A Comparison of Reactivity Schemes for the Prediction Skin

Chem. Res. Toxicol. 2008, 21, 521–541

521

A Comparison of Reactivity Schemes for the Prediction Skin Sensitization Potential Grace Patlewicz,*,† David W. Roberts,‡ and E. Uriarte§ European Commission, DG Joint Research Centre, Institute for Health and Consumer Protection, European Chemicals Bureau TP582, 21027 Ispra (VA), Italy, School of Pharmacy and Chemistry, LiVerpool John Moores UniVersity, LiVerpool L3 3AF, United Kingdom, and Department of Organic Chemistry, Faculty of Pharmacy, UniVersity of Santiago de Compostela, 15872 Santiago de Compostela, Spain ReceiVed September 14, 2007

Skin sensitization is an important toxic end point for both regulatory frameworks and safety assessment. There are many hurdles for a chemical to overcome in terms of inducing skin sensitization, although the binding of chemicals to skin protein is thought to be the rate-determining step. Current strategies to predict the skin sensitization potential of chemicals in silico is through the identification of electrophilic characteristics. A number of predictive schemes have been developed in recent years, some based on broad structural rules and some with a reaction chemistry mechanistic basis. This work compares two schemes that are based on reaction chemistry. The first scheme comprises a set of rules that characterize reaction mechanistic domains as proposed by Aptula and Roberts [(2006) Chem. Res. Toxicol. 19, 1097–1105]. The second is a set of structure-toxicity and structure-metabolism pathways that are encoded and embedded into the TIssue MEtabolism Simulator skin sensitization model (TIMES-SS) [(2005) Int. J. Toxicol. 24, 189–204]. Here, a comparison of these schemes has been made using a recently published data set of 210 chemicals that have been tested in the local lymph node assay. The similarities and differences of the schemes are highlighted, together with modifications that could be made to TIMES-SS to harmonize the two approaches. Introduction Skin sensitization is an important end point that needs to be evaluated in the context of regulatory frameworks such as Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH)1 (1) as well as for risk management purposes within company safety evaluation assessments. Current strategies in the prediction of skin sensitization rely on in vivo testingsprincipally the local lymph node assay (LLNA), which provides a measure of relative sensitizing potency, a critical requirement for risk management purposes. Alternative approaches encompass in vitro technologies and in silico techniques such as (Q)SARs, read-across, or chemical categories (2). It is worth mentioning here in brief the context of the skin sensitization mechanism that shapes the factors that are thought to be important in the induction of skin sensitization. Clearly, for induction to occur, a chemical must overcome a number of hurdles (3). These include penetration into the viable epidermis and binding to skin proteins followed by lymphocyte proliferation as part of the immune response. In this framework, the critical factor is really the binding step; that is, a chemical has to react with the skin protein to form an immunogenic complex in order for the rest of the immune response to occur. The nature * To whom correspondence should be addressed. Tel: +39 0332 789616. Fax: +39 0332 786717. E-mail: [email protected] or patlewig@ hotmail.com. † European Commission. ‡ Liverpool John Moores University. § University of Santiago de Compostela. 1 Abbreviations: REACH, Registration, Evaluation, Authorisation, and Restriction of Chemicals; RAI, relative alkylation index; LLNA, local lymph node assay; GP, guinea pig; EC3, estimated concentration required to produce a SI of 3; SI, stimulation index.

of this binding is not fully established, although the current paradigm states that the binding is covalent; that is, the chemical behaves as an electrophile, and the protein behaves as the nucleophile. No doubt, there are other pathways to consider, such as free radical binding and so on, but it is believed that in the majority of cases the binding is covalent; hence, a range of electrophilic-nucleophilic reaction pathways play a role in how a chemical will react (4). The early strategies in silico that attempt to predict the likely sensitizing behavior of new chemicals have very much relied on this paradigm of covalent bonding. The types of approaches used for predicting skin-sensitizing effects span both qualitative and quantitative methods. The first quantitative method was that proposed by Roberts and Williams in 1982 when the so-called relative alkylation index (RAI) was defined (5). The fundamental basis of the approach is simple; sensitization is treated as a function of hydrophobicity and reactivity. The approach has been successfully applied to evaluate a number of data sets of chemicals including sultones, aldehydes, ketones, sulfonates, etc (6). The approach has been traditionally geared toward specific chemical classes and hence, until recently, been of limited predictive coverage. The identification of electrophilic features in chemicals has been the focus of an array of work where qualitative associations between structure and sensitization response have been investigated. Workers such as Barratt et al. (7) derived structural alerts for sensitizers that were subsequently encoded into the Derek for Windows (DfW) expert system (see later). Work conducted at the same time by Payne and Walsh (8) identified similar alerts that were also incorporated into the same expert system. Ashby et al. (9) defined broad structural relationships based on an evaluation of a large data set of LLNA skin sensitization data. Other workers such as Gerner et al. (10) and Zinke et al. (11)

10.1021/tx700338q CCC: $40.75  2008 American Chemical Society Published on Web 01/12/2008

strong strong

106-50-3 95-70-5 1166-52-5 95-55-6 5307-14-2 55302-96-0 108-45-2 25646-71-3

55-55-0 35691-65-7 3775-21-1 818-61-1 13041-12-8 1337-81-1 97-54-1 29043-97-8 6358-09-4 2871-01-41 104-46-1 3913-71-1 111-80-8 104-55-2 591-27-5 154750-20-6 111-80-8 104-55-2 591-27-5

1,4-phenylenediamine

2,5-diamino-toluene

lauryl gallate (dodecyl gallate)

2-aminophenol

2-nitro-p-phenylenediamine

2-methyl-5-hydroxyethylaminophenol

3-phenylenediamine

4-(N-ethyl-N-2-methan-sulfonamido-ethyl)-2-methyl-1,4-phenylenediamine (CD3) isopropyl isoeugenol

metol

1,2-dibromo-2,4-dicyanobutane

3-methyl-4-phenyl-1,2,5-thiadiazole-1,1-dioxide (MPT) 2-hydroxyethyl acrylate

6-methylisoeugenol

vinyl pyridine isoeugenol

5,5-dimethyl-3-methylene-dihydro-2(3H)-furanone

2-smino-6-chloro-4-nitrophenol

HC red no. 3

trans-anethol

trans-2-decenal

methyl 2-nonynoate

cinnamic aldehyde

3-aminophenol

3-bromomethyl-5,5-dimethyl-dihydro-2(3H)-furanone

methyl 2-nonynoate

cinnamic aldehyde

3-aminophenol

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate

moderate moderate

moderate

moderate moderate

strong

strong

strong

strong

strong

strong

strong

strong

strong

strong

123-31-9

hydroquinone

extreme

sensitizing potency

106-51-4

CAS no.

p-benzoquinone

name

probably/possibly a pro-Michael acceptor

definite_Michael

probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor definite_Michael

definite_Michael

definite_Michael

probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor definite_Michael

probably/possibly a pro-Michael acceptor definite_Michael probably/possibly a pro-Michael acceptor definite_Michael

definite_Michael definite_Michael

probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor pro-Michael

probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor pro-Michael

pro-Michael

definite_Michael

Roberts assignment

Michael type nucleophilic addition Michael type nucleophilic addition,Schiff base formation no binding

no binding

Michael type nucleophilic addition,Schiff base formation Michael type nucleophilic addition Michael type nucleophilic addition,Schiff base formation no binding

no binding

no binding

Michael type nucleophilic addition no binding

no binding no binding

nucleophilic substitution of haloaliphatics no binding Michael type nucleophilic addition no binding

no binding

no binding

no binding

no binding

no binding

no binding

no binding

arylcarboxylate aminolysis

no binding

no binding


TIMES-SS/toolbox protein binding

free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone

Michael type nucleophilic addition



nucleophilic hetereocyclic ring opening, Schiff base formation

Michael type nucleophilic addition, N-hydroxylamine condensation with proteins


free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone nucleophilic hetereocyclic ring opening free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone





free radical protein adduct, Michael type nucleophilic addition Michael type nucleophilic addition, N-hydroxylamine condensation with proteins Michael type nucleophilic addition, N-hydroxylamine condensation with proteins arylcarboxylate aminolysis, Free radical protein adduct Michael type nucleophilic addition

TIMES-SS/toolbox metabolism

Table 1. List of Chemicals, Their Sensitizing Potency, Their Roberts Assignment, and Their TIMES-SS Assignment

522 Chem. Res. Toxicol., Vol. 21, No. 2, 2008 Patlewicz et al.

104-27-8 101-86-0 122-40-7 7492-44-6 97-53-0 186743-25-9 186743-24-8 140-67-0 104-54-1 140-88-5 97-90-5 186743-26-0

1-(p-methoxyphenyl)-1-penten-3-one

hexyl cinnamic aldehyde

amyl cinnamic aldehyde

a-butyl cinnamic aldehyde

eugenol

5-methyleugenol

6-methyleugenol

4-allylanisole

cinnamic alcohol

ethyl acrylate

ethylene glycol dimethacrylate

3-methyleugenol

weak

weak

weak

weak

weak

weak

weak

weak

weak

weak

weak

moderate

moderate

nonsensitizer

nonsensitizer

nonsensitizer

75-35-4

2111-75-3

perillaldehyde

moderate

vinylidene dichloride

116-26-7

safranal (1,1,3-trimethyl-2-formylcyclohexa-2,4-diene)

moderate

92-48-8

2785-87-7

dihydroeugenol

moderate

6-methylcoumarin

141-05-9

diethyl maleate

moderate

923-26-2

93-51-6

2-methoxy-4-methyl-phenol

moderate

2-hydroxypropyl methacrylate

6728-26-3

trans-2-hexenal

moderate

nonsensitizer

101-39-3

a-methyl cinnamic aldehyde

moderate

91-64-5

5910-85-0

2,4-heptadienal

moderate

coumarin

122-57-6

benzylidene acetone (4-phenyl-3-buten-2-one)

moderate

nonsensitizer

186743-29-3

3-methylisoeugenol

definite_Michael

definite_Michael

definite_Michael


definite_Michael

probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a pro-Michael acceptor probably/possibly a Michael acceptor definite_Michael

probably/possibly a Michael acceptor



definite_Michael

definite_Michael

probably/possibly a pro-Michael acceptor definite_Michael

definite_Michael

pro-Michael

definite_Michael

definite_Michael

definite_Michael


Table 1. Continued moderate

isopropyl eugenol

154750-20-6

3-bromomethyl-5,5-dimethyl-dihydro-2(3H)-furanone

Michael type nucleophilic addition Michael type nucleophilic addition Michael type nucleophilic addition no binding

no binding

Michael type nucleophilic addition Michael type nucleophilic addition Michael type nucleophilic addition no binding

no binding

no binding

no binding

Michael type nucleophilic addition, Schiff base formation Michael type nucleophilic addition, Schiff base formation Michael type nucleophilic addition Michael type nucleophilic addition, Schiff base formation Michael type nucleophilic addition, Schiff base formation Michael type nucleophilic addition, Schiff base formation no binding


Michael type nucleophilic addition Michael type nucleophilic addition,Schiff base formation Michael type nucleophilic addition,Schiff base formation Michael type nucleophilic addition,Schiff base formation no binding

no binding

no binding

free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone nucleophilic hetereocyclic ring opening

free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone nucleophilic hetereocyclic ring opening


nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition, Schiff base formation

free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition free radical protein adduct, Michael type nucleophilic addition, nucleophilic cycloaddition to diketone nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition, Schiff base formation


nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition, Schiff base formation


A Comparison of ReactiVity Schemes for Skin Sensitization Chem. Res. Toxicol., Vol. 21, No. 2, 2008 523

108-46-3 1086-00-6 100-11-8 57-57-8 77-78-1 100-39-0 2374-65-4 26452-48-2 1675-54-3 112-82-3 64-67-5 10520-81-7 3508-00-7 629-72-1 4276-49-7 3344-77-2 66-27-3 6938-66-5 51323-71-8 4860-03-1 112-71-0 51323-71-8 4860-03-1 112-71-0 111-25-1 765-09-3 4292-19-7 19218-94-1 112-89-0 3386-33-2 120-51-4 143-15-7 73367-80-3 544-77-4 693-67-4 2425-54-9 74036-97-8 4282-42-2

1-chloromethylpyrene

4-nitrobenzyl bromide

propiolactone

dimethyl sulfate

benzyl bromide

methyl dodecane sulfonate

methyl hexadecene sulfonate

bisphenol A-diglycidyl ether

1-bromohexadecane diethyl sulfate

2-bromotetradecanoic acid

1-bromoheptadecane 1-bromopentadecane 1-bromoeicosane 12-bromo-1-dodecanol methyl methanesulphonate

1-bromodocosane dodecyl methane sulfonate

1-chlorohexadecane

1-bromotetradecane dodecyl methane sulfonate

1-chlorohexadecane

1-bromotetradecane 1-bromohexane 1-bromotridecane 1-iodododecane

1-iodotetradecane

1-bromooctadecane 1-chlorooctadecane

benzyl benzoate 1-bromododecane 12-bromododecanoic acid 1-iodohexadecane

1-bromoundecane 1-chlorotetradecane

7-bromotetradecane 1-iodononane

CAS no.

resorcinol

name

weak weak

weak weak

weak weak weak weak

weak weak

weak

moderate weak weak weak

moderate

moderate moderate

moderate

moderate moderate

moderate moderate moderate moderate moderate

moderate

moderate moderate

moderate

strong

strong

strong

strong

strong

extreme

extreme

nonsensitizer

SN2 SN2

SN2 SN2

SN2H-polar SN2 SN2H-polar SN2

SN2 SN2

SN2

SN2 SN2 SN2 SN2

SN2

SN2 SN2H-polar

SN2

SN2 SN2H-polar

SN2 SN2 SN2 SN2H-polar SN2H-polar

SN2H-polar

SN2H-polar SN2H-polar

SN2H-polar

SN2H-polar

SN2H-polar

SN2

SN2H-polar

SN2H-polar

SN2H-polar

probably/possibly a pro-Michael acceptor SN2

Roberts assignment

Table 1. Continued sensitizing potency

nucleophilic substitution of haloaliphatics nucleophilic substitution of haloaliphatics nucleophilic heterocycle ring opening nucleophilic substitution of alkyl sulfates nucleophilic substitution of haloaliphatics nucleophilic substitution of alkyl sulfonates nucleophilic substitution of alkyl sulfonates nucleophilic heterocycle ring opening no binding nucleophilic substitution of alkyl sulfates nucleophilic substitution of haloaliphatics no binding no binding no binding no binding nucleophilic substitution of alkyl sulfonates no binding nucleophilic substitution of alkyl sulfonates haloalkane free radical protein adduct formation no binding nucleophilic substitution of alkyl sulfonates haloalkane free radical protein adduct formation no binding no binding no binding haloalkane free radical protein adduct formation haloalkane free radical protein adduct formation no binding haloalkane free radical protein adduct formation arylcarboxylate aminolysis no binding no binding haloalkane free radical protein adduct formation no binding haloalkane free radical protein adduct formation no binding haloalkane free radical protein adduct formation

no binding


free radical protein adduct

free radical protein adduct free radical protein adduct






free radical protein adduct free radical protein adduct free radical protein adduct


nucleophilic heterocycle ring opening



35709-09-2 2426-08-6 109-65-9 693-58-3 2473-01-0 638-45-9 629-93-6 4230-15-3 50-00-0 111-30-8 579-07-7 107-22-2 109-55-7 107-15-3 122-78-1 111-40-0 93-53-8 112-45-8 167998-73-4 167998-76-7 465-29-2 110-41-8 431-03-8 55846-68-9 502-67-0 5392-40-5 56290-55-2 5406-12-2 31906-04-4 80-54-6 326-06-7 103-95-7 2277-19-2

oleyl methane sulfonate

butyl glycidyl ether

1-bromobutane 1-bromononane 1-chlorononane

1-iodohexane 1-iodooctadecane

methyl hexadecyl sulfonate

formaldehyde glutaraldehyde 1-phenyl-1,2-propanedione

glyoxal

3-dimethylaminopropylamine

ethylenediamine free base

phenylacetaldehyde diethylenetriamine

a-methylphenylacetaldehyde undec-10-enal

1-(2′,3′,4′,5′-tetramethylphenyl) butane-1,3-dione

1-(2′,5′-diethylphenyl)butane-1,3-dione

camphoroquinone

2-methylundecanal 2,3-butanedione

1-phenyloctane-1,3-dione

farnesal

citral

1-(2′,5′-dimethylphenyl)butane-1,3-dione

p-methylhydrocinnamic aldehyde lyral lilial (p-tert-butyl-a-ethyl hydrocinnamal) 4,4,4-trifluro-1-phenylbutane-1,3-dione

cyclamen aldehyde cis-6-nonenal

weak weak

weak weak weak weak

weak

weak

weak

weak

weak weak

weak

moderate

moderate

moderate moderate

moderate moderate

moderate

moderate

moderate

strong strong moderate

nonsensitizer

nonsensitizer nonsensitizer

SB SB

SB SB SB SB

SB

SB

SB

SB

SB SB

SB

SB

SB

proposed pro-SB via oxidative deamination proposed pro-SB via oxidative deamination SB proposed pro-SB via oxidative deamination SB SB

SB

SB SB SB

SN2H-polar

SN2 SN2

SN2 SN2 SN2

SN2H-polar

SN2H-polar

Table 1. Continued

nonsensitizer nonsensitizer nonsensitizer

weak

weak

Schiff base formation Michael type nucleophilic addition, Schiff base formation nucleophilic addition to ketones, nucleophilic cycloaddition to diketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones nucleophilic cycloaddition to diketones Schiff base formation nucleophilic cycloaddition to diketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones Michael type nucleophilic addition, Schiff base formation Michael type nucleophilic addition, Schiff base formation nucleophilic addition to ketones, nucleophilic cycloaddition to diketones Schiff base formation Schiff base formation Schiff base formation nucleophilic addition to ketones, nucleophilic cycloaddition to diketones Schiff base formation Schiff base formation

Schiff base formation no binding

no binding

nucleophilic substitution of alkyl sulfonates nucleophilic heterocycle ring opening no binding no binding haloalkane free radical protein adduct formation no binding haloalkane free radical protein adduct formation nucleophilic substitution of alkyl sulfonates Schiff base formation Schiff base formation nucleophilic cycloaddition to diketones nucleophilic cycloaddition to diketones, Schiff base formation no binding


nucleophilic addition to ketones



Schiff base formation



nucleophilic cycloaddition to diketones, Schiff base formation

Schiff base formation nucleophilic addition to ketones


nonsensitizer

135099-98-8

100-52-7 15646-46-5 1154-59-2 3326-32-7 525-76-8 176665-02-4 149-30-4 764-85-2 176664-99-6

1-(3′,4′,5′-trimethoxyphenyl)-4-dimethylpentane-1,3-dione

benzaldehyde oxazolone

tetrachlorosalicylanilide fluorescein-5-isothiocyanate

2-methyl-4H,3,1-benzoxazin-4-one (product 2040)

C6-azlactone

2-mercaptobenzothiazole

nonanoyl chloride

C4-azlactone

moderate

57077-36-8 36727-29-4 176665-04-6

isononanoyl chloride

3,5,5-trimethylhexanoyl chloride

C9-azlactone

moderate

moderate

moderate

methyl 2-sulfophenyl octadecanoate

moderate

moderate

moderate

moderate

strong

extreme strong

nonsensitizer extreme

nonsensitizer

1-(2′,3′,4′,5′-tetramethylphenyl)-3-(4′-tertbutylphenyl) propane-1,3-dione

nonsensitizer

492-94-4

weak weak weak nonsensitizer

furil

107-75-5 620159-84-4 97-96-1 874-23-7

hydroxycitronellal 2-(4-tert-amylcyclohexyl)acetaldehyde (QRM 2113) diethyl acetaldehyde 2-acetylcyclohexanenone

weak

weak

nonsensitizer

170928-69-5

3-ethoxy-1-(2′,3′,4′,5′-tetramethylphenyl)propane-1,3-dione

94-02-0

6668-24-2

1-phenyl-2-methylbutane-1,3-dione

weak

ethyl benzoylacetate

1118-71-4

2,2,6,6-tetramethyl-heptane-3,5-dione

weak

nonsensitizer

13706-86-0

5-methyl-2,3-hexanedione

weak

bis-1,3-(2′,5′-dimethylphenyl)-propane-1,3-dione

106-24-1

CAS no.

geraniol

name

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer

acyl transfer acyl transfer

relatively_unreactive_SB acyl transfer

relatively_unreactive_SB





SB SB SB SB

SB

SB

SB

possibly pro-SB via oxidation of CH2OH to CHO, but possibly pro-SN2 via sulfation of allylic CH2OH to CH2OSO3SB

Roberts assignment


no binding Michael type nucleophilic addition, nucleophilic addition to azomethynes no binding nucleophilic addition to isothiocyanates nucleophilic addition to azomethynes nucleophilic addition to azomethynes nucleophilic substitution of dithiocarbamic acid esters nucleophilic substitution of acyl halides nucleophilic addition to azomethynes arylcarboxylate aminolysis, electrophilic substitution of arenesulfinic acids nucleophilic substitution of acyl halides nucleophilic substitution of acyl halides nucleophilic addition to azomethynes

nucleophilic cycloaddition to diketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones nucleophilic addition to ketones nucleophilic addition to ketones Schiff base formation Schiff base formation Schiff base formation nucleophilic addition to ketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones nucleophilic addition to ketones Michael type nucleophilic addition, nucleophilic cycloaddition to diketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones nucleophilic addition to ketones, nucleophilic cycloaddition to diketones

no binding


nucleophilic addition to azomethynes

electrophilic substitution of arenesulfinic acids


disulfide formation, Michael type nucleophilic addition

nucleophilic addition to azomethynes, Michael type nucleophilic addition nucleophilic addition to azomethynes

free radical protein adduct nucleophilic addition to isothiocyanates

Michael type nucleophilic addition, nucleophilic addition to azomethynes, Schiff base formation

nucleophilic cycloaddition to diketones, nucleophilic addition to ketones, Michael type nucleophilic addition, Schiff base formation, free radical protein adduct

Michael type nucleophilic addition,“nucleophilic cycloaddition to diketones”









514-10-3 144-62-7 78-70-6 110-27-0 5989-27-5 67-68-5 110-86-1 62-53-3 94-09-7 108-90-7 121-32-4 56-81-5 110-54-3 99-96-7 67-63-0 59-01-8; 8063-07-8

abietic acid

oxalic acid

linalool

isopropyl myristate

R-(+)-limonene

dimethylsulfoxide

pyridine

aniline

benzocaine

chlorobenzene

ethyl vanillin

glycerol

hexane

4-hydroxybenzoic acid

isopropanol

kanamycin

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

weak

weak

weak

weak

weak

weak

weak

moderate (false positive) weak

nonsensitizer

151-21-3

weak weak

sodium lauryl sulfate

93-99-2 39236-46-9

phenyl benzoate imidazolidinyl urea

weak

81-07-2

176665-11-5

C17-azlactone

weak

saccharin

176665-09-1

C15-azlactone

weak weak

weak

13557-75-0 176665-06-8

pationic 138C (sodium lauroyl lactylate) C11-azlactone

moderate

61-33-6

552-30-7

1,2,4-benzenetricarboxylic anhydride (trimellitic anhydride)

moderate

penicillin G

112-67-4

palmitoyl chloride

moderate

weak

94612-91-6

sodium 3,5,5-trimethylhexanoyloxybenzenesulphonate

non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive

non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive

non_reactive_or_pro_ reactive

acyl transfer

acyl transfer

acyl transfer


acyl transfer

acyl transfer


acyl transfer

acyl transfer

acyl transfer


Table 1. Continued moderate moderate

C19-azlactone

17369-59-4 119-84-6

3-propylidenephthalide 3,4-dihydrocoumarin

no binding

no binding

no binding

no binding

no binding

no binding

no binding


no binding

no binding

no binding

no binding

no binding

nucleophilic cycloaddition to diketones no binding

no binding

nucleophilic addition to azomethynes nucleophilic heterocycle ring opening nucleophilic heterocycle ring opening no binding

nucleophilic substitution of acyl halides nucleophilic substitution of cyclic dicarbonyls no binding nucleophilic addition to azomethynes nucleophilic addition to azomethynes nucleophilic addition to azomethynes arylcarboxylate aminolysis no binding

no binding arylcarboxylate aminolysis,Nucleophilic addition to lactones arylcarboxylate aminolysis

free radical protein adduct, Michael type nucleophilic addition, Schiff base formation, nucleophilic cycloaddition to diketones



nucleophilic hetereocyclic ring opening

nucleophilic heterocycle ring opening, nucleophilic cycloaddition to diketones

nucleophilic cycloaddition to diketones, nucleophilic substitution of cyclic dicarbonyls, Schiff base formation nucleophilic addition to azomethynes




arylcarboxylate aminolysis, electrophilic substitution of arenesulfinic acids



2634-33-5 137-26-8 90-15-3 97-00-7 108-77-0 87-86-5

tetramethylthiuram disulfide

1-naphthol 1-chloro-2,4-dinitrobenzene 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride)

pentachlorophenol

3810-74-0

streptomycin sulfate

1,2-benzisothiazolin-3-one (proxel active)

84-66-2

diethylphthalate

70-25-7 684-93-5 759-73-9 2682-20-4

71-36-3

1-butanol

1-methyl-3-nitro-1-nitrosoguanidine N-methyl-N-nitrosourea, toxic N-ethyl-N-nitrosourea 2-methyl-2H-isothiazol-3-one

121-33-5

vanillin

26172-55-4

121-57-3

sulphanilic acid

5-chloro-2-methyl-4-isothiazolin-3-one

63-74-1

sulphanilamide

57-97-6

69-72-7

salicylic acid

7,12-dimethylbenz[a]anthracene

94-13-3

propyl paraben

7778-50-9 50-32-8

57-55-6

propylene glycol

potassium dichromate benzo[a]pyrene

124-07-2

octanoic acid

87-69-4

119-36-8

methyl salicylate

23593-75-1

99-76-3

methyl 4-hydroxybenzoate (methylparaben)

clotrimazole

100-06-1

4′-methoxyacetophenone

tartaric acid

50-21-5

CAS no.

lactic acid

name

weak

moderate extreme extreme

moderate

moderate

extreme extreme moderate moderate

extreme

extreme

extreme extreme

moderate

moderate

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

nonsensitizer

SNAR (or other mechanism)

special_case SNAR SNAR

special_case

special_case

special_case special_case special_case special_case

special_case

special_case

special_case special_case

non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive special_case

non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive non_reactive_or_pro_ reactive

Roberts assignment


Michael type nucleophilic addition, Michael type nucleophilic ring opening no binding no binding no binding Michael type nucleophilic addition, Michael type nucleophilic ring opening nucleophilic heterocycle ring opening disulfide exchange, nucleophilic substitution of dithiocarbamic acid esters no binding no binding nucleophilic substitution of haloaromatics nucleophilic substitution of haloaromatics

no binding

nucleophilic addition to azomethynes no binding no binding

no binding



no binding

electrophilic substitution of arenesulfinic acids no binding

no binding

no binding


no binding

no binding


nucleophilic addition to ketones arylcarboxylate aminolysis

no binding

TIMES-SS/toolbox protein binding TIMES-SS/toolbox metabolism

nucleophilic substitution of haloaromatics, free radical protein adduct

nucleophilic substitution of dithiocarbamic acid esters

disulfide formation

Schiff base formation Schiff base formation

nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition nucleophilic hetereocyclic ring opening, Michael type nucleophilic addition


nucleophilic cycloaddition to diketones



free radical protein adduct, Michael type nucleophilic addition, Schiff base formation, nucleophilic cycloaddition to diketones

electrophilic substitution of arenesulfinic acids


nucleophilic cycloaddition to diketones


have poefrmed sm i alir evauloaitns totry and help otdienfiyt elcrotphcili feautres whtini chemcias.l

A Comparison of ReactiVity Schemes for Skin Sensitization

In the meantime, within the QSAR field, a shift in terms of the types of computational techniques began to evolve, and instead of exploring the mechanistic basis of groups of chemicals and trying to rationalize their behavior in light of chemistry, there was a tendency to develop empirical models that predicted a yes/no sensitizing outcome using descriptors that were at times hard to interpret physically. That is, a prediction could be made from a robust model, but the reason for why the chemical had that prediction was not interpretable. Many of those types of models have not been very well-characterized, as discussed in Roberts et al. (12) and Patlewicz et al. (6, 13). Expert systems are available for the prediction of skin sensitizationssome of the systems rely on a statistical/empirical approach; others rely on a knowledge-based mechanistic approach, and others rely on a hybrid of the two approaches. Examples include Derek for Windows (DfW), TOPKAT, MCASE, and TIMES-SS. For more information on these types of models, the reader is encouraged to consult other information sources, including refs 6 and 14. Owing to the sharp focus of legislation such as REACH (1) and the seventh Amendment to the Cosmetics Directive (2), there have been renewed attempts to explore skin sensitization (Q)SARs. To this end, the RAI approach (5) was re-examined to determine whether it possessed the ability to be extended and hence applied in a wider scope. The fundamental basis of linking sensitization to a function of hydrophobicity and reactivity remained the same, but instead of targeting chemical classes, a set of reaction mechanistic domains were defined to rationalize the behavior on the basis of reaction chemistry. The approach was described by Aptula et al. (15) where a set of 41 chemicals were evaluated and the original structurebased classification (16) was replaced by a reaction mechanistic domain classification. The approach was then characterized fully in 2006 (17), and since then, the rules for assigning chemicals to their reaction mechanistic domains have been updated and refined upon application to other data sets (18, 19). It is worth noting that these rules (17) are really just a statement of well-established reaction mechanistic principles, which are presented in the context of electrophile/nucleophile chemistry and are not derived from evaluating skin sensitization data sets per se. As such, they provide guidelines and an aide memoir, which human experts can use for reaction mechanistic domain classification in the context of QSAR, quantitative mechanistic modeling (QMM), or read across. They can also be used by human experts, collaborating with software developers, to refine mechanism-based computerized expert systems, and their potential for being used in this way is explored in the present paper. The analysis performed in Roberts et al. (19) is further considered here; specifically, it is compared, using the same data set (20), to the proposed reaction pathways contained within the expert system TIMES-SS (21–23). TIMES-SS is an expert system describing structure-toxicity and structure-metabolism relationships through a number of transformations simulating skin metabolism and interaction of the generated reactive metabolites with skin proteins. The skin metabolism simulator mimics metabolism using two-dimensional structural information. Metabolic pathways are generated based on a set of 236 hierarchically ordered principal transformations including spontaneous reactions and enzyme-catalyzed biotransformation reactions (phases I and II). The covalent reactions with proteins are described by 47 alerting groups. The associated mechanisms are in accordance with the existing knowledge on electrophilic interaction mechanisms of various

Chem. Res. Toxicol., Vol. 21, No. 2, 2008 529

structural functionalities. The transformations within TIMESSS are largely derived from previous chemical interpretations of sensitization data sets elsewhere such as those found in refs 7–9. This being so, the TIMES-SS approach and the reaction mechanistic domain approach are fundamentally based on the same principles. In this exercise, we compare and contrast the two schemes of reactivity rules to establish where they are similar and where there are differences. In particular, we use the mechanistic domain approach to help suggest potential refinements to TIMES-SS so as to identify the commonality between the two different schemes and to specify opportunities to harmonize the schemes or where a translation between the schemes is required. These aspects are important from an end user perspective.

Materials and Methods The approach taken for this comparison focused on a set of LLNA data for 210 chemicals recently published by Gerberick et al. (20). An evaluation of the data set into assigned mechanistic domains as per the rules described in ref 17 has recently been carried out (19). The strategy here was to use the domains defined and to compare for each of the groups where there was overlap with the rules identified by the TIMES-SS system and where there was not. Recently, the rules from TIMES-SS were implemented into the Organisation for Economic Co-operation and Development (OECD) Application Toolbox, which is currently under development (see h t t p : / / w w w . o e c d . o r g / d o c u m e n t / 2 3 / 0,3343,en_2649_34365_33957015_1_1_1_1,00.html for more details). The rules are the same (personal communication from LMC, the TIMES-SS developers), but the Toolbox enables chemical structures to be rapidly profiled on the basis of their protein-binding characteristics. For convenience, the rules from TIMES-SS as implemented in the OECD Application Toolbox (Version 0.3, release March, 2007) were used. The data set was introduced into the OECD Application Toolbox and profiled according to the protein-binding schema. An additional profiler was used to simulate the skin metabolism. For ease of evaluating the results, the mechanistic domains as defined in ref 19 were used as the basis of comparison, such that for each mechanistic domain, the binding profiles as determined by TIMES-SS were then compared. The overall outcomes of consensus are provided in brief. A more detailed explanation and rationale are proposed for the cases where there is a discrepancy between the two schemes. For convenience, we will refer to the mechanistic domains, as defined in ref 19, as the Roberts domains.

Results and Discussion The set of 210 chemicals tested in the LLNA (20) is provided in Table 1 together with the mechanistic pathways identified by the two schemes. The Roberts rules for assigning compounds to their reaction mechanistic domains can be found in refs 17 and 19. The TIMES-SS rules, while transparent and explicit within both the TIMES-SS and the OECD Toolbox software, are not so readily exportable from these programs. Examples of the types of rules that TIMES-SS contained are described in previous publications on TIMES-SS (21). If a rule was pertinent to rationalizing a discrepancy, it was presented forthwith. Initially, the data set was profiled using the OECD Application Toolbox using the protein-binding rules alone. Of the 210 chemicals profiled, 87 were flagged as nonbinders. This could be incorrectly interpreted to signify no sensitization activity, but on the contrary, of these 87 chemicals, some were likely to require activation to reactive species to cause skin sensitization.

530 Chem. Res. Toxicol., Vol. 21, No. 2, 2008

Patlewicz et al.

Figure 1. Categorization of the LLNA data using the TIMES-SS protein binding profiler.

Figure 2. Categorization of the LLNA data set according to the Roberts rules.

A proportion would be truly unreactive and therefore would not be expected to sensitize. The large number of predicted nonbinders provided an impetus to probe the data set further. Obviously, given the LLNA information available, the proportion of sensitizers relative to nonsensitizers was known, such that it was immediately obvious that a number of transformations must be missing. There were 42 nonsensitizers (20%) in the data set, as compared with 168 sensitizers; that is, the number of predicted nonbinding chemicals (87) was in fact twice as high as expected.

The set of chemicals was subsequently processed through the TIMES-SS metabolic transformations simulator contained within the Toolbox. This provided a more comprehensive view of the reactivity profile of the chemicals taking into account their activation potential. The number of reaction schemes for direct binders within TIMES-SS is quite extensive. Figure 1 depicts the number of chemicals and the reaction pathways expressed. At least 26 different reaction schemes were identified for the 210 data set (38 different pathways currently exist in the March 2007 release of the Toolbox). Many pathways were only triggered once or



Table 2. List of Proposed Michael Acceptors, Which Are Assigned Differently by TIMES-SS

twice due to a specific chemical. The dominating reaction pathways identified for this data set were that of Michael addition (MA) (13 chemicals) and Schiff base formation (SB)

(14 chemicals). Other reactions included haloalkane free radical protein adduct formation, nucleophilic addition to azomethynes (although azomethynes is not a commonly used or understood


term), nucleophilic addition to diketones, and arylcarboxylate aminolysis, which were found for nine chemicals in each case. In contrast, the scheme devised by Roberts contains six distinct domains, namely, Michael acceptors (MA), Schiff base electrophiles (SB), acyl transfer electrophiles, SN2 (H-polar and non-H-polar), SNAr, and non(pro)-reactives. The first five classes are well-characterized by specific structural rules and conditions (see refs 17 and 19). Figure 2 depicts the distribution of chemicals for each of the classes. As can be seen from Figure 2, there is a reasonably even distribution of chemicals in the six reaction mechanistic pathways with the exception of the SNAr set, which contains only three examples. As an initial comparison, it is clear that the number of MA and SB formers together with the number of non(pro)-reactives is markedly different between the two schemes. The Roberts classification assigns 55 as MA and 40 as SB, in contrast to the TIMES-SS scheme, which assigns 13 as MA, 14 as SB, and a set of 13 as “MA and SB”. The Roberts scheme accounts for metabolism within its rule description whereas metabolism is considered separately in the TIMES-SS rules. Here, after profiling for metabolism, the TIMES-SS profile dramatically changessinstead of 87 chemicals that are nonreactive or nonbinding, the number falls to 41 compounds [c.f. the Roberts scheme, which assigns only 32 as non(pro)-reactive]. The overall TIMES-SS classification with metabolism profiling assigns 41 compounds as definitely unreactive, 73 as reactive, 46 compounds as having reactive metabolites, and 50 cases where both the parent compound and its metabolite(s) are assigned as reactive. Thus, of the 87 original predicted nonbinders, 46 of those are now predicted reactive following activation. Since there are fewer domains in the Roberts scheme, this was chosen as the reference. The output from the TIMES-SS profile was then compared against each domain. Any discrepancies were discussed and rationalized by expert chemical judgment for each compound in turn. (Pro-)Michael Acceptors (MA). Fifty-five compounds in the data set were assigned as (pro)-Michael acceptors by the Roberts rules. TIMES-SS was in agreement for 43 of them; in 15 cases, these were exact matches, and in 28 cases, other pathways were also identified. There were discrepancies between the pathways for 12 compounds. These are listed in Table 2 and then discussed in turn. The Roberts rules assigned lauryl gallate as a pro-Michael acceptor. Upon oxidation to an o-quinone, a Michael addition could take place as shown in Scheme 1. TIMES-SS proposed three possible mechanisms for lauryl gallate: an aryl carboxylate aminolysis reaction on the parent compound (Scheme 2); or, after metabolic activation, a free radical route (not shown); or, after metabolic activation, an aryl carboxylate aminolysis (not shown). Inspection of the structure suggests that lauryl gallate in its parent form could, on paper, react via a free radical route (Scheme 3). The aryl carboxylate aminolysis rule within TIMES-SS appears to need some amendment to be mechanistically correct. It should trigger the -C(dO)O-Ar group (electrophilic because the OAr anion is a good leaving group) but not Ar-C(dO)O(not generally electrophilic). A rule describing compounds that are capable of undergoing oxidation to ortho-quinone Michael acceptors could also be added. Overall, a free radical route (although based on the parent compound rather than on a metabolite) cannot be ruled out, but the pro-Michael mechanism, analogous to that commonly

Patlewicz et al. Scheme 1. Proposed Michael Addition Mechanism for Lauryl Gallate

Scheme 2. Mechanism of Aryl Carboxylate Aminolysis for Lauryl Gallate

Scheme 3. Suggested Free Radical Route for Lauryl Gallate

accepted for poison ivy (24), seems likely. The aryl carboxylate aminolysis pathway does not seem plausible. 2-Methyl-5-hydroxyethylaminophenol is proposed as a potential pro-Michael acceptor by the Roberts rules, although not to the exclusion of other pathways. TIMES-SS flags no binding either directly or after metabolism. A transformation is clearly missing since this compound is a strong sensitizer in the LLNA, with an EC3 (estimated concentration required to produce a SI of 3) value of 0.4%. The chemical mechanism of substituted



Scheme 4. Proposed Mechanism for 2-Methyl-5-hydroxyethylaminophenol

Figure 3. Shortcomings in the alkyl halide alert within TIMES-SS.

Figure 4. Modification to TIMES-SS rule on nucleophilic substitution of haloalkanes.

Scheme 5. Proposed Mechanistic Pathway of Isopropyl Isoeugenol

Scheme 6. Proposed Mechanistic Pathways for 1,2-Dibromo-2-dicyanobutane

Scheme 8. Proposed Michael Addition Mechanism of Vinyl Pyridine

Scheme 9. Proposed Mechanism by TIMES-SS for Vinyl Pyridine

Scheme 10. Proposed Michael Addition Pathway for trans-Anethol

Scheme 11. MA Mechanism for 3-Bromomethyl-5,5-dimethyl-dihydro-2(3H)-furanone

Scheme 7. Proposed Mechanism for 3-Methyl-4-phenyl-1,2,5-thiadiazole

Scheme 12. TIMES-SS Mechanism for 4-Allylanisole

anilines, in the context of skin sensitization, is still somewhat poorly understood (18); some are sensitizers, and some are not. In this case, oxidation of some kind probably occurs, which facilitates nucleophilic attack (as suggested in Scheme 4). A transformation is required within TIMES-SS to account for the chemistry underlying the sensitization response observed for

this chemical, but implementation must await further work to better define the mechanistic principles of compounds of this type. Isopropyl isoeugenol has the potential to react via a Michael addition upon activation or else via a free radical route. The compound is a strong sensitizer; yet, no protein binding mechanism was identified by TIMES-SS. Isopropyl isoeugenol

534 Chem. Res. Toxicol., Vol. 21, No. 2, 2008 Scheme 13. Proposed MA Mechanism for Vinylidene Dichloride

Scheme 14. Mechanisms for Benzyl Benzoate

Scheme 15. (Cyclo)Addition Reactions Proposed by TIMES-SS for 1,2- and 1,3-Diketones

is thought to react similarly to isoeugenol, which is itself metabolically activated (15). The proposed pathway is shown in Scheme 5. TIMES-SS proposes that a nucleophilic substitution of a haloalkane for 1,2-dibromo-2,4-dicyanobutane is responsible for the strong sensitization response observed. Roberts et al. (19) propose that this compound reacts via a pro-Michael addition route involving 1,2-dehydrobromination. An alternative pathway is for a SN2 reaction to occur on the allylic bromide resulting from 2,3-dehydrobromination. Both pathways are shown in Scheme 6. Nucleophilic substitution of the primary alkyl bromide group of the parent compound is unlikely to occur in this case. While

Patlewicz et al.

-CH2-Br is generally an alerting group for SN2 reactions, in 1,2-dibromo-2,4-dicyanobutane, the alkyl bromide is analogous to neopentyl halides, strongly deactivated by steric hindrance to the approach of nucleophiles (25). A modification in the TIMES-SS could be incorporated to take account of this (Figures 3 and 4). 3-Methyl-4-phenyl-1,2,5-thiadiazole is categorized by the Roberts rules as a definite Michael acceptor, whereas TIMESSS identifies no binding despite the fact that this compound is a moderate sensitizer. The mechanism proposed in Roberts et al. (12) is shown in Scheme 7. Ideally, TIMES-SS’s Michael type rules should be extended based on the structural criteria presented in ref 17, specifically to include activating groups such as CdC-SO2 and CdN-SO2. Vinyl pyridine is a Michael acceptor and can react via nucleophilic attack at the double bond as shown in Scheme 8. TIMES-SS postulates a nucleophilic heterocylic ring opening for the epoxide metabolite (Scheme 9). The pathway proposed in Scheme 8 could be incorporated into TIMES-SS by extending the current Michael type rules to include ortho-pyridine and para-pyridine as activating groups. TIMES-SS flags no binding for trans-anethol; yet, this chemical is a moderate sensitizer in the LLNA, and as shown in Scheme 10, there is the possibility for at least a Michael addition pathway following demethylation and oxidation. Other pathways such as free radical or others may also exist. No modifications are proposed for TIMES-SS until further mechanistic work has made the situation clearer. 3-Bromomethyl-5,5-dimethyl-dihydro-2(3H)-furanone is a moderate sensitizer; yet, TIMES-SS identifies no protein binding. On the basis of inspection of the structure, it is surprising that TIMESSS does not trigger an alert for nucleophilic substitution of haloaliphatics, that is, an SN2 reaction at the aliphatic halide -CH2Br, bearing in mind that it does so, albeit in this case incorrectly, for 1,2-dibromo-2,4-dicyanobutane. The chemistry of this and related furanones with respect to skin sensitization have been extensively studied previously (26, 27), and a similar MA pathway to that proposed for 1,2-dibromo-2,4-dicyanobutane has been demonstrated in chemico (Scheme 11). An extension to the MA type rules for TIMES-SS, to cover pro-Michael activation by dehydrohalogenation, would be worthwhile. 4-Allylanisole (weak sensitizer) and isopropyl eugenol (nonsensitizing) are likely to undergo a similar pathway to that of isoeugenol and trans-anethole. TIMES-SS, in contrast, proposes a nucleophilic heterocylic ring opening for 4-allylanisole when converted to an epoxide metabolite (as shown in Scheme 12). No modifications are proposed for TIMES-SS at this time. However, we do see a need to establish guidelines for deciding

Scheme 16. Proposed Mechanisms for Geraniol


which double bonds are able to be activated by epoxidation and which are not, on the basis of which an appropriate modification to TIMES-SS could be formulated. Vinylidene dichloride, although tested and found to be a nonsensitizer in the LLNA, is proposed as a direct-acting MA (Scheme 13), although its reactivity is expected to be low. TIMES-SS indicates no binding. It is unnecessary to propose


any amendments to the TIMES-SS rules, since the issue of whether or not it reacts as a Michael acceptor is academic. The mechanism of resorcinol is not yet fully understood, although reaction after transformation via a Michael addition route is feasible. The likely mechanism still needs to be rationalized further. TIMES-SS, in contrast, identifies no binding. No modification is suggested for TIMES-SS at the current time.

Table 3. Table of Acyl Transfer Agents Where There Is a Potential Discrepancy between TIMES-SS and the Roberts’ Rules


Patlewicz et al. Table 3. Continued

SN2 Electrophiles. The Roberts rules identified 43 SN2 electrophiles. In 16 cases, there was consensus between TIMESSS and the Roberts rules. In these cases, the rules within TIMESSS were described in a greater level of detail, that is, effectively describing various structural subsets of SN2 reactions. Examples include nucleophilic substitution of haloaliphatics for 1-chloromethylpyrene, 4-nitrobenzyl bromide, benzyl bromide, and 2-bromotetradecanoic acid; nucleophilic substitution of alkyl sulfonates for methyl dodecane sulfonate and related sulfonates; and nucleophilic substitution of alkyl sulfates for methyl and ethyl sulfates. In 27 cases, there was disagreement, and in all of these cases aside from one, these chemicals represented linear haloalkanes or similar derivatives that could react directly via an SN2 reaction. Examples include 1-bromotridecane, 1-bromohexadecane, and 1-bromoheptadecane. In TIMES-SS, either no binding was flagged or a haloalkane free radical route was proposed instead. The final compound that showed a discrepancy was benzyl benzoate. This chemical can undergo an SN2 reaction [(1) in Scheme 14] although TIMES-SS wrongly identified the acyl transfer route via an arylcarboxylate aminolysis instead [(2) in Scheme 14] (see also lauryl gallate in the previous section on MA). The alternative SN2 pathway, well-established in the literature for these compounds (25), should be included in TIMES-SS. Schiff Base Electrophiles. The Roberts rules identified 40 SB electrophiles. For 20 chemicals, there was an apparent disagreement between the two schemes. Benzaldehyde was presented as a nonbinder by TIMES-SS. Aromatic aldehydes (i.e., compounds with a CHO group attached directly to the aromatic ring) are considered insufficiently reactive to act as SB sensitizers (28). For 18 1,2- and 1,3-diketones, no Schiff base mechanism was proposed by TIMES-SS; instead, a (cyclo)addition reaction as shown in Scheme 15 was proposed. Examples of the diketones triggered by this TIMES-SS rule include 1-phenyl-1,2-propanedione, camphoroquinone, 2,3butanedione, 2,2′,6,6′-tetramethyl-heptane-3,5-dione, and ethyl benzoylacetate. This is an unimportant discrepancy since the TIMES-SS mechanism is closely related to the Schiff base

mechanism and the rate determining step is probably the same for both mechanisms. In the remaining case, geraniol, TIMES-SS proposed no binding, whereas the Roberts rules suggest that there is a potential for metabolic transformation to result in a SB reactionsoxidation of the CH2OH to CHO [(1) in Scheme 16]. In addition, a pro-SN2 via sulfation of allylic CH2OH to CH2OSO3 [(2) in Scheme 16] may be applicable. These two potential pathways could be incorporated into TIMES-SS, but at this stage, until a larger number of similar examples have been assessed, this would probably be premature. Acyl Transfer Electrophiles. Twenty-six acyl transfer electrophiles were identified by the Roberts rules. Twelve look to be in agreement, but the rules in TIMES-SS are written to a greater level of detail, each rule specific to a structural subclass. Examples include the rule nucleophilic substitution of acyl halides, which is triggered for 3,5,5′-trimethylhexanoyl chloride; nucleophilic substitution of cyclic dicarbonyls for 1,2,4-benzenetricarboxylic anhydride; aryl carboxylate aminolysis for 3,4dihydrocoumarin; nucleophilic substitution of dithiocarbamic acid esters for 2-mercaptobenzothiazole; and nucleophilic heterocycle ring opening for saccharin. There were 14 discrepancies, which are described in turn. For convenience, these have been listed in Table 3. No binding was identified by TIMES-SS for tetrachlorosalicylanilide as the parent compound. A free radical route was proposed upon metabolic transformation. Inspection of the structure suggests that it could have the potential to behave as an acyl transfer electrophile [see Roberts et al. (18, 19)]. Tetrachlorosalicylanilide is itself an extreme sensitizer with an EC3 of 0.04%. Whether tetrachlorosalicylanilide is really an acyl transfer electrophile depends on the pKa of the dichloroaniline, acting as an acid by ionization of an N-H bond. Even if it is an acyl transfer agent, its reactivity is unlikely to be enough to rationalize its extreme potency. Free radical binding after photoactivation is another possible mechanism. No modification is proposed at the current time for TIMES-SS. TIMES-SS proposes that oxazolone either reacts directly or indirectly via a Michael addition route or nucleophilic addition



Scheme 17. Nucleophilic Addition to Azomethynes

Scheme 19. TIMES-SS Rule for Cycloaddition to Diketones

Scheme 18. TIMES-SS Mechanism for Fluorescein-5-isothiocyanate

Scheme 20. Mechanism of Autoxidation of Limonene

to azomethynes (see Scheme 17 for azomethyne nucleophilic addition). The Roberts rules scheme suggests that oxazolone will react via an acyl route at the carbonyl C of the cyclic ester functional group as illustrated in Roberts et al. (19) previously. Fluorescein-5-isothiocyanate is proposed by TIMES-SS to react via a nucleophilic addition to isothiocyanates as shown in Scheme 18. The Roberts rules assign this compound to react via an acylation route (strictly speaking thioacylation), but the two schemes are in fact consistent with each other. 2-Methyl-4H,3,1-benzoxazin-4-one (product 2040) was flagged by TIMES-SS to undergo a nucleophilic addition to azomethynes directly or indirectly, in addition to a Michael addition route following metabolic transformation. The Michael addition seems unlikely since the –OCOR group, unlike the –COOR group, is not a recognized Michael activator. Inspection of the structure highlights the potential for an acyl route where the ring is opened. Sodium lauroyl lactylate is identified by TIMES-SS to be a nonbinder despite being a weak sensitizer in the LLNA. In view of the weak sensitization potential of this compound, the discrepancy is not serious. C4,C6,C9,C11,C15,C17,C19-azlactones were flagged by TIMESSS to undergo a nucleophilic addition to azomethynes directly. The literature on azlactones reveals an alternative and more likely acylation pathway (18). TIMES-SS identified 3-propylidenephthalide to react via a nucleophilic addition to ketones after metabolic transformation. Inspection of the structure reveals a potential for acyl transfer. TIMES-SS identified imidazolidinyl urea to react via nucleophilic cycloaddition to diketones, nucleophilic substitution of cyclic dicarbonyls, or Schiff base formation following metabolic transformation. The rules flagged here suggest that TIMES-SS is mistaking amide carbonyls for keto groups. Nonreactives. Thirty-two chemicals were identified by the Roberts rules as nonreactive and not transformed to reactive derivatives. In 17 cases, there was agreement between the two schemes. In 15 cases, there was a discrepancy between the two schemes. These are listed in Table 4. Oxalic acid, lactic acid, and tartaric acid are flagged as reactive via a nucleophilic cycloaddition to diketones. In this case, the rule within TIMES-SS should be refined and modified to exclude acids, that is X(Y)CdO should only be a ketone (or aldehyde) where X and Y are either hydrogen or are groups bonding through C. The cycloaddition to diketones rule is shown in Scheme 19. TIMES-SS flags D-limonene to react via nucleophilic heterocyclic ring opening following metabolic transformation, that is, epoxidation followed by ring opening. Limonene is considered to be a nonsensitizer (indeed tests on the pure material have been found to be negative), which is able to give rise to sensitizing impurities with prolonged exposure to air. Matura et al. (29), on the basis of chemical studies, have proposed that it acts via abiotic autoxidation to a reactive hydroperoxide (Scheme 20).

Aniline reacts following metabolic transformation according to TIMES-SS. The very weak sensitization response observed (EC3 ) 89%) may indicate a false positive (possibly due to solvent action as a defatting agent) and in any case is so low as to be insignificant. Ethyl vanillin and vanillin are flagged by TIMES-SS to have the potential to react following metabolic transformation either via a free radical reaction with protein, Michael type nucleophilic addition, Schiff base formation, or nucleophilic cycloaddition to diketones. The observation that ethyl vanillin and vanillin are not sensitizers suggests that in practice these reactions do not appear to occur. Benzocaine, methyl salicylate, and propyl paraben were flagged by TIMES-SS as reacting via arylcarboxylate aminolysis. The aryl carboxylate aminolysis rule within TIMES-SS needs some amendmenttobemechanisticallycorrect.Itshouldtriggerthe–C(dO)O-Ar group instead of Ar-C(dO)O- (as discussed for lauryl gallate in an earlier section). 4-Methoxyacetophenone is flagged by TIMES-SS as reacting by nucleophilic addition. Simple monoketones are insufficiently good electrophiles to react in this way. The rule within TIMESSS needs to be refined to exclude simple monoketones. Sulphanilic acid is flagged by TIMES-SS to undergo an electrophilic substitution of arenesulfinic acids. This is incorrectly classified, as the structure is actually an arenesulfonic acid. No modifications are suggested for TIMES-SS; the mechanistic principles for these types of compounds are still not entirely clear (13). Streptomycin sulfate is experimentally a nonsensitizer. It contains two identical aldehyde groups, which should be able to react via the Schiff base mechanism, but it is very hydrophilic, and this reduces the likelihood that it will sensitize. The QMM for Schiff bases (30) has been applied to streptomycin sulfate as follows. The aldehyde group can be simplified to the general structure (ROCH2)2C(OH)·CHO. Using the method of Perrin et al. (31), the σ* value for the (ROCH2)2C(OH)- group has been determined to be 1.12. Adding σ* for the H atom (0.49), a value of 1.61 has been obtained for Σσ*. Log P has been calculated by ACD laboratories to be -2.5. Inserting these values in the Schiff base QMM (pEC3 ) 1.12 Σσ* + 0.42Log P – 0.62) gives a pEC3 value of 0.13 for streptomycin sulfate. The molecular weight of streptomycin is 1457, and it contains two identical aldehyde groups. Thus, the calculated EC3 value is 540%. This being well above 100%, the maximum possible test concentration, the QMM correctly predicts streptomycin sulfate to be a nonsensitizer. Special Cases. There were 12 special cases that could not be confidently assigned to reaction mechanistic domains via the Roberts rules. For clotrimazole, TIMES-SS proposed a nucleophilic addition to azomethyne reaction, which is unlikely since the double bonds in the imidazole ring are not Michael activated. The SN1 reaction proposed in Roberts et al. (19) seems more plausible.


Patlewicz et al.

Table 4. Nonreactives Where There Was a Discrepancy between the Roberts’ Rules and the TIMES-SS Assignment

1-Napthol is hypothesized to react via a keto-tautomer (Michael acceptor) as described in Roberts et al. (19), whereas TIMES-SS identified no binding.

No binding mechanisms are proposed by TIMES-SS for potassium dichromate. On the other hand, potassium dichromate has been discussed in Roberts et al. (19) as being not only an



Table 5. Summary of Discrepancies/Agreements for the Roberts’ and TIMES-SS Schemes Roberts’ rules

TIMES-SS


Schiff base formation nucleophilic addition to (di)ketones nucleophilic substitution of cyclic dicarbonyls nucleophilic cycloaddition to diketones nucleophilic addition to ketones Michael type addition nucleophilic substitution of alkyl sulfates nucleophilic substitution of alkyl sulfonates nucleophilic substitution of haloaliphatics nucleophilic substitution of dithiocarbamic acid esters disulfide exchange nucleophilic hetereocycle ring opening nucleophilic addition to isothiocyanates nucleophilic substitution of acyl halides arylcarboxylate aminolysis nucleophilic substitution of haloaromatics electrophilic substitution arenesulfinic acids haloalkane free radical protein adduct nucleophilic addition to azomethynes

Michael addition SN 2 SN2 at S SN2 (epoxides) acyl formation SNAr

oxidizing agent but also a source of electrophilic trivalent chromium ions. Transition state metal ions can behave as SN1 electrophiles, able to bind covalently to proteins in covalent coordination complexes. Michael type nucleophilic addition and nucleophilic ring opening were identified as possible pathways for 2-methyl-2H-isothiazol3-one and 5-chloro-2-methyl-4-isothiazolin-3-one within TIMESSS. For these compounds, there are in principle several electrophilic mechanisms available and it is more a matter of selecting which of them applies than deciding whether or not the compounds are reactive. The TIMES-SS rule (Michael type reactions) does not need modification. However, it may be useful to consider a new rule of –S-LG where LG is a potential leaving group, that is, one capable of stabilizing the negative charge. In the present case, the LG is NHCOR, so these compounds are able to react with a nucleophile (Nu) by an SN2 reaction at sulfur to form –S-Nu (32). 1-Methyl-3-nitro-1-nitrosoguanidine, N-methyl-N-nitrosourea, Nethyl-N-nitrosourea are all sensitizers (EC3 values were, respectively, 0.03, 0.05, and 1%). TIMES-SS, however, identified no binding. All three compounds have been discussed in extensive detail in Roberts et al. (19) where they were classified as oxophilic SN2 electrophiles. In this case, nucleophilic attack could occur at the central carbon atom, leading to direct protein modification with simultaneous release of a diazoalkyl cation that could alkylate the protein to give a second protein modification. A modification to account for the proposed reaction pathway could be added in TIMES-SS. TIMES-SS identified two possible reaction mechanisms for tetramethylthiuram disulfide, namely, disulfide exchange and nucleophilic substitution of dithiocarbamic acid esters. A SN2 reaction at the S atom has been discussed in Roberts et al. (18). In this case, the two schemes are consistent with each other. 7,12-Dimethylbenz[a]anthracene and benzo[a]pyrene are both well-recognized as being highly carcinogenic and mutagenic (33, 34). Their metabolism, involving cytochrome P450, to electrophilic species that can bind covalently to hard nucleophilic groups of DNA has been extensively investigated. 7,12-Dimethylbenz[a]anthracene has been shown to undergo oxidation of the methyl groups to hydroxymethyl groups that can be sulfated to give benzylic sulfates, which are hard SN2 electrophiles able to bind covalently to DNA (35). Evidence has also been presented to support the argument that these are the major “ultimate carcinogens” for 7,12-dimethylbenz[a]anthracene (36). The metabolic activation of benzo[a]pyrene is different in that the ultimate carcinogen is the diol epoxide (34), which is another hard SN2 electrophile (alternatively, it is possible that it acts by the SN1

comments

needs adjustment

mechanistically incorrect appears incorrect structuressulfonic not sulfinic disagreesshould be SN2 mechanistically incorrect

mechanism), able to bind covalently to DNA. Both compounds are very strong sensitizers in the LLNA. The skin has the capability to convert these polyaromatic hydrocarbons into their hard electrophilic metabolites, which in this case bind covalently to protein. These two compounds can be classified as pro-SN2 electrophiles (oxophilic). The TIMES-SS predictions for these compounds after metabolism are in fact consistent with the above. SNAr. There are only three SNAr electrophiles identified. Two concur with the schemes laid out in TIMES-SS; the third presents a discrepancy. 1-Chloro-2,4-dinitrobenzene is a very well-known strong sensitizer in humans and animals and is classed as an extreme sensitizer in the LLNA (EC3 ) 0.04%). The reaction pathway of sensitization is similar to that of the Michael acceptors, but instead of the anionic intermediate accepting a proton, the halogen atom is eliminated (see the scheme in ref 15). The negative charge in the intermediate is stabilized by the resonance and inductive effects of two nitro groups, which are much more electronegative than carbonyl groups. In contrast, TIMES-SS flagged no alerts. A revision in TIMES-SS as regards SNAr electrophiles is recommended, basing the amendment on published models in refs (37) and (38). 2,4,6-Trichloro-1,3,5-triazine (cyanuric chloride) and pentachlorophenol are both discussed in more detail in Roberts et al. (19); in any case, there is consensus, as nucleophilic substitution of haloaromatics is identified in both schemes.

Conclusions Here, a comparison has been made between two reactivity schemes using a published set of LLNA data for 210 compounds. In general, the agreement between the two schemes demonstrates a good degree of consistency. There were several areas where the rules within TIMES-SS could be usefully modified. Table 5 aims to summarize the commonalities and differences between the schemes. In brief, the aryl carboxylate aminolysis rule needs amending to be mechanistically correct. In several cases, extensions to the repertoire of Michael type reactions could be usefully carried out to flag compounds that are Michael activated. The rule for –CH2Br needs to be modified to account for scenarios when the attached groups are deactivating. A modification also needs to take place to correctly identify carbonyls that are SB electrophiles; for example, neither amide carbonyls nor carboxylic acids are keto groups. This should be the subject of further work.


The use of reaction chemistry principles in these two schemes has been shown to be effective in rationalizing skin sensitization behavior. This study has demonstrated that aside from some minor discrepancies, there is a great deal of consistency between these two reaction schemes. Future research should be directed into defining the mechanistic principles underlying those compounds still poorly understood, such as substituted anilines, phenols, and aromatic amines. Overall, our findings demonstrate the potential of the reaction mechanistic domain approach for synergistic interaction between human experts and mechanism-based computerized expert systems such as TIMES-SS.

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TX700338Q

A Comparison of Reactivity Schemes for the Prediction Skin

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