Toxicology in Vitro - COnnecting REpositories · 2016. 12. 9. · Classification and labeling of...
Transcript of Toxicology in Vitro - COnnecting REpositories · 2016. 12. 9. · Classification and labeling of...
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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/
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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
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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,
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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)
Metal pretreatmentproduct 2
Contains polymers, inorganic acidsand salts thereof (includingdihydrogenhexafluorotitanate(2�))
Not irritating(notclassified)
0.4 (R36)0.4 (R38)
2.0 Not irritating(acidreserve = 0.5)
Not corrosive(97.8/74.4)
Not irritating(105.3)
Not irritating(S = 6)
4 + 6 Not irritating toskin; irritating toeyes (GHS Cat 2(H319) or DPD Xi;R36)
Metal pretreatmentproduct 3
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)
Not corrosive(92.6/58.4)
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)
Metal pretreatmentproduct 4
Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))
Xi; R36/38 0.7 (R34)0.7 (R41)a
1.9 (R36)1.9 (R38)
0.6 Irritating(acidreserve = 2.7)
Not corrosive(95.2/65.4)
Irritating(4.4)
Severelyirritating(Q = 2.45)
3 + 5 Skinirritating + severelyirritating to theeyes (GHS Cat 1(H318), Cat 2(H315) or DPD Xi;R38-41)
Metal pretreatmentproduct 5
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)
1.3 Not irritating(acidreserve = 1.0)
Not corrosive(99.1/88.1)
Not irritating(52.9)[borderlineresult:individualtissues: 42.7;46.4; 69.6]
Severelyirritating(S = 17)
3 + 5 Skinirritating + severelyirritating to theeyes (GHS Cat 1(H318), Cat 2(H315) or DPD Xi;R38-41)
Metal pretreatmentproduct 6
Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))
Xi; R36/38 0.6 (R34)0.6 (R41)a
1.7 (R36)1.7 (R38)
1.1 Irritating(acidreserve = 2.4)
Not corrosive(86.2/41.2)
Irritating(3.9)
Irritating(Q = 1.76)
3 + 6 Irritating to skinand eyes (GHS Cat 2(H315, H319) orDPD Xi; R36-38)
Metal pretreatmentproduct 7
Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))
Not irritating(notclassified)
0.2 (R36)0.2 (R38)
1.3 Not irritating(acidreserve = 0.5)
Not corrosive(79.0/72.0)
Not irritating(60.3)
Not irritating(Q = 0.14)
4 + 6 Not irritating toskin; irritating toeyes (GHS Cat 2(H319) or DPD Xi;R36)
Metal pretreatmentproduct 8
Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))
Xi; R36/38 0.8 (R34)0.8 (R41)a
1.2 (R36)1.2 (R38)
2.0 Not irritating(acidreserve = 0.1)
Not corrosive(98.8/91.0)
Not irritating(50.5)[borderlineresult:individualtissues 40.9;43.5; 67.0]
Not irritating(Q = 0.71)
3 + 6 Irritating to skinand eyes (GHS Cat 2(H315, H319) orDPD Xi; R36-38)
Metal pretreatmentproduct 9
Contains inorganic acids and saltsthereof (includingdihydrogenhexafluorozirconate(2�))
Not irritating(notclassified)
0.3 (R36)0.3 (R38)
1.0 Irritating(acidreserve = 1.0)
Not corrosive(83.6/18.8)
Not irritating(56.8)[borderlineresult:individualtissues: 48.5;54.5; 67.5]
Not irritating(Q = 0.38)
3 + 6 Irritating to skinand eyes (GHS Cat 2(H315, H319) orDPD Xi; R36-38)
1438J.Scheel
etal./Toxicology
inV
itro25
(2011)1435–
1447
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Met
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sth
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f(i
ncl
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dih
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gen
hex
aflu
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Not
irri
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ng
(not
clas
sifi
ed)
0.0
(R36
)0.
0(R
38)
1.6
Not
irri
tati
ng
(aci
dre
serv
e=
0.3)
Not
corr
osiv
e(8
4.7/
97.8
)N
otir
rita
tin
g(8
3.6)
Not
irri
tati
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(Q=
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+6
Not
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tosk
in;
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Cat
2(H
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Met
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11C
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ican
dor
gan
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(in
clu
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g20
–40%
citr
icac
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Xi;
R36
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41)
2.0
(R36
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38)
0.8
Irri
tati
ng
(aci
dre
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e=
8.6)
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12.2
)N
otte
sted
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ed2
+5
Skin
irri
tati
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+se
vere
lyir
rita
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at1
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at2
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5)or
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-41)
d
Met
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mer
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ther
eof
(in
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dih
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gen
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2�))
Xi;
R36
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0.6
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1.9
(R36
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9(R
28)
1.9
Not
irri
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Not
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7.1/
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)N
otir
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[bor
derl
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resu
lt:
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ssu
es:
64.3
;59
.9;
48.8
]
Irri
tati
ng
(Q=
1.20
)3
+6
Irri
tati
ng
tosk
inan
dey
es(G
HS
Cat
2(H
315,
H31
9)or
DPD
Xi;
R36
-38)
Met
alpr
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atm
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prod
uct
13C
onta
ins
inor
gan
ican
dor
gan
icac
ids
(in
clu
din
g1–
5%su
lph
uri
cac
id)
Not
irri
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ng
(not
clas
sifi
ed)
0.6
(R36
)0.
6(R
38)
0.5
Irri
tati
ng
(aci
dre
serv
e=
0.1)
Not
corr
osiv
e(9
4.4/
56.9
)N
otir
rita
tin
g(9
3.5)
Not
irri
tati
ng
(Q=
0.10
)4
+6
Not
irri
tati
ng
tosk
in;
irri
tati
ng
toey
es(G
HS
Cat
2(H
319)
orD
PDX
i;R
36)
aTh
isca
lcu
lati
onco
nsi
ders
one
orm
ore
subs
tan
ces
wit
hsp
ecifi
ccl
assi
fica
tion
lim
its
(An
nex
1D
SD)
only
defi
ned
for
R34
but
not
for
spec
ifica
lly
for
R41
.Th
ese
con
cen
trat
ion
lim
its
wer
eal
sou
sed
for
the
dete
rmin
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R41
clas
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ith
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2ti
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low
50%
.As
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ecau
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ere
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ere
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nir
rita
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g’’i
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Add
itio
nal
info
rmat
ion
was
rele
van
tfo
rth
eW
oEcl
assi
fici
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nde
cisi
on.
dTh
ete
rmin
olog
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desc
ribe
eye
irri
tati
ng
effe
cts
isdi
ffer
ent
un
der
DSD
/DPD
and
GH
S(t
he
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term
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ogy
was
prim
aril
yu
sed)
.
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
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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)
Not irritating(95.1)
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
irritating or not irritating
Scenario 1
not corrosive4
Scenario 2
products in this study
Scenario 3
Scenario 4
Scenario 6
Scenario 5
irritating or not irritating
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
irritating or not irritating
Scenario 1
not corrosive4
Scenario 2
products in this study
Scenario 3
Scenario 4
Scenario 6
Scenario 5
irritating or not irritating
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.
J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447 1443
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.
J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447 1445
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.
References
BIBRA, 1989. (The British Industrial Biological Research Association) Toxicity profileof ethanolamine.
Carpenter, C.P., Smyth Jr., H.F., 1946. Chemical burns of the rabbit cornea. AmericanJournal of Ophthalmology 29, 1363–1372.
Cognis Deutschland GmbH & Co. KG, 2007. Material Safety Data Sheet according to91/155/EEC – ISO 11014-1.
ECB, 2000a. (European Chemical Bureau) IUCLID Data Sheet for monoethanolamine,CAS 141-43-5, Vers. 19 Feb., 2000.
ECB, 2000b. (European Chemical Bureau) IUCLID Data Sheet for 2-(2-butoxyethoxy)ethanol, CAS 112-34-5, Vers. 18 Feb., 2000.
ECB, 2000c. (European Chemical Bureau) IUCLID Data Sheet for citric acid, CAS 77-92-9, Vers. 19 Feb., 2000.
ECB, 2000d. (European Chemical Bureau) IUCLID Data Sheet for orthophosphoricacid, CAS 7664-38-2, Vers. 19 Feb., 2000.
ECB, 2000e. (European Chemical Bureau) IUCLID Data Sheet for trisodiumnitrilotriacetate, CAS 5064-31-3, Vers. 18 Feb., 2000.
EU, 1976. Directive 67/548/EEC of 27 June, 1967 on the approximation of laws,regulations, and administrative provisions relating to the classification,packaging and labeling of dangerous substances Official Journal of theEuropean Communities. L196, 1–98.
EU, 1999. Directive 1999/45/EC of the European parliament and of the council of 31May, 1999 concerning the approximation of the laws, regulations andadministrative provisions of the Member States relating to the classification,packaging and labelling of dangerous preparations Official Journal of theEuropean Communities. L200, 1–68.
EU, 2008. Regulation (EC) No. 1272/2008 of the European parliament and of thecouncil on classification, labelling and packaging of substances and mixturesOfficial Journal of the European Union. L353, 1–1354.
EU, 2009. Guidance to Regulation (EC) No 1272/2008 on classification, labelling andpackaging (CLP) of substances and mixtures. ECHA Reference: ECHA-09-G-02-EN, p. 245ff.
Henkel AG & Co. KGaA, 1977. Prüfung verschiedener Substanzen und Produkte aufHautverträglichkeit nach der vom VCI vorgeschlagenen Methode (unpublisheddata, internal reports TBD770118 and TBD770124).
Henkel AG & Co. KGaA, 1994a. Primary Skin Irritation/Corrosion Study in the Rabbit(unpublished data, internal report R9400637).
Henkel AG & Co. KGaA, 1994b. Acute Dermal Irritation on Rabbit (unpublished data,internal report R9400325).
HERA, 2005. (Human & Environmental Risk Assessment on ingredients of Europeanhousehold cleaning products) Soluble Silicates (CAS No.: 1344-09-8, 6834-92-0,10213-79-3, 13517-24-3, 1312-76-1) Draft for Public Comment.
CIR, 1983. Final report on the safety assessment of triethanolamine,diethanolamine, and monoethanolamine. Journal of the American College ofToxicology 2, 183–235.
Jacobs, G.A., 1992. Two dilutions of phosphoric acid tested on eye. Journal of theAmerican College of Toxicology 11, 724.
Karlsson, N., Lodén, M., 1984. A study of acute skin irritancy of some silicates.Forsvarets Forskningsanstalt. Project report FOAtox 402.
MatTek, 2010. Protocol for: In Vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT) foruse with MatTek Corporation’s Reconstructed Human Epidermal ModelEpiDerm™. (Epi-200) Rev. 1/19/2010.
Merck KGaA, 2009. Safety Data Sheet according to Regulation (EC) No. 1907/2006for ortho phosphoric acid 85%.
OECD, 2001a. SIDS Initial Assessment Report Sulfuric Acid, CAS 7664-93-9.OECD, 2001b. SIDS Initial Assessment Report Citric Acid, CAS 77-92-9.OECD, 2002a. OECD Guideline for the Testing of Chemicals 405: Acute Eye Irritation/
Corrosion. 2nd revision, adopted: 24 April, 2002.OECD, 2002b. OECD Guideline for the Testing of Chemicals 404: Acute Dermal
Irritation/Corrosion. Adopted: 24 April, 2002.OECD, 2004a. OECD Guideline for the Testing of Chemicals 431: In Vitro Skin
Corrosion: Human Skin Model Test. Adopted: 13 April, 2004.OECD, 2004b. OECD Guideline for the Testing of Chemicals 430: In Vitro Skin
Corrosion: Transcutaneous Electrical Resistance Test (TER). Adopted: 13 April,2004.
OECD, 2006. OECD Guideline for the Testing of Chemicals 435: In Vitro MembraneBarrier Test Method for Skin Corrosion. Adopted: 19 July, 2006.
OECD, 2009a. OECD Guideline for the Testing of Chemicals 437: Bovine CornealOpacity and Permeability Test Method for Identifying Ocular Corrosives andSevere Irritants, adopted: 7 Sep, 2009.
OECD, 2009b. OECD Guideline for the Testing of Chemicals 438: Isolated ChickenEye Test Method for Identifying Ocular Corrosives and Severe Irritants, adopted7th September 2009.
-
J. Scheel et al. / Toxicology in Vitro 25 (2011) 1435–1447 1447
OECD, 2010a. OECD Guideline for the Testing of Chemicals 439: In Vitro SkinIrritation: Reconstructed Human Epidermis Test Method. Adopted: 22 Jul,2010.
OECD, 2010b. OECD Draft Proposal for a new Guideline 4XX. The CytosensorMicrophysiometer Test Method: An In Vitro Method for Identifying ChemicalsNot Classified as Irritant, as well as Ocular Corrosive and Severe IrritantChemicals. Paris. 2 July, 2010.
Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alepee, N., Bufo, M., Clothier, R.,Facchini, D., Faller, C., Guest, R., Harbell, J., Hartung, T., Kamp, H., Varlet, B.L.,Meloni, M., McNamee, P., Osborne, R., Pape, W., Pfannenbecker, U., Prinsen, M.,Seaman, C., Spielmann, H., Stokes, W., Trouba, K., Berghe, C.V., Goethem, F.V.,Vassallo, M., Vinardell, P., Zuang, V., 2010. A proposed eye irritation testing
strategy to reduce and replace in vivo studies using Bottom-Up and Top-Downapproaches. Toxicology in Vitro 24, 1–9.
Steiling, W., Bracher, M., Courtellemont, P., de Silva, O., 1999. The HET-CAM, a usefulin vitro assay for assessing the eye irritation properties of cosmeticformulations and ingredients. Toxicology in Vitro 13, 375–384.
UN, 2003. Globally Harmonized System of Classification and Labelling of Chemicals(GHS). Published 2003.
UN, 2009. Globally Harmonized System of Classification and Labelling of Chemicals(GHS). Last (third) revision 2009 (published 2003).
Young, J.R., How, M.J., Walker, A.P., Worth, W.M.H., 1988. Classification as corrosiveor irritant to skin of preparations containing acidic or alkaline substances,without testing on animals. Toxicology in Vitro 2, 19–26.
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