Abstracts / Toxicology 231 (2007) 104–119 115

Table 1Acute toxicity classifications for pesticide active substances evaluated in the EU

Total Very toxic Toxic Harmful

Dermal Dermal not oral Dermal Dermal not orala Dermal Dermal not orala

195 5 1 9 0 15 1

a An equivalent or higher classification orally.

Table 2Acute toxicity classifications for pesticide products currently approved by PSD

Total Very toxic Toxic Harmful

Dermal Dermal not other Dermal Dermal not othera Dermal Dermal not othera

3 b

moavcatcfpt

ddeffsptt

d

111 9 0 7

a An equivalent or higher classification orally or by inhalation.b Only one confirmed as reliable.

ulated products produced a similar picture with only 9ut of 3111 products having a classification for dermalcute toxicity but no equivalent or higher classificationia other routes of exposure (Table 2). The data andlassifications for formulated products had been gener-ted over many years and under differing schemes, andhere was uncertainty about the basis for a number of thelassifications. Therefore, the results of the analysis forormulated products should only be taken as indicativeending a repeated exercise following the completion ofhe ongoing EU re-registration procedure.

From this analysis it is possible to conclude that theermal acute toxicity study adds little if anything to theatabase on pesticide active substances. In all cases anquivalent indication of hazard, although often by a dif-erent route, was present. A similar result was indicatedor formulated products. It is recommended that organi-ations responsible for setting the data requirements foresticides give consideration to removing acute dermal

oxicity studies from the core data requirements for pes-icide active substances and formulated products.

oi:10.1016/j.tox.2006.11.040

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Chemical insult and toll-like receptor ligands: Syn-ergistic effects on dendritic cells

Gavin Maxwell 2,, Marie Cumberbatch 1, CraigPortsmouth 1, Kirsty Clelland 1, Carolin Schramm 1,Carl Westmoreland 2, David A. Basketter 2, Rebecca J.Dearman 1, Ian Kimber 1

E-mail address: rebecca.dearman@syngenta.com(R.J. Dearman).

1 Syngenta CTL, Macclesfield, Cheshire, UK 2 Safetyand Environmental Assurance Centre, Unilever, Bedford,UK

There is considerable interest in the developmentof in vitro methods for the identification and charac-terisation of contact sensitisers. Dendritic cells (DC)play a central role in cutaneous immune responses andmethods are now available for their culture from bonemarrow precursors. In the current investigations, we haveexamined the influence of co-culture of murine bonemarrow derived DC with chemical (the allergen dini-trobenzene sulfonic acid [DNBS] or the skin irritantbenzene sulfonic acid [BS]) and selected toll-like recep-tor (TLR) ligands; the latter being potent activators ofDC. Twenty-four hour culture of DC with 100 ng/mlof the TLR1-2 ligand Pam3Cys-Ser-(Lys)4 (PAM) orthe TLR6-2 ligand macrophage-activating lipopeptide-2(MALP-2) resulted in marked up-regulation of cytokineexpression (including interleukin [IL]-6 and tumournecrosis factor � [TNF-�] and the up-regulation of mat-

uration markers. In contrast, treatment with the TLR5ligand flagellin (500 ng/ml) caused similar increasesin membrane markers but failed to induce vigorouscytokine secretion. In subsequent experiments, DC were

116 Abstracts / Toxicology 231 (2007) 104–119

Table 1Influence of BS and DNBS on TLR-induced cytokine expression by murine DC

TLR ligand Medium TLR alone BS BS + TLR DNBS DNBS + TLR

MALP-2 IL-6 0.06 (0.03) 12.6 (1.4) 0.09 (0.01) 37.3 (9.0) 0.10 (0.02) 18.3 (2.8)MALP-2 TNF 0.14 (0.02) 2.8 (0.2) 0.26 (0.06) 12.7 (4.3) 0.12 (0.02) 2.1 (0.6)PAM IL-6 0.11 (0.08) 26.5 (6.4) 0.11 (0.04) 58.0 (8.4)* 0.11 (0.04) 31.5 (2.1)PAM TNF 0.18 (0.04) 4.9 (2.4) 0.42 (0.15) 19.2 (2.4)* 0.12 (0.04) 3.3 (0.5)Flagellin IL-6 0.06 (0.01) 0.13 (0.04) 0.09 (0.02) 0.19 (0.04) 0.13 (0.03) 0.46 (0.06)**

Flagellin TNF 0.18 (0.02) 0.32 (0.08) 0.47 (0.12) 0.78 (0.16) 0.24 (0.07) 0.43 (0.1)

Cytokine expression (ng/ml); mean and S.E.M. (in parentheses) of 3–5 experiments. Statistical significance of differences between TLR alone and

TLR plus chemical groups analysed by Student’s t test.

* p < 0.05.** p < 0.01.

cultured with subtoxic doses of DNBS (0.5 mM) or BS(50 mM), together with doses of MALP-2 (10 ng/ml),PAM (50 ng/ml) and flagellin (100 ng/ml) that were sub-optimal with respect to cytokine induction.

Clear synergistic effects were observed for IL-6 andTNF-� production when DC were co-cultured with theTLR1-2 and TLR6-2 ligands together with the skinirritant BS, that reached statistical significance for theformer (Table 1; p < 0.05). Co-culture with DNBS didnot enhance MALP-2 or PAM-induced cytokine secre-tion. In contrast, a synergistic effect on IL-6 secretionwas observed when DC were cultured with subtoxicdoses of the chemical allergen DNBS (0.5 mM) and flag-ellin (Table 1; p < 0.01). Treatment with doses of BSthat were of equivalent toxicity (50 mM) or equimolardoses of BS (0.1–1 mM; data not shown) were with-out effect on flagellin-induced IL-6 expression. Theseresults suggest that chemical allergens and skin irritantsmay interact differently with DC and that TLR ligandsmay prime DC for enhanced reactivity to chemical insult.Such differences may provide for the basis of improved

DC-based approaches for the in vitro identification ofskin sensitising chemicals.

doi:10.1016/j.tox.2006.11.041

Effect of arsenic on DNA damage, glutathione con-tent, apoptosis and necrosis in HaCat cells

Ghazalla Benhusein, Elaine Mutch, Faith M. WilliamsE-mail address: ghazalla.benhusein@ncl.ac.uk(G. Benhusein).

The Toxicology Unit, Devonshire Building, University ofNewcastle upon Tyne, Newcastle upon Tyne NE1 7RU,United Kingdom

Arsenic is an environmental chemical of toxicologicalconcern today since it is a human genotoxin and chronicexposure is associated with development of cancers,including skin. Inorganic arsenate (As5+) is metaboli-cally reduced to arsenite (As3+) by glutathione (GSH)prior to methylation. The aim of this study was to deter-mine the relative toxic effects of arsenate and arsenite inHaCat cells (immortalised human keratinocytes) in vitroby measuring cytotoxicity, DNA damage, depletion ofglutathione and apoptotic and necrotic events.

Multi-well plates were seeded with HaCat cells(approximately 2.5 × 105) in DMEM with 10% fetalcalf serum. When approximately 90% confluency wasreached the cell surface of the monolayers were washedto waste and then arsenate or arsenite were added inDMEM and DNA damage (Comet assay) (10 �M) andcytotoxicity (10, 60 and 100 �M) measured at 24 h. Insome experiments arsenate (10 �M) or arsenite (10 �M)was added at the same time as BSO (10 �M) for 24 h(n = 3), and GSH levels were measured by HPLC withfluorescence detection. Two colour (Annexin V and PI)flow cytometry was used to investigate apoptotic andnecrotic events following arsenate (10 �M) or arsenite(10 �M) treatment (n = 3) for 24 h. One-Way ANOVA

compared treated cells with controls.

Arsenate and arsenite at 60 and 100 �M, but not10 �M, reduced the number of adherent viable cellswith time (data not shown). Therefore, DNA damage