 �0.5� irritating, if pH + 1/6 alkali reserve P 13 or pH � 1/6 acid

reserve 6 1.

2.4. The EpiDerm™ human epidermis model

The EpiDerm™ skin model, produced by MatTek Corporation(Ashland, MA, USA), consists of normal human keratinoctyes(NHEK) cultured to form a multilayered, highly differentiated mod-el of the human epidermis in vitro. The model consists of organizedbasal, spinous, granular and cornified layers analogous to thosefound in vivo. The EpiDerm™ Tissues (surface area 0.63 cm2) werecultured on specially prepared cell culture inserts and shipped askits containing 24 tissues on agarose. Each batch was controlledby the manufacturer. Both the tissues and the provided culturemedia were tested for viral, bacterial, fungal, and mycoplasma con-tamination. The manufacturer also provides information on theET50 (50% reduction in tissue viability at a given time) for the stan-dard test chemical Triton X-100, and on tissue viability (testedwith MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide)) for each lot. All tests were performed according to GLP.

2.4.1. EpiDerm™ skin corrosion testThe experiments were performed according to OECD guideline

431 (OECD, 2004a). In these assays, the irritation potential of a testmaterial is typically determined by measuring cell viability in thetreated tissues by means of the colorimetric MTT reduction assayafter topical application onto the tissue surface. Cell viability isdetermined by evaluating enzymatic reduction of the yellow MTTtetrazolium salt to a blue formazan salt. Reduced MTT is quantified

Table 1Products with extreme pH: testing results and WoE assessments.

Product type Chemical description Classificationfor skin & eyecorrosion/irritationaccording toCCM (DPD)

CCM sum ofquotients

pH Alkali/acidreserve(Young)

HSM corrosivitytest (mean cellviability after3 min/1 h [%])

HSMirritation test(mean cellviability [%])b

HET-CAM WoEconclusions(scenariossee Fig. 1)

Classification

Alkaline cleaner forindustrial use 1

Contains organic compounds; salts oforganic and inorganic acids,

Table 1 (continued)

Product type Chemical description Classificationfor skin & eyecorrosion/irritationaccording toCCM (DPD)

CCM sum ofquotients

pH Alkali/acidreserve(Young)

HSM corrosivitytest (mean cellviability after3 min/1 h [%])

HSMirritation test(mean cellviability [%])b

HET-CAM WoEconclusions(scenariossee Fig. 1)

Classification

Metal pretreatmentproduct 1

Contains polymers, inorganic acidsand salts thereof (includingdihydrogenhexafluorotitanate(2�))

Not irritating(notclassified)

0.2 (R36)0.2 (R38)

2.0 Not irritating(acidreserve = 0.5)

Not corrosive(97.8/64.2)

Not irritating(91.4)

Not irritating(S = 10)

4 + 6 Not irritating toskin; irritating toeyes (GHS Cat 2(H319) or DPD Xi;R36)


Contains polymers, inorganic acidsand salts thereof (includingdihydrogenhexafluorotitanate(2�))


0.4 (R36)0.4 (R38)




Not irritating(S = 6)



Contains organic acids, inorganicacids and salts thereof (includingdihydrogenhexafluorozirconate(2�))

Xi; R36/38 0.7 (R34)0.7 (R41)a

1.9 (R36)1.9 (R38)

0.6 Irritating(acidreserve = 2.6)


Irritating(30.1)

Severelyirritating(Q = 1.76 at 25%dilution; 100%strongreactions < 10s)

3 + 5 Skinirritating + severelyirritating to theeyes (GHS Cat 1(H318), Cat 2(H315) or DPD Xi;R38-41)


Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))

Xi; R36/38 0.7 (R34)0.7 (R41)a

1.9 (R36)1.9 (R38)



Irritating(4.4)

Severelyirritating(Q = 2.45)



Contains organic acids, inorganicacids (including 1–5% phosphoricacid) and salts thereof, alcohols/ethers

Xi; R36/38 0.1 (R41)a

2.1 (R36)1.6 (R38)



Not irritating(52.9)[borderlineresult:individualtissues: 42.7;46.4; 69.6]

Severelyirritating(S = 17)




Xi; R36/38 0.6 (R34)0.6 (R41)a

1.7 (R36)1.7 (R38)



Irritating(3.9)

Irritating(Q = 1.76)

3 + 6 Irritating to skinand eyes (GHS Cat 2(H315, H319) orDPD Xi; R36-38)




0.2 (R36)0.2 (R38)




Not irritating(Q = 0.14)




Xi; R36/38 0.8 (R34)0.8 (R41)a

1.2 (R36)1.2 (R38)



Not irritating(50.5)[borderlineresult:individualtissues 40.9;43.5; 67.0]






0.3 (R36)0.3 (R38)



Not irritating(56.8)[borderlineresult:individualtissues: 48.5;54.5; 67.5]



1438J.Scheel

etal./Toxicology

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J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447 1439

photometrically with the results expressed as% viability in the testmaterial treated tissues relative to the negative control. The proce-dure will be described in brief. Initially, the ability of a test sub-stance to directly reduce MTT was assessed. The liquid testsamples (30 lL) were added to the MTT solution and incubatedfor 60 min at room temperature. If the MTT solution turned toblue/purple, it was assumed that the test chemical had reducedthe MTT. Since none of the test items reacted with the MTT solu-tion, an additional check with freeze-killed controls to checkwhether residual test compound binds to the tissue was not per-formed. On the day of receipt, tissues were aseptically removedfrom the transport agarose and transferred into cell culture plates.The tissues were pre-incubated at 37 �C in 5% CO2/95% air for atleast 1 h. After pre-incubation the tissues were transferred tonew cell culture plates containing fresh medium and were exposedtopically to the test chemicals. Liquids (50 lL) were applied with amicropipette. In addition to the test item a negative control (dis-tilled water) and a positive control (8 N KOH) was tested. The testitems and each control were tested in four tissues per sample, i.e.in duplicate for 3 and 60 min. After the treatment the tissues werestringently rinsed with buffered salt solution in order to removeresidues from the test item. Subsequently the viability of the tis-sues was determined using the MTT assay: Tissues were exposedto the MTT solution for 3 h at 37 �C in 5% CO2/95% air. After rinsing,the tissues were transferred into new cell culture plates and weresubmerged in isopropanol in order to lyse the cells and release theformazan salt. After at least 2 h extraction the optical density of theisopropanol extracts was determined photometrically at 570 nm.

The relative viability was calculated as percentage of the meanviability of the negative controls for each treatment interval. Themean of the two values from identically treated tissues for eachtreatment interval was then used to classify the test item. A testitem was considered to be not corrosive to the skin if the mean via-bility value after 3 min is P50% and/or the viability after 60 min isP15%. In case of a viability of

Table 2Selected compounds: testing results and literature data.

Type ofcompound

Chemical name andtest concentration

CAS Suppliera Classificationneatsubstance(DSD/suppliers)

Classificationtesteddilution(DSD)

pH (asmeasuredbeforetesting)

Alkali/acidreserve(Young)

HSMcorrosivitytest (meancellviabilityafter3 min/1 h[%])

HSM skinirritationtest(meancellviability[%])

HET-CAM Skin effects, in vivoanimal data

Eye effects, in vivoanimal data

Inorganicacid salt

Sodiumsilicate � 5H2O(molar ratio 1) 5%

10213-79-3

SilmacoN.V.,Belgium

C; R34 Xi; R36/38 13.1 Irritating(alkalireserve = 1.26)

Not tested Irritating(19.7)

Severelyirritating(Q = 2.14(25%); strongreactions (telquel,

caused slightkeratitis ECB(2000b)20% no testinformationNo OECD guidelinetest available

Organic acid Citric acid � 1H2O20%

5949-29-1

BrenntagGmbH,Germany

Xi; R36 Xi; R36 1.3 Irritating (acidreserve = 4.14)

Not tested Irritating(21.9)

Irritating(Q = 1.62)

Analogy to citric acid:not irritating (OECD 404)ECB (2000c)30% slightly or notirritating in three rabbitstudies OECD SIDS(2001b)20% no test information

Analogy to citricacid: highlyirritating (OECD405) ECB (2000c)and OECD SIDS(2001b)20% no testinformation

Detergent Alkyl ethersulphate C12-C14with EO, sodiumsalt 7%

68891-38-3

CognisGmbH &Co. KG,Germany

Xi; R38/41 Xi; R36 7.7 Not tested (noextreme pH)

Notcorrosive(90.6/107.0)


Severelyirritating(Q = 1.25 incombinationwith strongand earlycoagulationwhich alsoimpairedassessmentof H and L)

Undiluted irritating(OECD 404) Henkel AGand Co. KGaA (1994a &1994b) and CognisDeutschland GmbH andCo. KG (2007)7% no test information

Undilutedseverely irritatingto eyes (analogy totest with dilution)(OECD 405) CognisDeutschlandGmbH and Co. KG(2007)7% no testinformation

Inorganicacid 2a

Phosphoric acid10%

7664-38-2

BK-Giulini,Germany

Xi; R36/38(P10–

Table 3HET-CAM prediction model.

Reactiontimemethod [Q]score

Endpointassessment[S] score

Not irritatinga Irritating Severelyirritating(R41/Cat1)

Slightlyirritating

Moderatelyirritating

60.8 0–5 x>0.8–

Conventional Calculation Method (CCM)

classify as corrosive3

alkaline/acid reserve (Young et al.)

In vitro Human Skin Model Test: Corrosion (OECD 431)

not corrosive

pH ≤2.0 or ≥11.51/2

irritating or not irritating

corrosive

default

In vitro Human Skin Model Test: Irritation

(OECD 439)

irritating to the skin7

not irritating to the skin /

not classified

classify as irritating to the skin

SKIN EYE

HET-CAM5

thorough evaluation of existing information on ingredients

no severe effects expected

classify as irritating to the eye

severe effects expected

severe effects

classify as severely irritating to the eye / serious eye damage

additional information / WoE6

corrosive

corrosive


Scenario 1

not corrosive4

Scenario 2

products in this study

Scenario 3

Scenario 4

Scenario 6

Scenario 5


Conventional Calculation Method (CCM)

classify as corrosive3

alkaline/acid reserve (Young et al.)

In vitro Human Skin Model Test: Corrosion (OECD 431)

not corrosive

pH ≤2.0 or ≥11.51/2


corrosive

default

In vitro Human Skin Model Test: Irritation

(OECD 439)

irritating to the skin7

not irritating to the skin /

not classified

classify as irritating to the skin

SKIN EYE

HET-CAM5

thorough evaluation of existing information on ingredients

no severe effects expected

classify as irritating to the eye

severe effects expected

severe effects

classify as severely irritating to the eye / serious eye damage

additional information / WoE6

corrosive

corrosive


Scenario 1

not corrosive4

Scenario 2

products in this study

Scenario 3

Scenario 4

Scenario 6

Scenario 5


Fig. 1. Testing and evaluation scheme for skin and eye corrosion/irritation for industrial products with extreme pH values. Footnotes: 1If no overriding information isavailable (like human or animal data); this scheme is designed for product assessment w/o animal tests with the product. 2Exemptions possible for dilutions of substanceswith defined properties. 3Corrosivity as determined in the skin model is also considered relevant for the eye. 4Based on additional (more relevant) information. This can inprinciple result in a classification as skin irritating or not irritating. For eye effects, further evaluations are made. 5Since the HET-CAM is not formally validated, the result isonly used as supportive evidence for the evaluation in case of non-severe effects. Other in vitro tests may be appropriate as well. 6As a precaution the minimum classificationis ‘‘irritating’’. 7As a precaution, borderline results were considered irritating if CCM or AR indicated a classification as irritating to skin.


products 5, 8, 12) the CCM results in less or equally severe clas-sifications than AR and HSM. In ten cases CCM and AR showedthe same results (alkaline cleaners 1 and 3; acid cleaner 3; MPTproducts 1–4, 6, 7, 10), in another 10 cases CCM and HSM (acidcleaner 3; MPT products 1–4, 6, 7, 9, 10, 13). In eight cases allthree methods (CCM, AR and HSM) provided the same classifica-tion outcome all of which were acid products (acid cleaner 3;MPT products 1–4, 6, 7, 10). In addition, from the test results ofthe 17 acid products, a majority of 12 have the same classificationin AR and HSM (acid cleaners 1 and 3; MPT products 1–8, 10, 12).CCM most frequently (eleven times) lead to a classification as notirritating (alkaline cleaner 2; acid cleaners 1, 2 and 4; MPT prod-ucts 1, 2, 7, 9, 10, 11, 13), AR seven times and HSM nine times

(AR: MPT products 1, 2, 5, 7, 8, 10, 12; HSM: MPT products1, 2, 5, 7–10, 12, 13 provided a strict interpretation of HSMresults according to OECD criteria; the number would be reducedto five products if borderline results were qualified as possiblyirritating). Four products were equally detected as not irritatingin CCM, AR and HSM (MPT products 1, 2, 7, and 10).

Five products (MPT products 6–10) contain varying concentra-tions of dihydrogen hexafluorozirconate(2�) and hydrogen fluo-ride, which are presumed to be the major constituentsresponsible for corrosive/irritating effects. A systematic compari-son of these products shows that overall the difference in concen-tration is reflected quite well in the results of the in vitro methods(Table 5).

Table 4Testing results for products (skin corrosion/irritation) grouped according to individualclassification outcomes.

Number ofproducts

Notirritating

Irritating Corrosivea Product category

2 CCM, AR pH, HSM Alkaline cleaners 1, 31 CCM AR pH, HSM Alkaline cleaner 22 CCM AR pH, HSM Acid cleaners 2, 41 CCM, AR,

HSMpH Acid cleaner 3

1 CCM AR, HSM pH Acid cleaner 14 CCM, AR,

HSMpH Acid MPT products 1,

2, 7, 103 CCM, AR,

HSMpH Acid MPT products 3,

4, 63 AR, HSM CCM pH Acid MPT products 5,

8, 122 CCM, HSM AR pH Acid MPT products 9,

131 CCM AR pH, HSM Acid MPT product 11

a Extreme pH as such would result in a default classification as corrosive.

Tabl

e5

Syst

emat

icco

mpa

riso

nof

five

MPT

prod

ucts

wit

hdi

ffer

ent

conc

entr

atio

nsof

dihy

drog

enhe

xaflu

oroz

irco

nate

(2�

)an

dhy

drofl

uori

cac

id.

Dih

ydro

gen

hex

aflu

oroz

irco

nat

e(2�

)[%

]H

F[%

]O

ther

ingr

edie

nts

wit

hco

rros

ive/

irri

tan

tpr

oper

ties

[%]

pHC

CM

You

ng

(aci

dre

serv

e)H

SM,c

orro

sion

(cel

lvi

abil

ity

[%])

HSM

,irr

itat

ion

(cel

lvi

abil

ity

[%])

HET

-CA

M

Cor

rosi

ve[%

]Ir

rita

nt

[%]

Met

alpr

etre

atm

ent

prod

uct

65–

80.

06–

0.10

Non

e3–

41.

1X

i;R

36/3

8Ir

rita

tin

g(a

cid

rese

rve

=2.

42)

Not

corr

osiv

e(8

6.2/

41.2

)Ir

rita

tin

g(3

.9)

Irri

tati

ng

(Q=

1.76

)

Met

alpr

etre

atm

ent

prod

uct

81.

6–4

0.02

–0.

05N

one

Non

e2.

0X

i;R

36/3

8N

otir

rita

tin

g(a

cid

rese

rve

=0.

05)

Not

corr

osiv

e(9

8.8/

91.0

)N

otir

rita

ting

(50.

5)/b

orde

rlin

eN

otir

rita

tin

g(Q

=0.

71)

Met

alpr

etre

atm

ent

prod

uct

91.

2–1.

5A

ppro

x.0.

010.

5–1

1–2

1.0

Not

irri

tati

ng/

not

clas

sifi

edIr

rita

tin

g(a

cid

rese

rve

=0.

98)

Not

corr

osiv

e(8

3.6/

18.8

)N

otir

rita

ting

(56.

8)/b

orde

rlin

eN

otir

rita

tin

g(Q

=0.

38)

Met

alpr

etre

atm

ent

prod

uct

70.

6–1.

1A

ppro

x.0.

01N

one

0.1–

0.5

1.3

Not

irri

tati

ng/

not

clas

sifi

edN

otir

rita

tin

g(a

cid

rese

rve

=0.

46)

Not

corr

osiv

e(7

9.0/

72.0

)N

otir

rita

ting

(60.

3)N

otir

rita

tin

g(Q

=0.

14)

Met

alpr

etre

atm

ent

prod

uct

100.

1–0.

5A

ppro

x.0.

010.

1–0.

5

Table 6Testing results for substances (skin corrosion/irritation) grouped according toindividual classification outcomes.

Number ofsubstances

Notirritating

Irritating Corrosivea Chemical(cf. Table 2)

1 DSD, AR,HSM

pH Inorganic acid 2a

1 pH, DSD, AR,HSM

Inorganic acid 2b

1 HSM DSD, AR pH Inorganic acid 11 DSD, AR,

HSMpH Inorganic acid salt

1 DSD AR, HSM pH Organic acid1 DSD AR, HSM pH Organic acid salt1 AR, HSM DSD pH Alkanolamine1 DSD,

HSMSolvent (noextreme pH)

1 DSD,HSM

Detergent (noextreme pH)

a Extreme pH as such would result in a default classification as corrosive.


acid 10% and 25%) the in vivo information was in line with the HET-CAM result.

3.5. Product classification decisions

As described in Section 3.2 and Fig. 1, a tiered testing andassessment scheme was used. Regarding the WoE outcomes, sixscenarios were possible in this study. In Table 1 the results of theWoE assessments and resulting classifications are listed in the lasttwo columns.

Scenarios 1–4 are related to skin irritation/corrosion:

scenario 1: ‘‘corrosive’’ based on results of the HSM corrosiontest (4�: alkaline cleaners 1–3; acid cleaner 2)scenario 2: ‘‘non-corrosive’’ despite a positive outcome in theHSM (2�: acid cleaner 4; MPT product 11)scenario 3: ‘‘irritating’’ based on HSM irritation test (9�: acidcleaners 1 and 3; MPT products 3–6, 8, 9, and 12)scenario 4: ‘‘not irritating’’ based on HSM irritation test (5�:MPT products 1, 2, 7, 10, and 13).

Scenarios 1, 5, and 6 are related to eye effects:

scenario 1: ‘‘corrosive’’ based on results of the HSM corrosiontest (4�: alkaline cleaners 1–3; acid cleaner 2)scenario 5: ‘‘severely irritating/serious eye damage’’ based onWoE (7�: acid cleaners 1, 3, and 4; MPT products 3, 4, 5 and 11)scenario 6: ‘‘irritating’’ based on WoE (9�: MPT products 1, 2,6–10, 12, 13).

As a precaution, classification for eye irritation was never set below‘‘irritating’’ even if all testing results were clearly negative.

For the scenarios of skin vs. eye irritation/corrosion the follow-ing observations were made: scenario 2 for skin effects was in bothcases combined with scenario 5 for eye effects (acid cleaner 4; MPTproduct 11); scenario 3 for skin effects was either combined withscenario 5 (five times, acid cleaners 1 and 3; MPT products 3–5)or scenario 6 for eye effects (four times, MPT products 6, 8, 9,and 12); scenario 4 for skin effects happened to be always com-bined with scenario 6.

For the two products for which scenario 2 was followed (acidcleaner 4 and MPT product 11) the clearly predominant substancewith a irritating/corrosive potential was citric acid for which in vivostudies were available that demonstrated no irritating propertiesto the skin (see Table 2). In-house data with similar products sup-ported this assumption. Due to very low amounts of other acids

and/or surfactants from which a slight impact on the irritatingproperties could not be fully excluded, these products were classi-fied as skin irritating. With regard to eye effects, a classification asseverely irritating/serious eye damage was chosen as a worst caseassumption since the combined data for eye irritation were notclear without ambiguity.

4. Discussion

In this study we have investigated the corrosive and irritatingproperties of 20 products with extreme pH values by making useof different in vitro methods in a tiered testing and assessmentstrategy. Nine individual compounds (dilutions) were tested inparallel. The tiered approach that was used has proven to be apragmatic tool to produce data suitable to support classificationsaccording to chemicals law. As soon as a solid classification is de-rived for a series of products, the properties of similar productscan be ‘‘bridged’’ based on expert judgment. The use of such bridg-ing principles is outlined under GHS and CLP.

The way how the tiered testing strategy was applied in thisstudy represents partially a worst case approach, since productsclassified as corrosive according to the CCM were excluded fromtesting and classified/labeled as corrosive. ‘‘CCM = corrosive’’ how-ever must not necessarily mean that the product is indeed corro-sive due to the fact that the generic cut-off limits are usually notbased on experimental data of individual compounds and thatthe additivity approach may not always be justified with regardto the real physiological situation in human skin. Further testingin such cases is also possible to verify or falsify the initial out-comes. Since such products were excluded from this study, nojudgment can be made from the available data about a possiblecorrelation between CCM classifications as corrosive in comparisonto the respective in vitro results.

Human skin model tests have undergone extensive formal val-idation and acceptance procedures in order to be broadly applica-ble. Since the validation was performed with a specific and limitedset of compounds, it seems useful to further substantiate theirapplicability by practical experience.

Since there are no in vivo studies available for the productstested in this study, a direct comparison to in vivo data is not pos-sible. For the individual compounds, a comparison to in vivo data ispossible only in a limited way since testing conditions may havebeen different, or were not available in detail (e.g. pH adjustment).A crude plausibility check shows that the in vitro results in somecases seem to be matching or may have overestimated or, in veryfew cases (skin and eye effects of monoethanolamine), may haveunderestimated the effects in vivo. This study is not a direct fol-low-up of the validation where well-documented in vivo datawas available for the tested reference compounds. Nevertheless,valuable information could be obtained by comparing the resultsfrom the various non-animal methods. For example, the results ob-tained with a subset of products with varying contents of zirconateand hydrofluoric acid indicate that discrimination between the de-grees of irritancy is possible by in vitro methods.

With regard to eye irritation, the situation is still more complexsince there are no validated and accepted methods available for thewhole range of irritancy. Therefore, additional information to thein vitro results is needed within a weight of evidence assessment.In cases were the overall knowledge of the ingredients is consid-ered insufficient to allow for a WoE assessment, data from otherassays like the BCOP test can be useful in addition to the HET-CAM. It has previously been discussed that combination with addi-tional methods (e.g. models with stroma like the BCOP) in a batteryapproach could be a solution (Scott et al., 2010). An observationform our study was also that from the 14 products that were tested

1446 J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447

in the HET-CAM, only in two cases the HET-CAM resulted in a lesssevere classification than the AR.

Though the number was small (three products) it strikes that allalkaline cleaners in this study were tested as corrosive in the HSMwhich was in contrast to the results of the AR, and also of the CCM.The question remains if the test system properly reflects the physi-ological effects in these cases, and what might be the trigger for theseeffects (e.g. the combination of strong alkaline pH and detergents). Atypical detergent which was tested in dilution did not per se prove tobe corrosive in the HSM. In case the corrosive result for these kinds ofproducts would be supported by in vivo data, testing could finally beabandoned and pH alone may serve as reliable classification crite-rion. Further systematic investigations with combinations of con-stituents in various concentration ranges and with different pHvalues could provide more insight, including possible thresholds ofirritancy/corrosivity related to product composition and pH.

Further knowledge on such issues is expected from a projectinitiated in 2010 by The European Detergent Association (A.I.S.E.)to investigate the applicability of validated and adopted in vitroeye and skin irritation/corrosion methods to reliably classify deter-gent and cleaning product formulations. Product categories includehand dishwashing liquids, laundry detergents, all purpose cleanersand extreme pH products. A review of existing literature and datashared by A.I.S.E. member companies, and the practical testing inselected in vitro test methods of representative formulations sup-ported by existing animal and/or human data is envisaged(A.I.S.E., personal communication; initial results were presentedat regulatory meetings in 2010 in Germany and Switzerland and2011 in the US (Eskes C, Cazelle E, Hermann M, Jones P, McNameeP, Strutt A. Applicability of validated and adopted in vitro methodsto assess detergents and cleaning products. Poster presented at theICCVAM Workshop series on best practices for regulatory safetytesting: assessing the potential for chemically induced eye injuries.Bethesda, USA)).

As more data is expected to become available from this and pos-sibly other sources the approach might be refined for its domain ofapplicability in the future based on additional experience.

5. Conclusions

The tiered testing and assessment approach used in this studyhas proven to be a pragmatic tool to derive classifications accord-ing to chemicals regulations. The approach includes several ‘‘worstcase’’ assumptions. In vitro tests can be used to qualify initial eval-uations based on the pH value and the alkali or acid reserve. In par-ticular, the usefulness of the inclusion of the human skin modeltests and the HET-CAM in the tiered approach was shown. HSM re-sults match in most cases with the AR results but overall ratherpredict a comparatively higher skin corrosive/irritating potential.A final judgment whether the in vitro results correctly reflect thephysiological effects regarding irritating or corrosive propertiesof pH extreme products or if they may lead to over- predictionscannot be made based on the current data. Further investigationsmight help to systematically evaluate the reliability and physiolog-ical relevance of in vitro testing results for mixtures. A specific fo-cus should be on alkaline cleaners and potential interactions oftheir components. For eye effects, further efforts are needed toachieve approved test systems for the whole range of irritating/corrosive effects. If suitable information is available, properties ofsimilar formulations can be ‘‘bridged’’ based on expert judgmentas outlined under GHS and CLP.

6. Conflicts of interest statement

The authors declare no conflict of interest.

7. Funding source statement

The work was funded by Henkel AG & Co. KGaA.

Acknowledgements

We would like to thank Frederike Wiebel for manuscript reviewand all other colleagues who have supported our work.

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Classification and labeling of industrial products with extreme pH by making use of in vitro methods for the assessment of skin and eye irritation and corrosion in a weight of evidence approach1 Introduction2 Materials and methods2.1 Test samples2.2 Conventional calculation method (CCM) according to DPD2.3 Determination of pH and the acid or alkali reserve2.4 The EpiDerm™ human epidermis model2.4.1 EpiDerm™ skin corrosion test2.4.2 EpiDerm™ skin irritation test (EPI-200-SIT)

2.5 HET-CAM (Hen‘s egg test – chorioallantoic membrane)

3 Results3.1 Composition of the dataset3.2 Testing and evaluation scheme3.3 Test results: products3.4 Test results: compounds3.5 Product classification decisions

4 Discussion5 Conclusions6 Conflicts of interest statement7 Funding source statementAcknowledgementsReferences

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Toxicology in Vitro - COnnecting REpositories · 2016. 12. 9. · Classiﬁcation and labeling of industrial products with extreme pH by making use of in vitro methods for the assessment