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Protocol no. 37
RED BLOOD CELL TEST SYSTEM
An in vitro red blood cell assay is presented
which allows the estimation of the irritation potential of tensides and
tenside containing materials such as shampoos, shower gels, cleaning products,
etc. The estimation is based on the fact that surfactants interact strongly
with cellular membranes and proteins. Both effects are measured photometrically
by use of the inherent native dye, oxyhemoglobin. Unlike other cell-based
systems, the RBC assay is able to differentiate between membrane damage
(haemolysis) and protein damage (denaturation).
CONTACT
Dr Wolfgang Pape Beiersdorf AG 4232, Dept of
Biocompatibility & Immunology Unnastrasse 48 D-2000 Hamburg 20 Germany
Tel: Germany - 40 569-0 2833 Fax: Germany - 40 5693434
RATIONALE
This protocol describes an approach based on
the use of red blood cells to quantify adverse effects of surfactants and
detergent products on the cytoplasmic membrane (haemolysis) in combination
with the damage of liberated cellular proteins (denaturation), and which
may be sensitively detected by following changes in the photometrical absorbance
of oxyhemoglobin, an indicator of both processes. Since, safety testing
of such products is primarily related to the injury of accidental applications
in particular to mucous membranes, the predominant interest is directed
to the first step of its elicitation which is known to be the damage of
cellular membranes and structural and functional proteins.
BASIC PROCEDURE
Fresh calf blood samples are obtained directly
from the slaughterhouse. Red blood cells (RBCs) are washed and centrifuged
several times, to remove white cells and any traces of plasma. a) Haemolysis
Various concentrations of test sample in PBS are incubated with a defined
quantity of RBC suspension (determined via the concentration of oxyhemoglobin)
for 10 minutes, with constant shaking, at room temperature. The incubation
period is terminated by rapid, high-speed centrifugation, which removes
intact cells and debris from the medium. The resulting supernatant is then
monitored photometrically at 530nm or 560nm against blank. The total hypotonic
release of oxyhemoglobin is set to 100%, whilst the fractional release
caused by each tenside sample is expressed as a relative percentage. The
half-maximal effective concentration is then calculated from the resulting
dose-response curve. b) Denaturation A 1% solution of test sample or 0.1%
solution of surfactants in PBS is incubated with a defined quantity of
RBC suspension for 10 minutes, with constant shaking, at room temperature.
Again the incubation period is terminated by a rapid, high-speed centrifugation
which stops the denaturation of just released oxyhemoglobin. The resulting
supernatant is determined photometrically at 575nm and 540nm against a
blank containing test sample only. The relation between the effective concentration
of 50% haemolysis and the protein denaturation (related to SDS denaturation)
known as the Lysis/Denaturation Ratio is then calculated and may be compared
with acute eye irritancy data.
CRITICAL ASSESSMENT
In the authors opinion, the RBC assay can be
used routinely to assess irritancy in safety evaluation of surfactants
and tensidoactive consumer goods. The test system is simple and characterised
by defined and objective end-points using the inherent indicator, oxyhemoglobin.
The assay is inexpensive, does not require special equipment, and needs
only 1 hour per sample. The test can be used as a rapid screening assay
in a first-order in vitro test battery for the assessment of acute eye
irritation potential. Haemoglobin release is an excellent end-point of
cytoplasmic membrane integrity. However, it should be borne in mind that
liberated cellular proteins, such as cytoplasmic enzymes, may be strongly
affected by toxicants, in particular by ionic surfactants, which inactivate
them. This protein interaction can easily be quantified as carried out
in the Hb-denaturation test. This is an essential part of the assay, because
protein interaction (related to corneal opacity) may also be related to
the main part of the Draize test score. Oxyhemoglobin is also denatured
by surfactants, therefore, to take this into account, measurements are
made at 575 and 540nm to monitor (or quantify) the spectral changes of
the protein as a result of tenside denaturation. Such denaturation has
the advantage of being a sensitive indicator for damaging protein interactions.
The RBC test is not proposed as a global alternative to the Draize test,
but as part of a practical in vitro test battery. It is far less expensive
than other cell culture tests and commercial systems. On the contrary,
the haemolysis and denaturation of erythrocyte protein has been designed
as a test for chemicals, for which lysis of membranes and/or the denaturation
of proteins constitutes their principal mechanism of action. Thus, RBC
test is proposed as a bioassay for predicting the lytic and damaging effects
of tensides or surfactants at large in the plasma membrane and cellular
protein. The haemolytic potency alone may not be sufficient to characterise
the irritation potential of tensides. In addition, the denaturation index
and lysis/denaturation relationship has been derived to further characterise
the irritation potential of surfactants. Comparison with other tests Internal
studies have demonstrated that the RBC test shows a good correlation with
the CAM or HET-CAM test for tensides and cleaning products. It appears
that haemolysis might be comparable to the measurement of haemorrhage and
lysis in the CAM test whilst denaturation might be comparable to the measurement
of coagulation. Good correlations have also been found with the neutral
red uptake test.
TEST STATUS
This test has been evaluated in-house and is
presently undergoing interlaboratory validation. The RBC test will be evaluated
in phase III of the CTFA evaluation study, when the irritation potential
of cleaning products will be assessed.
OTHER ORGANISATIONS USING THE TEST
About 20 laboratories from raw materials suppliers,
cosmetic companies, and research institutes are already using this test,
or plan to do so.
CHEMICALS TESTED
A large number of anionic, amphoteric, cationic,
and non-ionic surfactants have been tested, examples of which are given
below: PEG-24-glycerylstearate Polysorbate-20 PEG-10-nonylphenol Cocodimethylbetaine
Cocodimethylaminoxide Cocoamphocarboxylglycinate a-Olefin sulfonate sec-Alkane
sulfonate Alkylbenzene sulfonate Na-Lauryl sulfate MEA-Lauryl sulfate NH4-Lauryl
sulfate Na-Laureth 2-sulfate NH4-Laureth 2-sulfate TEA-Lauryl sulfate Mg-Laureth
2-sulfate TEA-Laureth 2-sulfate sec-Alkyl ether sulfate Na-Soap (unbuff.)
Laureth sulfosuccinate Na-Amidether sulfate Mg-Amidether sulfate Cocoylprotein
condensate TEA-dodecanoate Laureth-10-carboxylate Cetyltrimethylammonia-Cl
Benzalkonium chloride
REFERENCES
- Kondo, T. (1976) Mechanisms of haemolysis by
surface active agents. Adv. Colloid & Interface Sci., 6, 139-172 Kondo,
T. & Tomizawa, M. (1968) Haemolysis by nonionic surface-active agents.
J. Pharm. Sci., 57, 1246-1248.
- Gloxhuber, Ch. (1974) Toxicological properties
of surfactants. Arch. Toxicol., 32, 245-270.
- Pape, W.J.W., Pfannenbecker, U. & Hoppe,
U. (1987) Validation of the red blood cell test as an in vitro assay for
the rapid screening of irritation potential of surfactants. Molecular Toxicology,
1, 525-536.
- Pape, W. & Hoppe, U. (1988) Second World
Surfactants Congress, Paris. Evaluation of acute irritation potentials
of tensides using the in vitro alternative red blood cell test system.
Proceedings, IV, 414-428.
- Pape, W.J.W. (1990) In vitro methods for the
assessment of local effects of cosmetics on skin and mucous membranes.
Presented at: In-cosmetics 1990, Birmingham, UK.
- Pape, W.J.W. & Hoppe, U. (1991) Standardisation
on an in vitro red blood cell test for evaluating the acute cytotoxic potential
of tensides. Arzneimittel-Forschung/Drug Research, 40(I), 4, 498-502.
- Pape, W.J.W. & Hoppe, U. (1991) In vitro
methods for the assessment of primary local effects of topically applied
preparations. Skin Pharmacology (in press).
IP-37 January 1992
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