S138 Abstracts / Toxicology Letters 211S (2012) S43–S216
P20-22
Use of in vitro assays for the prediction of the skin sensitizing
potential of chemicals
Pierre-Jacques Ferret, Marie-Pierre Gomez-Berrada, Carole Berge
Pierre Fabre Dermo Cosmétique, France
Purpose: Cutaneous sensitization results from the induction of
an immune response following the exposure of the skin to a sub-
stance. Currently, the only validated alternative method remains
the Murine Local Lymph Node Assay (LLNA). Progress in the com-
prehension of cutaneous sensitizing mechanisms and the need for
conforming to the current legislation requirements supported the
development of new alternative methods to the animal experimen-
tation. These methods will have to summarize the whole complex
interactions of the allergen with the immune system and need to
be exploitable for any type of compound. Methods: The results pre-
sented here were achieved with 40 substances of known sensitizing
potential (25 cosmetic raw materials and 15 control substances)
using various methods: (1) the direct peptide reactive assay (DPRA),
(2) a dentritic cell activation assay (h-CLAT) and (3) the ARE-
dependent gene expression in SENS-IS. The results are expressed in
4 classes (Slightly; Moderately; Strongly and Very strongly sensitiz-
ing) allowing to compare them. The correlation of individual tests
was obtained by comparison to human Repeated Insult Patch Test
(hRIPT) and LLNA data. Results and conclusion: We have observed
a good correlation between the results obtained with DPRA and
in vitro models (75–90%) and a good correlation with those result-
ing from the LLNA and hRIPT (75–85%). It appears obvious that
only the combination of several alternative methods belonging to
a strategy of evaluation could replace the LLNA. Moreover it would
be interesting to supplement these data by realization of studies
relating to the Nrf2/Keap1 pathway.
doi:10.1016/j.toxlet.2012.03.503
P20-23
Human dendritic cell based model (DC-100) for
immunotoxicity studies
Silvia Letasiova 1 , Amy Hunter 2 , Maureen Spratt 2 , Alex
Armento 2 , Mitchell Klausner 2 , Seyoum Ayehunie 2
1MatTek IVLSL, Slovakia, 2 MatTek Corporation, Ashland, MA, United
States
The immune system can be the target of a broad variety of
chemicals in the form of environmental contaminants, medica-
ments, fabrics, pesticides, food additives and preservatives, and
day-to-day household products with adverse effects to human
health. Immunotoxicants can induce suppressive or stimulatory
responses and DNA damage in components of the immune sys-
tem. In this study, dendritic cells (DC) were used to monitor
immunotoxicant induced phenotypic changes (surface marker
expression), functional alteration (cytokine release), and DNA dam-
age (comet assay). We evaluated the effect of 5 immunotoxic
compounds (ITC) and 2 reproductive hormones, estradiol and pro-
gesterone, on DC responses. Finally, we checked DNA damage
of DC following treatment with the carcinogenic agent, methyl
methanesulfonate (MMS).DC were exposed to non-cytotoxic con-
centrations of the ITC or hormones for 24 h and then to LPS
for an additional 18 h. FACS analysis of ITC-exposed DC showed
effects in the rank order of immunotoxicity (severe to low effect):
Tributyltin chloride > Cyclosporine A > Benzo(a)pyrene > Fursemide
and Urethane. Dose-dependent decreases in the secretion of
immuno-stimulatory cytokines including interleukin (IL)-12, IL-
6, and IL-1␤ were observed. Furthermore, treatment of DC with
the reproductive hormones, estradiol and progesterone, showed
a decrease in lipopolysaccharide-induced CCR7 expression and
IL-12 secretion. Exposure of DC to MMS also showed dose depen-
dent DNA damage. Conclusion: The release of immunostimulatory
cytokines and DNA damage may serve as endpoints to predict
immunotoxicity. Due to concerns with animal models in terms of
cost, ethical issues, and relevance to hazard assessment in humans,
the human primary DC based assay is an attractive in vitro model
to predict the immunotoxicity of compounds and formulations.
doi:10.1016/j.toxlet.2012.03.504
P21: Metabolism and Kinetics
P21-01
Inhibition of hepatic mixed function oxidase system by
natamycin
Arturo Anadon, Maria A. Martinez, Victor Castellano, Marta
Martinez, alejandro Romero, Irma Ares, Eva Ramos, Maria R.
Martinez-Larrañaga
Universidad Complutense de Madrid, Spain
Natamycin (pimaricin), a polyene macrolide antibiotic, has been
used as a topical antimycotic agent in animals and humans for over
40 years and it is also used as an antifungal agent in food processing.
Natamycin appears to be an extremely effective inhibitor of mould
growth and mycotoxin production. The low solubility of natamycin
makes it suitable for use as a surface treatment on foods because
does not readily migrate into the interior and does not adversely
affect flavour or appearance. For risk assessment, it is of prime
interest to obtain relevant information on the natamycin oxidative
metabolism.
The objective of this study is to establish if natamycin interacts
with hepatic microsomal metabolising enzymes. Animals (male
Wistar rats) were treated with natamycin (0.8, 2 and 8 mg/kg/day
i.p. 4 days). Natamycin-treated and control animals were sacri-
ficed 24 h after the last administration and livers were removed.
The livers were individually homogenized and microsomal pellets
prepared and stored at −80 ◦ C for enzyme assays.
Natamycin decreased significantly the content of cytochrome
P450, and the activities of aniline hydroxylase, erythromycin N-
demethylase (CYP3A1), ethoxyresorufin O-deethylase (CYP1A1)
and pentoxyresorufin O-depenthylase (CYP2B1). Natamycin also
produced a significant decrease in the hydroxylation of lauric acid
associated with the CYP4A subfamily. These data suggest reduc-
tion in the formation of toxic products of oxidative metabolism,
but natamycin as inhibitor of a number of cytochrome P450 medi-
ated metabolic pathways, might cause clinically pharmacokinetic
drug interactions.
Acknowledgements: This work was supported by projects
Ref. BSCHGR58/08(UCM), Ref. No. S2009/AGR-1469 (CAM) and
Consolider-Ingenio 2010 No.CSD2007-063 (MEC), Spain.
doi:10.1016/j.toxlet.2012.03.506