InSilicoTox – D4.

Report on Reaction Mechanisms in Toxicology

InSilicoTox Project

Deliverable 4 (D4)

Report on Reaction Mechanisms in Toxicology

October 2009

European Union Sixth Framework Programme Marie Curie Actions

Project No.: 42328
Project Acronym: InSilicoTox
Project Full Name: In Silico Tools to Reduce the Use of Animals in
Toxicity Testing for Bioreactive Chemicals

Deliverable Prepared by

Johannes Schwöbel, Mark Cronin, Yana Koleva, Judith Madden,

School of Pharmacy and Chemistry
Liverpool John Moores University
Byrom Street
Liverpool
L3 3AF
England

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Important Information
This document is a deliverable of the InSilicoTox Marie Curie Transfer of Knowledge
Project. The copyright of this material belongs to Liverpool John Moores University.
This document was prepared in good faith, Liverpool John Moores University or the
authors of this document are not responsible for any inaccuracies or errors.

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

D4. Report on Reaction Mechanisms in Toxicology

Summary

The toxicity of a compound, related to different modes of action, is linked to

the specific chemical reactivity of this compound. Therefore, a mechanistic
understanding is needed for a sound assessment and prediction of toxicological and
ecotoxicological behaviour.1 In the framework of the EU regulations Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH), risk assessment of
industrial chemicals is a very important issue in the upcoming decade.2

Depending on the specific target site of a toxicological mode of action, the

compound shows a variation in the underlying biochemical mechanism.3 This comes
along with Pearson’s Hard and Soft Acid and Bases (HSAB) concept. 4, 5 Molecular
interactions between electrophiles and nucleophiles are governed by their properties,
which follow hardness and softness patterns in general. Biological nucleophiles,
such as the DNA molecule, are predominantly hard chemicals, acting via rather hard
oxygen or nitrogen containing moieties. Softness of a nucleophile can be associated
with easy oxidisability and low electronegativity, for example thiol residues of
peptides and proteins with its sulphur atom. As many environmental pollutants are
soft agents, this is a first clue to understand their toxicity. Dissimilar hardness leads
to a higher potential energy barrier of reactions between electrophiles and
nucleophiles.

As an example for dissimilar reactivity, nitroaromatics do not show any

reactivity in skin sensitisation,6 while they form reactive DNA attacking
hydroxylamine intermediates, conducted by the enzyme nitroreductase, 6 but they
follow nucleophilic substitution (SN1 or SN2) in acute aquatic toxicity.7 In contrast,
,-unsaturated carbonyl compounds follow the same mechanism in protein binding

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

initiating skin sensitisation, in DNA binding, and in acute aquatic toxicity, namely
Michael-type addition.8

As a promising tool in alternative testing and in silico strategies, qualitative

QSAR models evaluate the presence or absence of a specific substructure in a
molecule, called structural alerts. They imply a specific mechanism connected with a
substructure. The substructures should be specific enough for reliable prediction of a
particular mechanistic pathway.

A systematic compilation of substructures, following different toxicological

modes of action, is provided by this report. For the first time, similarities and
dissimilarities in their biochemical mechanisms are assembled systematically in the
following table. Namely, the toxicological endpoints are acute aquatic toxicity, skin
sensitisation, and mutagenicity. All of them are considered as important endpoints,
according to REACH legislation. Briefly, for the ecotoxicological endpoint acute
aquatic toxicity,9 testing submerges species of fish to certain environmental
concentrations of a substance as a single dose expose to determine the lethality
level. Skin sensitisation is the induction of allergic responses in the skin. 10, 11

Exposure of a hapten to the skin leads to formation of a hapten-protein adduct,

following induction of allergen-specific lymphocytes and antibodies, both being ready
to respond to the next exposure of the hapten. Agents causing mutagenicity, 12 react
either directly with DNA and cause structural changes, or indirectly by cell
metabolites that have the direct mutagenic effect. Apart from that, some mutagens
may act as base analogues and get inserted into the DNA strand.

Literature

1. Cronin, M. T. D.; Livingstone, D. J., Predicting Chemical Toxicity and Fate.

CRC Press: Boca Raton, FL, USA, 2004.
2. REACH, Regulation (EC) No 1907/2006 concerning the Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a
European Chemicals Agency, amending Directive 1999/45/EC and repealing
Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No
1488/94 as well as Council Directive 76/769/EEC and Commission Directives
91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. 2006.

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

3. Borgert, C. J.; Quill, T. F.; McCarty, L. S.; Mason, A. M., Can mode of action
predict mixture toxicity for risk assessment? . Toxicol. Appl. Pharmacol. 2004,
201, 85-96.
4. Pearson, R. G.; Songstad, J., Application of the principle of hard and soft acids
and bases to organic chemistry. J. Am. Chem. Soc. 1967, 89, 1827-1836.
5. Ho, T.-L.; Ho, H. C.; Hamilton, L. D., Biochemical significance of the hard and
soft acids and bases principle. Chem. Biol. Interact. 1978, 23, 65-84.
6. OECD Organization for Economic Co-operation and Development (Q)SAR
Application Toolbox, 1.1; OASIS, Laboratory of Mathematical Chemistry:
Bourgas, Bulgaria, 2009.
7. Roberts, D. W., An analysis of published data on fish toxicity of nitrobenzene
and amine derivatives. In QSAR in Environmental Toxicology, Kaiser, K. L. E.,
Ed. Reidel: Dordrecht, The Netherlands, 1987; Vol. II., pp 295-308.
8. Koleva, Y. K.; Madden, J. C.; Cronin, M. T. D., Formation of categories from
structure-activity relationships to allow read-across for risk assessment: Toxicity
of α,β-unsaturated carbonyl compounds. Chem. Res. Toxicol. 2008, 21, 2300-
2313.
9. Russom, C. L.; Bradbury, S. P.; Broderius, S. J.; Hammermeister, D. E.;
Drummond, R. A., Predicting modes of toxic action from chemical structure:
acute toxicity in the fathead minnow (Pimephales promelas). Environ. Toxicol.
Chem. 1997, 16, 948-967.
10. Roberts, D. W.; Aptula, A. O.; Patlewicz, G., Electrophilic chemistry related to
skin sensitization. Reaction mechanistic applicability domain classification for a
published data set of 106 chemicals tested in the mouse local lymph node
assay. Chem. Res. Toxicol. 2007, 20, 40-60.
11. Roberts, D. W.; Patlewicz, G.; Kern, P. S.; Gerberick, F.; Kimber, I.; Dearman,
R. J.; Ryan, C. A.; Basketter, D. A.; Aptula, A. O., Mechanistic applicability
domain classification of a local lymph node assay dataset for skin sensitization.
Chem. Res. Toxicol. 2007, 20, 1019-1030.
12. Lutz, W. K., Quantitative evaluation of DNA binding data for risk estimation and
for classification of direct and indirect carcinogens. Cancer Res. Clin. Oncol.
1986, 112, 85-91.

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Table 1: Substructures and associated mechanisms for different endpoints

Substructure Acute Aquatic Toxicity Skin Sensitisation Mutagenicity

Description 2D Fragment Mechanism Ref. Mechanism Ref. Mechanism Ref.

Benzene rings, NO2 Various (including 1, No binding 9 Ambiguous mechanisms; 63-71,

without aniline or SN1+SN2) 17-21 Compared models for reduction of 79
phenol compounds by xanthine oxidase
substructures, that NO2 (because the crucial activation step
have two nitro for mutagenic activity of these
chemicals is the metabolic
groups on one ring
reduction by one or more cytosolic
nitroreductases).

9, 105,
Nitroreductase; 106

Halogenated Hal Nucleophilic 9, 29 Nucleophilic 9 Nitroreductase 9

nitroaromatic substitution substitution of
haloaromatics
NO2

Aliphatic nitro RNO2 Basesurface narcotics 1, 9, No binding 9 Ambiguous mechanisms; 9

10, 29 Nitroreductase

Acetamidophenols NH Unknown; 1, 10, No binding 9 No binding 9

O (Pro-Michael-type 22
HO
addition)

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Quinolines Unknown 1, 10 No binding 9 Unknown; 72, 73

N
No binding

Chlorodiesters Arylcarboxylate 9, 107 No binding 9

aminolysis

Acrylates O Michael-type 1, 2, Michael-type 11, 61 No binding 9

nucleophilic addition; 4, 6, nucleophilic addition;
O SN2 10,
23-27,
57, 58

Diesters Unknown 28

Ketenes C C O Acylation 29 No binding 9 No binding 9

Acid halides (Acyl O Acylation 29 Nucleophilic 9, 107 Electrophilic mechanism 9

halide) R substitution of acyl
Hal halides

Dialkyl O Acylation 29 Nucleophilic 9 Electrophilic mechanism 9

carbomylchlorides N substitution of acyl
Hal halides

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Carboxylic acid O Acylation 29 Nucleophilic 9 No binding 9

anhydrides substitution of cyclic
R O dicarbonyls;
O
R = CH3, C2H5

Isocyanates N C O Acylation 1, 29 Nucleophilic addition to 9, No binding 9

isocyanates 108,
109

Isothiocyanates N C S Acylation; Michael- 1, 8, Nucleophilic addition to 9 No binding 9

type nucleophilic 29, isothiocyanates
addition 30, 31
Two mechanisms, which are
likely to take place
concurrently: (a) addition
across the C=N triple bond,
and (b) nucleophilic attack at
the benzylic sulfur with
displace-ment of the cyanide
leaving group. In (a), these
compounds act as
electrophiles, but not as
cyanogenic toxicants; in (b),
they are both electrophiles
and cyanogenic toxicants.

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Lactone (α-, β-, and O Schiff base formation; 1, 2, Michael-type 11, Direct alkylating agents 9
unsaturated) 8, 29 nucleophilic addition; 59,
R' O Strained ring
60,
electrophiles: Cyanide
R release via Nucleophilic
R' = H, CH3 cyanohydrin-like heterocycle ring 9
toxicant opening

Aldehydes C=O Schiff base formation; 1- 3, Schiff base formation 9, No binding 9

5, 25, 11-13,
Benzaldehydes:
28-29, 62
unknown
32- 35

Epoxides O Alkylation 1, 10, Nucleophilic 9 Ambiguous mechanisms; 76-78,

29, heterocycle ring Direct alkylation 9
36-38 opening

Aziridines H Alkylation 29 Nucleophilic 9 Direct alkylation 9

N heterocycle ring
opening

Sulfonic acid esters O O Alkylation 29, Nucleophilic 9 No binding 9

S 39, 40 substitution of alkyl
O sulfonates

Sulfuric acid esters O O Alkylation 29 Nucleophilic 9 No binding 9

S substitution of alkyl
O O sulfates

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Phosphoric acid O Alkylation 29 Nucleophilic 9 No binding 9

esters substitution of alkyl
P phosphonates and
O O
(thio)phosphates

Sulfonyl halide Miscellaneous reactions 29 Nucleophilic 9 No binding 9

substitution of sulfonyl
halides

Sulfonium salt Basesurface narcotics 9 No binding 9 Carbenium and episulfonium 9

ions: act directly on the DNA
bases

Haloacetamides Nucleophilic 29 Nucleophilic 9 Haloalkanes and compounds, 9

substitution substitution of containing labile halogen:
haloaliphatics electrophilic mechanism

Haloacetates Nucleophilic 29 Nucleophilic 9 Haloalkanes and compounds, 9

substitution substitution of containing labile halogen:
haloaliphatics electrophilic mechanism

Haloethyl amines Narcotic amines; 9, 29 No binding 9 Electrophilic mechanism 9

Nucleophilic
substitution

Haloethyl Nucleophilic 29 Disulfide formation 9 Electrophilic mechanism 9

substitution
sulfides

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Haloethers Basesurface narcotics; 9, 29 No binding 9 Electrophilic mechanism 9

Nucleophilic
substitution

Allylic alcohols Alcohol dehydrogenase 1, 8, No binding 9 No binding 9

activation 10, 41

Propargylic alcohols Alcohol dehydrogenase 1, 10, No binding 9 No binding 9

activation 42,43,
44-46

Quinones Michael-type 1, 10, Pro- Michael-type 9, 11 No binding 9

nucleophilic addition 29, acceptors, activated by
47, 48 oxidation (Michael-type
nucleophilic addition);

Unsaturated amides Addition to activated 1, 10, Michael-type 9 No binding 9

carbon-carbon double 29 nucleophilic addition
bond

Allylic cyano group Various; 1, 8, No binding 9 No binding 9

10, 29
Addition to activated
carbon-carbon double
bond

Allylic nitro group Addition to activated 1, 10, No binding 9 Nitroreductase 9

carbon-carbon double 29
bond

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Allylic sulfone Addition to activated 1, 10, No binding 9 No binding 9

group carbon-carbon double 29
bond

Allylic carboxy Addition to activated 1, 10, No binding 9 No binding 9

group carbon-carbon double 29
bond

Allylic carbonyl Michael-type 1-10 Michael-type 9, 11, Michael-type nucleophilic 9, 14,

group (aldehydes nucleophilic addition; nucleophilic addition; 12, 13 addition 15, 16
and ketones) ; Schiff base formation Schiff base formation
α,β-Unsaturated
carbonyl
compounds;

Styrenes Basesurface narcotics 1, 10 No binding 9 No binding 9

Allylic halides Nucleophilic 1, 10, Nucleophilic 9 Electrophilic mechanism 9

substitution 29, substitution of
49-51 haloaliphatics

Benzylic halides Nucleophilic 1, 10 Nucleophilic 9 Electrophilic mechanism 9

substitution substitution of
haloaliphatics

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Diazo compounds Nucleophilic 1, 9, No binding 9 Electrophilic mechanism 9

substitution 10, 29

Aromatic azo No binding 9 Decomposition of azoarenes 9

to active species (radicals) –
Radical mechanism

Mustards (Nitrogene Narcotic amines 1, 10 No binding 9 Electrophilic mechanism 9

mustard)

Disulfides Miscellaneous reactions 1, 10, No binding 9 No binding 9

29

Sulfenyl halides Miscellaneous reactions 1, 10, Disulfide formation 9 No binding 9

29

Peroxides 1, 10 Peroxide free radical 9 No binding 9

decomposition

Thiocyanates 1, 10 Disulfide formation 9 No binding 9

Hydrazines 1, 10 No binding 9 Unknown 74,

Nucleophilic addition 9
Reactions

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

N-nitroso Monooxygenase- 1,8, No binding 9 Unknown 104

compounds mediated N- 10
demethylation
transformation followed
by tauto-merism from
p-nitrosoaniline to the
corresponding Michael-
type acceptor quinoid
oxime structure.

C-nitroso 1, 10 No binding 9 No binding 9

compounds

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

1) Allylic and Cyanide release via 1, 10, 1) No binding; 9 1) No binding; 9

propargylic nitriles, cyanohydrin-like 29, 2) Nucleophilic 2) Electrophilic agent
or toxicant; 52-54 substitution of
2) -halogenated The resulting intermediate haloaliphatics
substituted nitriles liberates formaldehyde, a
Schiff-base electrophile, and
p-chloroaniline on
hydrolysis;
Two different mechanisms
acting concurrently: (a)
Michael-type addition across
the double bond conjugated
to two cyano-groups with
covaent bond formation, and
(b) corresponding addition
of water with ready loss of
malononitrile and o-chloro-
benzaldehyde.
Malononitrile can
undergo further
Metabolic transformation to
generate two moles of cya-
nide; o-chlorobenzaldehyde
can act as an electrophile by
Schiff-base formation;
Alkylation, SN2

Oximes 1, 10 Oxime group 9 No binding 9

condensation

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

β-Halogenated 1, 10 No binding 9 Electrophilic agent 9

alcohols

Halogenated 1, 10 1. Nucleophilic addition 9 Electrophilic agent 9

acetophenones to ketones;
2. Nucleophilic
substitution of
haloaliphatics

Pyridiniums Various 1, 10, No binding 9 No binding 9

ammonium 55, 56
compounds

Quaternary 1, 10 Electrostatic interaction 9 No binding 9

ammonium with proteins
compounds

Diketones Unknown 1, 2, Schiff base formation; 13, 62 No binding 9

8, 10 Nucleophilic
cycloaddition to 9
diketones

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Primary alkyl Basesurface narcotics; 9 No binding 9 Ambiguous mechanisms; 75, 80,

monohalide Nucleophilic Electrophilic agent 9
substitution

Secondary alkyl Basesurface narcotics; 9 No binding 9 Ambiguous mechanisms; 75, 80,

monohalide Nucleophilic Electrophilic agent; 9
substitution

Tertiary alkyl Basesurface narcotics; 9 No binding 9 Ambiguous mechanisms; 75, 80,

monohalide Nucleophilic Electrophilic agent; 9
substitution

Alkyl multihalide Basesurface narcotics 9 No binding 9 Ambiguous mechanisms; 75, 80,

Electrophilic agent; 9

Ethylene sulfide Nucleophilic 9 No binding 9

heterocycle ring
opening

Cyclopropane No binding 9 No binding 9

Oxetane Basesurface narcotics 9 No binding 9 No binding 9

Trimethyleneimine Basesurface narcotics 9 No binding 9 No binding 9

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Aromatic primary No binding 9 ambiguous mechanisms; 81-102,

amine electrophilic reactants 9

Aromatic secondary ambiguous mechanisms; 81-102,

amine electrophilic reactants 9

Aromatic tertiary ambiguous mechanisms; 81-102,

amine electrophilic reactants 9

Aromatic No binding 9 No binding 9

sulfonamide

Aliphatic primary Narcotic amines 9 No binding 9 No binding 9

amine

Hydroxylamine No binding 9 No binding 9

Pyridine-N-oxide No binding 9 No binding 9

Diazonium salt No binding 9 No binding 9

Hydrazone No binding 9 Nucleophilic addition 9

Reactions

Nitryl No binding 9

Anilide No binding 9 No binding 9

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

Carboxylic acid Basesurface narcotics 9 No binding 9 Unknown 103

amide

Sulfamide No binding 9 No binding 9

Halogenated Basesurface narcotics; 9 No binding 9 No binding 9

aromatic ring
Nucleophilic
substitution

Sulfonic acid No binding 9 No binding 9

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

References to Table 1:

1. Russom, C. L.; Bradbury, S. P.; Broderius, S. J.; Hammermeister, D. E.; Drummond, R. A., Predicting modes of toxic action
from chemical structure: acute toxicity in the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 1997, 16,
948-967.
2. Cronin, M. T. D.; Sinks, G. D.; Schultz, T. W., Modelling of toxicity to the ciliate Tetrahymena pyriformis: the aliphatic
carbonyl domain. In Forecasting the Environmental Fate and Effects of Chemicals, Rainbow, P. S.; Hopkins, S. P.; Crane, M.,
Eds. John Wiley & Sons: Chichester, United Kingdom, 2000; p 113.
3. Schultz, T. W.; Netzeva, T. I.; Roberts, D. W.; Cronin, M. T. D., Structure-toxicity relationships for the effects to Tetrahymena
pyriformis of aliphatic, carbonyl-containing, a,b-unsaturated chemicals. Chem. Res. Toxicol. 2005, 18, 330-341.
4. Schultz, T. W.; Yarbrough, J. W., Trends in structure-toxicity relationships for carbonyl-containing α,β-unsaturated
compounds. SAR QSAR Environ. Res. 2004, 15, 139-146.
5. Dimitrov, S. D.; Koleva, Y. K.; Schultz, T. W.; Walker, J. D.; Mekenyan, O. G., Interspecies quantitative structure-activity
relationship model for aldehydes: aquatic toxicity. Environ. Toxicol. Chem. 2004, 23, 463-470.
6. Yarbrough, J. W.; Schultz, T. W., Abiotic sulfhydryl reactivity: a predictor of aquatic toxicity for carbonyl-containing α,β-
unsaturated compounds. Chem. Res. Toxicol. 2007, 20, 558-562.
7. Schultz, T. W.; Sink, G. D.; Hunter, R. S., Structure-toxicity relationships for alkanones and alkenones. SAR QSAR Environ.
Res. 1995, 3, 27-36.
8. Lipnick, R. L., Outliers: their origin and use in the classification of molecular mechanisms of toxicity. In QSAR in
Environmental Toxicology-IV, Hermans, J. L. M.; Opperhuizen, A., Eds. Elsevier: Amsterdam, The Netherlands, 1991; pp
131-154.
9. OECD Organization for Economic Co-operation and Development (Q)SAR Application Toolbox, 1.1; OASIS, Laboratory of
Mathematical Chemistry: Bourgas, Bulgaria, 2009.
10. Verhaar, H. J. M.; van Leeuwen, C. J.; Hermens, J. L. M., Classifying environmental pollutants.1: Structure-activity
relationships for prediction of aquatic toxicity. Chemosphere 1992, 25, 471-491.

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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

11. Aptula, A. O.; Roberts, D. W., Mechanistic applicability domains for nonanimal-based prediction of toxicological end points:
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genotoxic activity through structure-activity relationship models. Environ. Mol. Mutagen. 2005, 46, 268-280.
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18. Yan, X.-F.; Xiao, H.-M.; Gong, X.-D.; Ju, X.-H., Quantitative structure-activity relationships of nitroaromatics toxicity to the
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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

23. Jaworska, J. S.; Hunter, R. S.; Schultz, T. M., Quantitative structure-activity relationships and volume fraction analyses for
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InSilicoTox – D4. Report on Reaction Mechanisms in Toxicology

36. Deneer, J. W.; Sinnige, T. L.; Seinen, W.; Hermens, J. L. M., A quantitative structure-activity relationship for the acute toxicity
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39. Hodges, G.; Roberts, D. W.; Marshall, S. J.; Dearden, J. C., The aquatic toxicity of anionic surfactants to Daphnia magna-A
comparative QSAR study of linear alkylbenzene sulphonates and ester sulphonates. Chemosphere 2006, 63, 1443-1450.
40. Roy, K.; Ghosh, G., QSTR with extended topochemical atom indices. Part 5: Modeling of the acute toxicity of phenylsulfonyl
carboxylates to Vibrio fischeri using genetic function approximation. Bioorg. Med. Chem. 2005, 13, 1185–1194.
41. Bradbury, S. P.; Christensen, G. M., Inhibition of alcohol dehydrogenase activity by acetylenic and allylic alcohols:
Concordance with in vivo electrophilic reactivity in fish. Environ. Toxicol. Chem. 1991, 10, 1155-1160.
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