comparative cesio study in surfactants: inconsistent...

European Committee of

Organic Surfactants and

their Intermediates

LRI Workshop

“ Applicability of skin sensitization testing methods for

regulatory purposes"

Comparative CESIO study in

surfactants:

Inconsistent results between the

LLNA and Guinea Pig

sensitization tests

J.-C. Carrillo; N. Ball; S. Cagen; H. Certa; D. Eigler; H. Esch; C. Garcia; C.

Graham; C. Haux; R. Kreiling; A. Mehling



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OVERVIEW

Background

Allergy and test methods for skin sensitization

Specificity, selectivity and accuracy

Industry data

Conclusions



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Background:

Skin sensitization tests (e.g. OECD 406 or 429) are performed for hazard

assessment and C&L purposes.

Under REACH LLNA (OECD 429) is the prescribed test

New LLNA-generated data led to „unexpected positive‟ results for surfactants

“Word of mouth” revealed the unexpected results were not limited to

surfactants

Past experience and weight of evidence „unexpected‟ positive results

could be „false‟ positives

The CESIO-LLNA Task Force was formed as an inter-industry effort to

investigate these findings

The CESIO-LLNA TF is currently investigating the reliability of the LLNA for

predicting skin sensitization potential of surfactants and their intermediates

Why was the CESIO-LLNA-Task Force formed?

INTRODUCTION



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OVERVIEW

Introduction



Industry data

Conclusions



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VALIDATION in a Nutshell

The LLNA was the first regulatory test to be formally

validated.

Validation was based on comparative data (e.g. LLNA vs.

Guinea Pig Tests)

Comparative data: The same substances tested in the LLNA

and GPT systems allows comparing the methods to one

another. Are results concordant (yes/no) ?



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DEFINITIONS (I)

Selectivity/Sensitivity: The proportion of all positive

chemicals that are correctly classified as positive in a test

Specificity: The proportion of all negative chemicals that are

correctly classified as negative in a test

Accuracy: The closeness of agreement between a test result

and an accepted reference value; (b) the proportion of correct

outcomes of a method

Dean et al., Regulatory Toxicology and Pharmacology 34, 258–273 (2001)



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DEFINITIONS (II)

In the following (as in the ICCVAM validation):

Positive/(negative) results mean that in a regulatory

context, labeling would be required (or not)

“False” positive means that a LLNA result is positive,

the result of the guinea pig test is negative

“False” negative means that a LLNA result is

negative, the result of the guinea pig test is positive



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CHEMICAL

IDENTIFICATION

GUINEA PIG

POSITIVES

GUINEA PIG

NEGATIVES

UNCLEAR TOTAL

LLNA Positives 86 6 0 92

LLNA Negatives 10 28 0 38

total 96 34 0 130

DATA SUBMITTED TO ICCVAM (METHOD VALIDATION)

COMPARISON OF LLNA AND GUINEA PIG CLASSIFICATIONS

Sensitivity (86 LLNA positives of 96 true positives): 90%

Specificity (28 LLNA negatives of 34 true negatives): 82%

Accuracy (correctly predicted results 86+28=114 of 130): 88%

http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarepAppx/llnac1.htm

But: Not all chemical classes were represented



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CHEMICAL IDENTIFICATION GUINEA PIG LLNA

Ammonium thioglycolate negative positive

Benzalkonium chloride negative negative

C12-13 –ß-branched alcohol sulfate negative positive

Cocoamidopropyl betaine positive positive

Dodecylthiosulphonate positive positive

Methyl dodecane sulphonate positive positive

Sodium lauryl sulfate negative positive

Tween 80 negative negative

3 of 8 => 38%

have discordant results

COMPARISON OF LLNA AND GPT RESULTS: SURFACTANTS

(ICCVAM n=8)



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CHEMICAL IDENTIFICATIONGUINEA PIG

TESTLLNA

OTHER

INFORMATION

Fatty acid glutamate negative positive

Lauralkonium chloride negative positive

Triisobutylphosphate positive negative

Surfactant 1 positive positive

Surfactant 2 positive positive

5 Sugar lipid surfactants negative negative HRIPT negative

5 Sugar lipid surfactants negative positiveSome irritation (ear-swelling)

HRIPT negative

3 Alkyl ethoxylates negative positive

Iso-nonyl - glucopyranoside positive positive

Heptyl –thioglucopyranoside negative positive

Nonanesulfonic acid, Na-salt negative negative

Alkylphenol- ethoxylate negative positive

COMPARISON OF LLNA AND GPT RESULTS: SURFACTANTS

(INDUSTRY n= 22)

TNO report; 2007; Eurotox Congress

2009 Industry (22) + ICCVAM (8) = 16/30 = 53%



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SPECIFITY & SELECTIVITY: SURFACTANTS

GUINEA PIG

POSITIVES

GUINEA PIG

NEGATIVES

TOTAL

LLNA Positives 6 15 21

LLNA Negatives 1 8 9

total 7 23 30

Surfactants ICCVAM

Sensitivity (6 LLNA positives of 7 true positives): 86% (90%)

Specificity (8 LLNA negatives of 23 true negatives): 35% (82%)

Accuracy of LLNA (6+8=14 of total 30) 47% (88%)



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OVERVIEW

Introduction



Industry data: CESIO surfactant comparative testing

Conclusions



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STUDY 1: Sugarlipid Surfactants




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Industry data: STUDY 1 – Sugarlipid surfactants

STUDY 1: Sugarlipid surfactants:

For comparison, 10 non-ionic sugarlipid surfactants were tested in the

GPMT and the LLNA

These surfactants were also tested in volunteers using the Human Repeat

Insult Patch Test (Marzulli & Maibach).

Fatty alcohol Glucose Xylose

C8-C10 C8-C10 glucoside C8-C10 xyloside

C10-C14 C10-C14 glucoside

C12-C18 C12-C18 glucoside A

C12-C18 C12-C18 glucoside B

C14 C14 glucoside

C16-C18 C16-C18 glucoside

C18 branched C18 branched glucoside

> C18 > C18 glucoside

>C18 branched >C18 branched xyloside

Sugar der ivates

Fatty alcohol

CATALYST

Fatty alcohol

In excess



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CONDITION LLNA GPMT HRIPT

Protocol OECD 429 (GLP) OECD 406 (GLP) Marzulli & Maibach

Principle Measurement of lymphocyte T

act ivat ion after a 2 phase

period (induct ion and rest)

Macroscopic evaluat ion of skin

react ions after a 3 phase period

(induct ion, rest and challenge)

Clinical evaluat ion of skin

react ions after a 3 phase

period (induct ion, rest and

challenge)

Number per group 4 10 50

Solvent DMF or acetone/ olive oil Dist illed water or olive oil Dist illed water

Concentrat ion At least 3 between 1.25% to

25%

1 for the induct ion between

1.25% to 10% and 2 for the

challenge between 2.5% to 100%

Concentrat ions previously

determined according to

necrosis (induct ion) or irritat ion

(challenge)

1 (use dose) for both

induct ion and challenge

between 1% to 6%

Parameter St imulat ion Index (3H

thymidine)

Ear thickness for skin irritat ion

Readings of skin react ions after

challenge

Readings of skin react ions

after challenge




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STUDY 1: Sugarlipid surfactants:

Irritation was assessed by:

LLNA increase in ear thickness (%)

GPMT control animals (% of positive animals)

HRIPT irritation index (induction phase)

Mostly, good overlap between concentrations in the GPMT and LLNA

In the GPMT in only one example (C8-C10 glucoside), 22% of the test

animals gave a positive response 24 hours after the first challenge,

however at 48 hours all animals were negative.

Except for C8-C10 glucoside, irritancy in GPMT was not consistent with

that observed in the LLNA.

Tested concentrations in the LLNA associated with increases over 10% in

ear thickness did not appear to cause an irritant response in the GPMT.



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- 5/10 are classified

according to LLNA

- 0/10 are classified

according to GPMT

- 0/8 are positive

according to HRIPT

-50% are discordant (LLNA vs. GPMT)

- HRIPT suggest discordances in classification

are related to overestimation by the LLNA

- Positive reactions with LLNA are well correlated

with skin irritation (R2=0.71).

- LLNA able to sufficiently distinguish skin

sensitization from skin irritation?




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STUDY 2: Surfactants




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STUDY 2: First attempt to use alternative endpoints to distinguish irritation from

sensitization

Seven model substances representing surfactant like compounds where

investigated in a comparative setting (GPMT and LLNA).

In the LLNA, additional to thymidine incorporation, alternative endpoints were

evaluated which would help assess the irritation potential of these substances.

Assessment included (but not limited to): ear weights and thickness, B220+.

Increase in ear weights and thickness can be indicators of the irritation properties

of the substance.

B220+ has been proposed as an alternative marker to differentiate sensitizers from

irritants.

The SI cut off for these endpoints has been suggested at SI >1.25* to be

considered biologically and statistically relevant.

Industry data: STUDY 2 – Surfactants

*. OECD, Draft proposal for an update to the test guideline 429. Skin Sensitisation: Local Lymph Node Assay. 2009.

* Gerberick, G.F., et al., Use of a B cell marker (B220) to discriminate between allergens and irritants in the local lymph node assay. Toxicol Sci, 2002. 68(2): p. 420-8.



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Based on SI thymidine incorporation alone the following is true:

The alternative endpoints assessed in this study did not help explain the role of

irritation in obtaining discordant results.

Irritation might have been a factor but not always.

A clear dose response was not always observed

Both OECD methods are equally accepted but in these cases which ones are

correct?

5/7 are discordant




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OVERVIEW

Introduction




Conclusions



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CONCLUSIONS

No test is without limitations.

The LLNA is suitable for many substance classes, but for surfactants:

Comparative data on GPMT and LLNA point to a possible false positives in

the LLNA.

Based on data collected and generated, accuracy is 47%

Is the LLNA the method of choice for these chemistries?

Alternative endpoints are available but are probably not suited for all type of

substances. More data needs to be generated to assess applicability

A clear need exists to better understand the underlying mechanisms for the

nature of false positives/negatives to further develop alternative endpoints in the

LLNA.

We need reliable data base in order to develop reliable non-animal tests.



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THANK YOU

http://www.clariant.com/C1256A42004A5FC8/vwWebPagesByID/08F20B4C4C43F258C1256FE0004AD18F?OpenDocument

http://infonet.americas.cognisgroup.net/Cognis/GLOBAL/

http://www.seppic.com/index.asp



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Backup



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Alkylpolyglycoside LLNA GPMT HRIPT

(concentrat ion tested):

% of increase in ear

thickness

(concentrat ion

tested): St imulat ion

Index (SI)


% of posit ive animals in

control group = irritat ion


% of posit ive animals in

t reated group =

sensit izat ion

(concentrat ion

tested): Irr itat ion

Index (induct ion

phase)


% of volunteers with posit ive

skin react ion (challenge

phase)

C8-C10 glucoside

(1.25%): 9.5%

(2.5%): 7.8%

(5%): 6.9%

(10%):12.6%

(25%):5.8%

(1.25%): 0.77

(2.5%): 1.2

(5%): 0.7

(10%):0.84

(25%):1.67

(2.5%): 0%

(5%): 0%

(2.5%): 0%

(5%): 0% (5%): 0.04 (5%): 0%

C8-C10 xyloside

(1.25%): 8.6%

(2.5%): 14.3%

(5%): 7.7%

(10%):8.6%

(25%):14.6%

(1.25%): 1.93

(2.5%): 1.2

(5%): 1.02

(10%): 3.04

(25%): 6.62

(12.5%): 0%

(25%): 25% (25% at 48h)

(12.5%): 0%

(25%): 22% (0% at 48h)

Not tested

Not tested

C10-C14 glucoside

(1.25%): 0%

(2.5%): 0%

(5%): 0%

(1.25%): 0.8

(2.5%): 0.9

(5%): 0.6

(2.5%): 0%

(5%): 0%

(2.5%): 0%

(5%): 0% (5%): 0 (5%): 0%

C12-C18 glucoside A

(2.5%): 4.8%

(5%): 6.2%

(10%): 2.7%

(2.5%): 2.1

(5%): 2.1

(10%):1

(5%): 0%

(10%): 0%

(5%): 0%

(10%):0%

(5%): 0.10

(5%): 0%

C12-C18 glucoside B

(1.25%): 4.1%

(2.5%): 4.1%

(5%): 9.4%

(10%): 8.4%

(1.25%): 1.32

(2.5%): 1.6

(5%): 1.81

(10%): 3.52

(5%): 0%

(10%): 0%

(5%): 0%

(10%):0%

(1%): 0.15

(1%): 0%

C14 glucoside

(1.25%): 17.5%

(2.5%): 12.4%

(5%): 25%

(10%): 35%

(1.25%): 2.32

(2.5%): 2.66

(5%): 2.72

(10%): 3.64

(25%): 0%

(50%): 0%

(25%): 0%

(50%): 0%

(5%): 0.62

(5%): 0%

C16-C18 glucoside

(2.5%): 2%

(5%): 2%

(10%): 2.8%

(2.5%): 0.9

(5%): 1.1

(10%): 0.7

(5%): 0%

(10%): 0%

(5%): 0%

(10%): 0%

(5%): 0.03

(5%): 0%

C18 branched glucoside

(2.5%): 46.9%

(5%): 35.6%

(10%): 55.3%

(25%): 94.7%

(50%): 112.9%

(2.5%): 6.12

(5%): 4.64

(10%): 10.41

(25%): 9.41

(50%): 28.4

(6.25%): 0%

(12.5%): 0%

(6.25%): 0%

(12.5%): 0%

(6%): 0.04

(6%): 0%

> C18 glucoside

(2.5%): 0.7%

(5%): 2%

(10%): 2.7%

(2.5%): 0.9

(5%): 0.8

(10%): 1.1

(2.5%): 0%

(5%): 0%

(2.5%): 0%

(5%): 0% (5%): 0.21 (5%): 0%

> C18 branched xyloside

(2.5%): 11.9%

(5%): 20.4%

(10%):23%

(25%): 48.5%

(50%): 57.6%

(2.5%): 2.08

(5%): 2.93

(10%): 9.5

(25%): 20.85

(50%): 21.97

(50%): 0%

(100%): 0%

(50%): 0%

(100%): 0%

Not tested

Not tested

Irritation parameter




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OVERVIEW




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REGULATORY DERMAL SENSITIZATION TEST METHODS

Guinea pig tests (OECD 406;

adopted 1992) have been used for

sensitization testing for many

years

The Local Lymph Node Assay

(LLNA, OECD 429; adopted 2002) is

now the preferred method for

chemical safety testing under new

European chemicals legislation

(REACH)



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SOME PROs AND CONs OF THE GPTs

Pros

Give information on both sensitization and induction phase

Allow clarification of equivocal results via rechallenge

Have been used for decades; large amounts of historical data are available

There is a long experience with marketed products

Cons

Animal test

Not time efficient

Visual (not “objective”) assessments of reactions

No dose response measured (generally gives no measure of potency)



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SOME PROs AND CONs OF THE LLNA

Pros

Minimizes stress and injury (3 Rs)

Dose response measured (potency) via objective measurements

Formally validated

Cons

Gives information on the induction phase only (no challenge)

Clarification of equivocal results via rechallenge not possible

Limited data available for some substance classes



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COMPARISON OF THE DIFFERENT TEST METHODS

Test

System

Number of

animals

(treat/cont.)

Induction

procedure

Time

required

Cut-off for

Positives*

Assess-

ment

GPMT 20 + 10

(10 + 5)

Intra- and

epidermal

23 days 30% Visual

Buehler 20 + 10 Epidermal 34 days 15% Visual

LLNA 15 + 5

(12+4)

Epidermal 7 days SI>3 Radiolabel

(pretest

visual)

* e.g. OECD 429, Directive (67/548/EEC); cut-offs now valid for EU GHS (3.4.2.2.4.1)



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Buehler Test Method (Buehler, 1965)

Repeated epidermal induction Challenge

~ 28 days

Appr. 30 h and 54 h

after challenge

Reading

optional

Rechallenge

Day 0, Day 6-8 and Day 13-15

GUINEA PIG TESTS (OECD 406)

6 h

occl.

6 h

occl.

Day 27-29



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GUINEA PIG TESTS (OECD 406)

Guinea Pig Maximation Test (Magnusson & Kligman,1969)

Intradermal induction Epidermal induction Challenge

InjectFreund„s Complete Adjuvant

+ test

substance

~ Day 6-8 ~ Day 20-22

24 and 48 h

after challenge

Reading

optional

Rechallenge

48 h

occl.

24 h

occl.



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Epicutaneous induction: Application

of the chemical on days 1, 2 and 3 (3

dose groups/1 vehicle group)

Remove

lymph nodes 5

hours later;

make a cell

suspension

Inject 3H-thymidine

on Day 6

Determine 3H-thymidine

incorporation via liquid

scintillation counting

THE LOCAL LYMPH NODE ASSAY (OECD 429)



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HUMAN REPEATED INSULT PATCH TEST (HRIPT)

The HRIPT is the most relevant for humans despite the fact that:

The HRIPT is not used to generate primary data on the sensitization

potential of a chemical in humans; not used for classification (ethical

aspects)

The HRIPT is generally used to confirm a lack of risk at levels of use

Therefore, lower concentrations are often used than in the animal tests



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HUMAN REPEATED INSULT PATCH TEST (HRIPT)

Repeated epidermal induction Challenge

Reading

9 applications

during 3

weeks

14 days

24 and 48 hours

Various protocols, e.g. Draize, Voss Griffith, Shelanski

comparative cesio study in surfactants: inconsistent...

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