May 20 , 2009 Page 1

Assessment of skin sensitizing potential of capsules

Available data in animals and humans on the skin sensitization potential of monomers

used in PMMA capsules have been reviewed . For the skin sensitizing monomers

a risk assessment has been developed to ensure the safe use of

capsules in fabric softener. A margin of safety has been calculated by means of

the ratio between the derived no-observed-adverse-effect-level (NOAEL) for skin

sensitization and the estimated consumer exposure :

MMA BUDA

NOAEL skin sensitization 11750 1Jg/cm 2 78.7 1Jg/cm 2

Est i mated consumer exposure 0.09 ng/cm 2 0.05 ng/cm 2

Margin of safety 130 X 10 6 2 X 10 6

has been reported conclusively to cause sk i n sensitization in animals and

humans . However , cou ld not be detected as residual monomer in

capsules. To provide a qualitative risk assessment it was assumed that and

are similar regarding skin sensitizing potency . Consumer exposure based on the

detection limit of the analytical method used for determination (1 00 ppm)

accounts for 0.03 ng /cm 2 which gives a maro1n of safety of 3 x 10 6 when

compared to the NOAEL for skin sensitisation ofPage 2

The margin of safety of all three monomers is estimated to be

well above 10 6

. In consequence , it is considered very unlikely that skin sensit i zat i on to

any of the three monomers occurs by contact with capsules used in fabric

softeners . Yours sincerelv .

Annex 1 : Derivation of a No-Adverse-Effect-Level from experimental data

Annex II: Exposure AssessmentPage 3

Annex 1: Derivation of a No-Adverse-Effect-Level from experimental data

has been tested extensively for skin sensitization in guinea pig mode l s and in the

murine Local Lymph Node Assay (LLNA) . Positive reactions in guinea pig tests were

obtained in tests using adjuvants , especially when h i gh induction and challenge

concentration were used . The purity of the used and its stabilizer content were not

specified in most of the guinea pig studies . In the LLNA of high purity (99.96 % )

was used , why data from the LLNA study have been chosen as point of departure for

derivation of a no-observed-adverse-effect-concentration (NOAEC) .

In humans , no skin sensitization has been tested by Human Repeat Insult Patch Test or

Human Maximizat i on Test ; however human pat c h tests gave clear evidence that

is skin sensitizing to humans . Howeve r, in terms of potency it is considered a weak skin

sensitizer.

In the LLNA was tested in two vehicles, acetone : o l ive oil 4:1 (AOO) and acetone

alone (Betts et al. 2006). To err on the safe side only data obtained with acetone as a

vehicle will be considered since stimulation indices with this vehicle were higher than

those obtained with AOO . From the published data an EC3 of 60 % an a NOAEC

(defined as concentration which did not exceed a stimulation inde x of 3) of 50 % can be

derived.

The NOAEC ( % )can be converted to dose per skin area (j.Jg/cm 2 ) by considering the

dose volume of 25 j.JI (standard LLNA protoco l ), and an est i mated app l ication area of 1

cm 2 for the mouse ear , and the density of 1 ,4-butanediol diacry l ate (0 . 94 mg/cm 3 ) :

NOAEC (%) x 10 x density x 25 j.Jg/cm 2 =dose per skin area [1Jg/cm 2 ]

Thus , a NOAEC of 50 % MMA accounts for 50 % x 10 x 0.94 x 25 = 11750 1-19 MMA/cm 2

Data from two pub l ished gu 1 nea p1g max 1 m1zation tests show discordant results for skin

sensitizing potential of In the first study concentrations of 1 % (w/w) in olive

oi l/ acetone (9 : 1) for the intradermal induction and 25 % (w/w) in petrolatum for the

epicutane induction were chosen . After challenge with 0.2 % (w / w) in petrolatum

10 out of 15 (67 %) tested gu i nea p i gs showed a positive reaction for skin sensitization

(Bjorkner , 1984). A decreased rate of positive reactions (7 out of 15 ; 47 %) was

observed when 0.2 % (w/w) in acetone was used for challenge after induction

with the same concentrat i ons aescribed above . The discrepancy between the vehicle

dependent test results was attributed to the polymerization process of the acrylic

compounds in petrolatum and acetone and it was assumed that the petro l atum vehicle

increased the rate of positive challenge results by preventing the polymerization of the

a c rylic monome r (Bjor k ner and Niklasson , 1984) .Page4Inasecondguineapigmaximizationtest10animalsweretreatedwith10%(w/w)narachisoilforintradermalinductionand2%(w/w)inpetrolatumor80%ethanol(notfurtherspecified)fortheepicutaneinduction.Afterchallengewith0.6%(w/w)inmethylethylketone/arachisoil(2:1)nopositiveskinreactionwasobservedinall10testedguineapigs(vanderWalleetal.,1983).AdditionaldataareavailablefromFreund'sCompleteAdjuvantTest(vanderWalleetal.,1983).Ondays0,2,4,7and9intradermalinjectionswith0.1mlcontaining10%(w/w)inFreund'sCompleteAdjuvantwereperformedforinductioninupto8guineapigs.Challengewasperformedepicutaneouslyonday21with0.6%(w/w)inmethylethylketone/arachisoil(2:1).Dependingonthepurityofthetestsubstanceapositivereactionforskinsensitizationwasobservedin1outof7(14%)or5outof8(63%)testedanimalsin98%or95%puretestsubstance,respectively(vanderWalleetal.,1983).Overthepast20yearstheskinsensitizingeffectsofinhumanhasbeendocumentedinnumerouscasereportsandstatisticalanalysisofpatchtestsperformedonpatientswithoccupationalcontactdermatitisorcontactdermatitisacquiredawayfromtheworkplace.TheInformationNetworkofDepartmentsofDermatologyreported10positivepatchtestresultsoutof483testedpatients(IVDK2000)andKanervaetal.reported11positiveresultsoutof242testedpatients(Kanervaetal.1997).Neitheroftheavailablepositivetestresultsfromguineapigtestingorhumanpatchtestsallowtoderiveaconcentrationofonwhichariskassessmentcanbebased.ForthispurposeamurineLocalLymphNodeAssay(LLNA)hasbeenconductedtoderiveaBUDAconcentrationatwhichaskinsensitisationisconsideredlikely.TheLLNAwasperformedaccordingtotheOECDtestguideline429.FiveCBA/Jmiceperdosegroup(1%,0.3%,and0.1%inmethylethylketone)plusavehiclecontrolgrouphavebeentreatedonthesameapplicationsiteforthreeconsecutiveThreedaysafterthelastinjectionalltestgroupsreceivedanintravenousinjectionofHthymidineintothetailvein.Fivehoursafterthethymidineinjectionthestudywasterminatedantheearweight,lymphnodeweightaswellasthecellcountandtheincorporatedradioactivityinthecellpopulationofthedrainingauricularlymphnodeswasdetermined.inducedadosedependentincreaseinboth,cellcountandthymidineincorporationinthelocallymphnodesofthetreatedmice.Maximumstimulationindex(7.9foldoverthecontrol)wasachievedwith1%whereasthestimulationindexaftertreatmentwith0.1%and0.3%1,4-butanedioldiacrylatestayedwellbelow3(cut-offforpositivesensitisationreaction).Asasurrogateforalowestobserved-effect-leveltheconcentrationtoinducea3-foldstimulationoverthevehiclecontrolwasdeterminedbylinearregressionandwasreportedtobe0.5%.Thenoobserved-adverse-effectconcentration(NOAEC)fromexperimentaldatawas0.3%Page 5

The NOAEC from the LLNA is converted to dose per skin area (1Jg/cm 2 ) by considering

the dose volume of 25 1-11 (standard LLNA protocol} , and an estimated application area

of 1 cm 2 for the mouse ear , and the density of ( 1 .049 mg/cm 3 ) :

NOAEC (%) x 10 x density x 25 j.Jg/cm 2 =dose per skin area [1Jg /cm 2 ]

Thus , the NOAEL of 0 . 3% BUDA accounts for

0 . 3% X 10 X 1.049 X 25 = 78.71JQ

Numerous studies in guinea pigs show that 1as a skin sensitizing potential.

When a concentration of 1% (w/w) in olive oi l /acetone (9: 1) was used for the

intradermal induction and 25% (w/w) in petrolatum for the epicutane induction were

chosen 10 out of 15 (67%) tested guinea pigs showed a positive reaction for skin

sensitization after challenge with 0.2% (w/w) in petrolatum (Bjorkner, 1984). No

positive reaction (0 out of 15) was observed when 0.2% (w/w) in acetone was

used for challenge after induction with the same concentrations described above. The

discrepancy between the vehicle dependent test results was attributed to the

polymerization process of the acrylic compounds in petrolatum and acetone and it was

assumed that the petro latum vehicle increased the rate of positive challenge results by

preventing the polymerization of the acrylic monomer (Bjorkner and Niklasson, 1984).

Over the past 30 years the skin sensitizing effects of in humans has been

documented in numerous case reports and statistical analysis of patch tests performed

on patients with occupational contact dermatitis or contact dermat i tis acquired away

from the workplace . The Information Network of Departments of Dermatology report ed

5 positive patch test results out of 369 tested patients (IVDK 1997) and Kanerva et al.

reported 2 positive results out of 124 tested patients (Kanerva et al. 1995) .

None of the available studies allows for the derivation of a NOAEC . For the sake of this

risk assessment it is assumed that may have the same skin sensitizing potential

to human as . Based on the available human patch test data and guinea pig

maximization test data this assumption seems justifiable .Page 6

References

Betts CJ, Dearman RJ, Heylings JR, Kimber I , Basketter DA (2006). Skin sensitization

potency of methyl methacrylate in the local lymph node assay : comparisons with

guinea-pig data and human experience . Contact Derm, 55 , 140-147 .

Bjorkner B (1984). The sensitizing capacity of multifunctional acrylates in the guinea

pig. Contact Derm , 11, 236- 246.

Bjorkner Band Niklasson B ( 1984) . Influence of the vehicle on elic itatio n of contact

allergic reactions to acrylic compounds in the guinea pig . Contact Derm , 11 , 268-

278. IVDK (lnfonnationsverbund Dennatologischer Kliniken) (1997). Auswertung der

zwischen 1992 und 1995 vom IVDK erfassten Daten zu Pentaerythrittiracrylat,

16.01.1997 , IVDK , Gottingen.

IVDK (lnfonnationsverbund Dennatologischer Kliniken) (2000) . Auswertung der

zwischen 1992 und 1998 vom IVDK erfassten Daten , 7.2.2000 , IVDK , Gottingen .

Kanerva L , Estlander T , Jolanki R , Tarvainen K (1995). Statistics on allergic patch test

reactions caused by acrylate compounds , inclu ding data on ethyl methacrylate. Am.

J . Contact Dermatitis 6, 75-77.

Kanerva L , Jolanki R , Estlander T ( 1997) 10 years of patch testing with the

(meth)acrylate series . Contact Dermatitis 37, 255-258.

Van der Walle HB , Waegemaekers T , Bensink T ( 1983 ). Sensitizing potential of 12

di(meth)acrylates in the guinea pig . Contact Derm 9 , 10 - 20 .Page?AnnexII:ExposureAssessmentEstimatedconsumerexposureresultingfromtheuseof:apsuleinTheconsumerexposuremodels(equations)andconsumerexposurefactorsusedinthisdocumentaretakenfromtheAppendixDandAppendixG,respectively,oftheguidancedocumentmethodologyforhuman &environmentalriskassessmentoningredientsofhouseholdcleaningproducts(HERA,2005).Useof.capsulecapsulewillbeusedinfabricsoftenersataconcentrationof2.5%.Mostconservativeassumptionshavebeenused(HERA,2005;EU,2003)toestimatetheconsumerexposures,namelythedermalexposurewhilewearingmachinelaunderedgarmentwithPMMAcapsuledepositedontheinnersurface.Dermalexposureofadultswearingmachine-washedfabricDermalexposure(EXP)tocapsulesviaindirectskincontactfromwearingmachine-washedclothesiscalculatedinmg::apsuleperdaybytheformula:F1percentage(%)weightfractionofsubstanceinproduct:2.5%.C'productload,inmg/cm2:Datanotavailable,butcan beestimatedasdescribedbelow:EstimationofproductloadC'whenproductisappliedtoskinbywearingmachine-washedfabrics:C'=(MxF'xFD)IW1Mmaximum amountoffabricsoftenerusedperwashingcycle,inmg:140g=140x103mg(EU,2003).F'percentage(%)weightfractionofsubstance(PMMAcapsule)depositedonfabricduringthewashingprocess:100%(worstcase).FDfabricdensity,inmg/cm2;;;23mg/cm2Approximateweightofmediumweightcottonfabricw1totalweightfabric,inmg:3.1x106mg.Theaverageamountofclothingwashedpercycleis3.1kg=3.1x106mgC'=(140x103mgx 1 x23mg/cm2)/3.1x106mg=1.04mg/cm2Sdersurfaceareaofexposedskin,incm2:17600cm2(HERA,2005).nexposurefrequency,innumberofeventsperday:1evenVday.Clothesarechangeddailyandnotreused(worsecase).F2percentage(%)weightfractiontransferredfromclothestoskin:1%(HERA,2005).F3percentage(%)weightfractionremainingonskin:100%(worstcase).EXP=2.5/100x1.04mg/cm2x17600cm2x 1evenVdayx1/100x1 x 1=4.6mgcapsule/dayPage 8

capsule are very unlikely to be absorbed through the sk i n , therefore risk for skin

sensitisat i on will be calculated based on the migrat i on rate of the residual starting

material of the c apsule .

Based on the calculated exposure to capsule pe r day the e x posure to the

residual starting material can be estimated based on their residual levels and migration

rate. Estimated exposure for

For MMA a residual monome r level of 330 ppm has been determined . To err on the

safe side it is assumed that MMA penetrates to 1 00 % through the skin

= 4 . 6 mg PMMA capsule / day x 330 ng MMA/mg PMMA capsule

= 1518 ng fday

Given a total exposed skin area of 17600 cm 2 the topical concentration of MMA

migrating out of PMMA capsules results in

1518 ng MMA/day / 17600 cm 2 = 0.086 ng ' cm 2

Estimated exposure for

For BUDA a residual monomer level of 200 ppm has been determined . To err on the

safe side it is assumed that penetrates to 100% through the skin

EXPsuoA = 4.6 mg

= 920 ng capsule / day x 200 ng

' day rmg capsule

Given a total exposed skin area of 17600 cm 2 the topical concentration of

migrating out of PMMA capsules results i n

920 ng BUDA/day / 17600 cm 2 = 0 . 052 ng rcm 2

Estimated exposure for

For no residual monomer has been found . For the purpose of this risk

assessment the limit of detection of the analyltical method 100 ppm has been chosen

as virtua l concentration.

Furthermore it is assumed that penetrates to 100 % through the skin

= 4 . 6 mg

= 460 ng capsule / da y x 100 ng

ctay mg : apsule

Given a total exposed skin area of 17600 c m 2 the topical concentrat i on of

migrating out of capsules results in

460 ng /day/17600 cm 2 = 0 . 026 ng cm 2Page 9

References HERA (2005). Guidance Document Methodology for Human & Environmental Risk

Assessment on Ingredients of Household Cleaning Products (HERA), Februa ry

2005. Accessible through the website of the International Association for Soaps,

Detergents and Maintenance Products ( http : //www . aise-net.orgQ.

EU (2003). Technical Guidance Document on Risk Assessment in support of

Commission Directive 93/67/EEC on Risk Assessment for new notified substances

[ ... ] Part I, lspra , Italy 2003.