Vox Sanguinis (2003) 85, 171–182

© 2003 Blackwell Publishing

ORIGINAL PAPER

Preclinical safety profile of plasma prepared using the

Blackwell Publishing Ltd.

INTERCEPT Blood System

V. Ciaravino, T. McCullough, G. Cimino & T. Sullivan
Cerus Corporation, Concord, California, USA

Background and Objectives The newly developed INTERCEPT Blood System for
plasma uses the addition of a new psoralen, amotosalen HCl (AMOTOSALEN), followed
by illumination with ultraviolet A light, to inactivate viruses, bacteria, protozoa and
leucocytes that may contaminate fresh-frozen plasma (FFP). Extensive toxicology
studies were performed to characterize the safety of the photochemical treatment
process for its intended use with plasma.
Materials and Methods The studies of general toxicology, safety pharmacology,
phototoxicity, reproductive toxicity and venous irritation, summarized in this review,
provide a comprehensive toxicology profile for photochemically treated 100% plasma.
Results No specific target organ toxicity (based on clinical or histological pathology),
phototoxicity, or reproductive toxicity was observed.
Conclusions The results of an extensive series of studies have demonstrated no toxi-
cologically relevant effects of photochemically treated 100% plasma prepared using
Received: 23 January 2003,
revised 25 June 2003,
the INTERCEPT Blood System for plasma.
accepted 28 June 2003 Key words: amotosalen, INTERCEPT Blood Systems, pathogen inactivation, toxicology.

factor deficiencies may require plasma on a chronic basis

Introduction
Clinical uses of fresh-frozen plasma
The demand for fresh-frozen plasma (FFP) has increased as
a response to the growing needs of patients who require FFP
and cryoprecipitate derived from FFP in support of coagulop-
athy, either with or without bleeding, during large-volume
blood transfusion, and for rapid reversal of anticoagulation
[1]. In addition, FFP is indicated for the treatment of anti-
thrombin III, Protein C or Protein S deficiency when a con-
centrate is not available, and for therapeutic plasma exchange
to treat thrombotic thrombocytopenic purpura (TTP) [2].
Assessment of preclinical safety requires the design of
studies to cover the anticipated clinical exposures. Patients
with severe (i.e. < 1% of normal) congenital coagulation
during their lifetimes. In contrast to severely affected patients,
patients with mild congenital coagulation deficiencies (5–
25% of normal) may be treated intermittently with FFP, as
prophylaxis, in preparation for invasive procedures or during
active bleeding. Correction of acquired complex coagulation-
factor deficiencies generally requires short-term treatment
with 15 ml/kg (4 units for a small adult, each unit containing
250–300 ml from a single blood-donor). As many as 50 FFP
units may be required to treat a single transfusion episode
associated with a major surgical procedure, such as liver
transplantation [3]. Use of FFP in total plasma exchange
(TPE) therapy for the treatment of TTP has been reported to
average 21·5 ± 7·8 l in a single cycle of TPE for an acute
episode of TTP [4].
Risk of transfusion-transmitted illness

Correspondence: Tim McCullough, PhD, Cerus Corporation, 2411 Stanwell Despite improvements in donor screening and testing, trans-
Drive, Concord, CA 94520, USA mission of viral diseases by labile blood products cannot be
E-mail: tim_mccullough@cerus.com totally excluded. Current testing is unreliable during the

171
172 V. Ciaravino et al.
Table 1 Test materials used for preclinical safety evaluation programme
Photochemically treated 100% plasma
a a
With CAD No CAD
Acute toxicity X X
≤ 1-month toxicity X X
3-month toxicity X X
Safety pharmacology X X
Reproductive toxicity X X
Genotoxicity
Carcinogenicity
Vein irritation X X
Phototoxicity X (X)b
a
Compound adsorption device (CAD) used to remove residual amotosalen and free photoproducts.
b
(X) = partial CAD treatment.
window period between infection and seroconversion of a
donor; however, window periods have been shortened by the
use of genomic testing. In current practice, in many European
countries where state-of-the-art donor screening and selection
procedures have been introduced, ‘window’ donations are rare.
The frequencies of seroconversion from repeat donations in
Europe have been reported to be 1 : 2·3 × 106 for human
immunodeficiency virus (HIV), 1 : 0·4 × 106 for hepatitis B
virus (HBV) and 1 : 0·62 × 106 for hepatitis C virus (HCV) [5].
Estimates for the risk of viral transmission were higher in
Germany and France (e.g. 1 : 1 × 106 for HIV). Similar, albeit
somewhat higher, values have been found in the United
States [6–9].
In view of the probable exposure to multiple donors asso-
ciated with the use of plasma in the treatment of congenital
and acquired coagulopathies, especially in the case of TPE
therapy for TTP, the risk for a patient to incur a transfusion-
transmitted illness increases accordingly.
Several measures have been adapted to prevent or elimi-
nate potential infectious risks in the blood supply and they
have been introduced in the production process of blood and
plasma products [10–12]. In the case of FFP, the treatment
used for viral reduction of pooled plasma products is solvent/
detergent; for single-donor products methylene blue is used
[10]. The solvent/detergent method is used to treat pools of
plasma products. The pool is initially exposed to chemicals
that denature the coats of enveloped viruses, the chemicals
are subsequently removed by chromatography, and then
individual plasma units are prepared and allocated from the
pool. This methodology is very effective for lipid-enveloped
viruses, but has limited or no effect on non-enveloped
pathogens. Photodynamic viral inactivation of plasma can be
mediated by visible light in the presence of the phenothiazine
dye, methlyene blue. The commercial procedure is applied to
individual plasma units, from single donors, in their storage
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171–182
Photochemically treated 35% plasma
With CADa No CADa Amotosalen
X X
X
X X
X
X X
X X
X X X
X (X)b X
containers. As methylene blue is a planar molecule, it has
affinity for binding to nucleic acids. After activation with
visible light, methylene blue transfers its energy to molecular
oxygen, generating singlet oxygen. Singlet oxygen is the
active virucidal agent in the methylene blue process. It is
known to modify guanosine residues in nucleic acids, as well
as some viral envelopes, through reactions with amino acids.
These methods are in contrast to Helinx technology, which
results in monoadducts and crosslinks between nucleic acids
and the intercalated amotosalen (see the Materials and
Methods, below).
INTERCEPT Blood System for plasma
More recently, the combination of the new psoralen, amoto-
salen, and ultraviolet A (UVA) (320–400 nm) has been shown
to be highly effective for inactivating bacteria, viruses,
protozoa and passenger leucocytes that may contaminate
plasma [13,14]. This procedure has been developed for single-
donor products and has been shown to preserve the thera-
peutic effectiveness of the plasma product [15–22].
Addition of chemicals to blood products for the inactiva-
tion of contaminants is a potential cause for concern because
of the possible risks associated with the introduction of
chemical agents into therapeutic preparations that will be
administered to patients. With respect to psoralens [23], the
combination of some compounds in this class and UV light
has been associated with genotoxicity, carcinogenicity,
phototoxicity and other adverse effects [24–30].
Preclinical safety
This article reviews results of the preclinical toxicology
programme carried out in support of the INTERCEPT Blood
System for plasma (Table 1). The programme consists of
 Preclinical safety profile 173

studies that not only evaluated photochemically treated 100%

plasma, but also evaluated the safety of the following test
articles: amotosalen alone and photochemically treated 35%
plasma (conducted to support the safety of the INTERCEPT
Blood System for platelets). In the photochemical treatment
process for platelets, a transfusion unit of platelets is sus-
pended in 35% plasma and 65% PAS III (sodium chloride/
acetate/citrate in phosphate buffer). In the photochemical
treatment process for plasma, a transfusion unit contains 100%
plasma. Studies conducted with photochemically treated 35%
plasma include: single-dose (acute) and multiple-dose (up to
13 weeks) intravenous toxicity, safety pharmacology (central
nervous system [CNS], renal and cardiovascular), male and
female reproductive toxicity, a series of in vitro and in vivo
mutagenicity studies, carcinogenicity in transgenic mice
heterozygous for the p53 tumour-suppressor gene mutation,
vein irritation and phototoxicity. A complete review of the
studies with amotosalen and photochemically treated 35%
plasma has been described in previous publications [31,32].

Materials and methods
Photochemical treatment
The INTERCEPT Blood System for plasma employs Helinx™
technology (Fig. 1), which uses amotosalen (a psoralen) and
UVA light, in an ex vivo photochemical treatment process,
to inactivate viruses, bacteria, protozoa and leucocytes in
plasma. The structure and photochemistry of amotosalen
has been described previously [33]. The photochemical treat-
ment process begins with the preparation of a plasma unit in
a plastic storage container (250-ml plasma transfusion unit).
After addition of amotosalen to a nominal concentration
of ≈ 50 µg/ml (150 µM), the mixture is illuminated with
3 Joule/cm2 UVA (320–400 nm) and horizontal agitation
is performed concurrently. This causes monoadduct and
crosslink formation between nucleic acids and intercalated
amotosalen, and the resultant inactivation of pathogens that
depend on nucleic acid replication for proliferation. In the
absence of UVA light, the amotosalen behaves as a reversible
intercalator and no chemistry occurs. The photochemically
treated plasma is then passed through a Compound
Adsorption Device (CAD) to remove residual amotosalen and
unbound photoproducts and then transferred to the final
container for storage at −18 °C, according to standard
procedures for FFP. After treatment with the CAD, the amount
of residual amotosalen and free photoproducts in a 250-ml
plasma transfusion unit are ≈ 60 µg and 170 µg, respectively.
The clinical exposure to amotosalen and its free photo-
products for a 1000-ml plasma transfusion would therefore be
≈ 4 µg/kg and 11 µg/kg, respectively, on a body weight basis
for a 60-kg individual. The toxicology programme support-
ing the INTERCEPT Blood System for plasma was conducted
Fig. 1 The INTERCEPT Blood System for plasma. Collected plasma is mixed
with amotosalen and the mixture is placed in an ultraviolet A (UVA)
illumination device. The contents are then passed through a Compound
Adsorption Device (CAD) for reduction of amotosalen and free
photoproducts. After CAD treatment, the plasma is placed
in a final storage container until transfusion.
in laboratory animals and in in vitro systems. Study selection
and design were in accordance with International Conference
on Harmonization (ICH) guidelines on technical requirements
for registration of pharmaceuticals for human use. All studies
were conducted in compliance with Good Laboratory Practices
or in accordance with prevailing professional standards. All
studies employing animals were perfored in laboratories that
fully complied with the requirements of the US Department
of Agriculture and the American Association for the Accred-
itation of Laboratory Animal Care.
Establishment of safety margins
Intravenous (i.v.) infusions of photochemically treated
plasma, with and without CAD treatment, were carried out to
evaluate potential toxicity. Analysis of the plasma level of
amotosalen was conducted to evaluate toxicokinetic para-
meters in toxicology studies. To establish large safety margins
for amotosalen and photoproduct exposure over clinical
applications, toxicology studies were designed to increase
the dose volumes administered, increase frequency of admin-
istration, and to include groups given dose formulations
prepared without CAD treatment. For toxicology studies
conducted for the platelet programme, transfusions were
administered over a 1-h period. For the 100% plasma studies,
transfusions were administered over a 2-h period.

© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171– 182
174 V. Ciaravino et al.
The maximum dose volumes of photochemically treated test
articles employed in the toxicology studies were 25–80 ml/kg
for single-dose studies and 25–60 ml/kg in multidose studies.
In the plasma programme, a dose volume of 60 ml/kg was
given to rats in a 1-month study. Owing to vehicle effects
observed in that study, a lower dose volume was selected for
subsequent studies. Therefore, depending upon the frequency
and duration of dosing, 50 ml/kg was selected for use in the
28-day phototoxicity study (where rats were dosed three
times per week), 50 ml/kg was selected for use in the teratology
study (where rats were dosed daily for 12 days), and 40 ml/kg
was selected for daily dosing in the 3-month study. In com-
parison, the plasma volumes of rats, rabbits and monkeys are
≈ 35–40 ml/kg, and the plasma volume of dogs is ≈ 50 ml/
kg. Therefore, the acute doses were approximately twice the
plasma volume of the species used (greater than twice for
rats, and less than twice for dogs), and the daily doses for the
multidose studies were ≥ 50% of the plasma volume of the
species used in the various studies. The maximum dose
volumes employed in the toxicology studies were considered
the maximum feasible dose volumes of a protein-containing
formulation that would not cause undue stress to the renal
and cardiovascular systems (especially in consideration of
the protein load) and thereby compromise the studies.
Rationale for toxicology studies
The following sections summarize the strategy for each study
category.
Rationale for 100% plasma studies
To support the clinical use of photochemically treated 100%
plasma, specific toxicology studies were chosen that were
considered critical in the overall evaluation of the programme
and used 100% plasma, in contrast to the previous studies
conducted with 35% plasma. As a 100% plasma test article
was being administered in the present toxicology studies, an
evaluation of potential vein irritation was important. Acute
and safety pharmacology studies were performed to evaluate
overall potential acute systemic toxicity or any effects on
cardiovascular or renal physiology. Even though no photo-
toxicity or reproductive toxicity was elicited with photo-
chemically treated 35% plasma, it was important to evaluate
the higher residual amotosalen levels after photochemical
treatment. Teratology was specifically selected because of
the importance of potential toxicity on the developing
fetus. Finally, a study of the longest duration, 3 months,
was selected to evaluate the potential toxicity of prolonged
administration of photochemically treated 100% plasma.
Acute and repeated-dose toxicity
Early in the toxicology programme, many technical challenges
had to be overcome, as the feasibility of the sterile preparation
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171–182
and i.v. administration of large volumes of species-specific
homologous plasma was not known. While there is some
knowledge about canine blood types, very little is known
about rodent blood types. Moreover, sterile rodent plasma and
sterile, blood-typed canine plasma are very difficult to obtain
in the quantities needed to conduct toxicology studies. Accord-
ingly, the toxicology programme progressed step-wise through
7-, 14-, 28- and 90-day toxicity studies in rats and dogs.
Based on the clinical use of plasma, the maximum duration
of toxicity studies for the toxicology programme was set at
3 months. Clinically, plasma units are primarily administered
in support of a single medical event. While Cerus Corporation
is not aware of any published comprehensive review of the
demographics of FFP use, a Cerus survey of the literature
indicated that most patients (> 97% of the patients given FFP)
receive a total of 3–5 units of plasma (600–1000 ml, assum-
ing 200 ml as an average volume for a unit of FFP), given as
a single dose in support of an acute event such as surgery,
trauma, or coumadin reversal (W. Greenmore, personal com-
munication). There is a small patient population (< 2% of the
patients given FFP) that receives multiple doses of FFP over
2–4 weeks in support of liver transplantation. At two centres
surveyed by Cerus, these patients received an average total
of 30 or 38 units of FFP, respectively (6–8 l). There is also a
very small patient population (< 1% of the patients given
FFP) that receives plasma exchanges (approximately 10 units
of FFP) on several occasions over a 2–4-week period (4–22 l
total) for chronic disorders, including TTP, Guillain–Barré
syndrome and myasthenia gravis. Of these patients, a very
small number may have recurrent therapy.
In acute studies, i.v. infusions of up to 80 ml/kg/h of
photochemically treated 100% plasma was administered as a
single 2-h dose, and the rats were evaluated for a 14-day
period. At the end of the 14 days, the rats were killed and
examined grossly. A set of tissues was preserved but not
processed for histopathological examination. In repeated-
dose studies, i.v. infusions of up to 60 ml/kg of photochemi-
cally treated 100% plasma was administered over 2 h daily
for up to 13 weeks. Clinical observations, body weights, food
intakes and clinical pathology evaluations (haematology,
chemistry and urinalysis) were conducted at scheduled inter-
vals. Ophthalmoscopic examinations (indirect and slit lamp)
were conducted at scheduled intervals. At the completion of
scheduled duration of treatment, animals were killed and
subjected to gross pathological examinations. In the 13-week
study, a subset of the animals were assigned to a 1-month
observation period and then killed. A set of tissues from each
animal was preserved and evaluated histopathologically.
Safety pharmacology
Safety pharmacology studies were conducted to investigate
any potential adverse effects of amotosalen and associated
photoproducts on physiological functions. In the renal safety

pharmacology study, a single i.v. infusion (80 ml/kg for 2 h)
of a vehicle control (100% rat plasma) or photochemically
treated 100% plasma with or without CAD treatment, was
given to rats. Urine and blood were collected both before
and after dosing. Creatinine clearance, urinalysis and clin-
ical chemistry parameters were measured. The kidneys were
removed and weighed.
In the cardiovascular study, an i.v. infusion (80 ml/kg for
for 2 h) of vehicle control (100% dog plasma) or photochem-
ically treated 100% plasma, with and without CAD treatment,
was given to groups of three male beagle dogs. Each dog
received control or treated formulation in random order with
1 h between infusions. Electrocardiogram recordings were
obtained at predetermined intervals throughout the study.
Mean cardiac output, stroke volume, total peripheral resistance
and pulmonary wedge pressure were recorded throughout
the testing period.
Reproductive toxicology
Clinically, women of childbearing potential and pregnant
women may receive plasma transfusions. In a developmental
toxicity (teratology) study, daily intravenous infusions of
50 ml/kg plasma, with and without CAD treatment, were given
to groups of 25 pregnant rats daily during organogenesis (days
6 –17 of gestation). On gestation day 21, dams were euthanized
and their fetuses were given teratological examinations.
Genotoxicity testing
Three in vitro and two in vivo genotoxicity assays were used
to assess the genotoxicity of photochemically treated platelets
and amotosalen alone; the results have been presented
previously [31,32]. No specific studies were completed using
photochemically treated 100% plasma. In vitro genotoxicity
studies were conducted in the presence and absence of
metabolic activation. This activation involves co-treatment
of the test article with a rat liver microsome preparation to
assess whether the test article is metabolized into products
resulting in more or less genotoxicity. The studies carried out
are described below.
Ames assay. The Ames Salmonella/microsome assay detects
point mutations in vitro in a series of histidine-requiring
auxotrophs of Salmonella typhimurium and Escherichia coli.
Tester strains TA98 and TA1537 are reverted from histidine
dependence (auxotrophy) to histidine independence (pro-
totrophy) by frameshift mutagens. These two strains have
different mutations in their histidine genes and thus have
different sensitivities to mutagens. Tester strain TA1535 is
reverted by mutagens that cause base pair (bp) substitutions.
Tester strain TA100 is reverted by mutagens that cause both
frameshift and bp substitutions. E. coli strains WP2 uvrA
[pKM101] and WP2 [pKM101] are sensitive to bp substitution
mutations. Positive controls are included in each study.
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171– 182
Preclinical safety profile 175
Chromosome aberration assay. This in vitro assay detects
chromosome damage. The Chinese hamster ovary (CHO) cell
line, with a well-defined karyotype, was used. The cells were
assessed for structural chromosome aberrations (chromosome
and chromatid gaps, breaks, rings, deletions, rearrangements,
etc.) after exposure to the test article and to positive controls.
Mouse lymphoma assay. This in vitro assay detects point
mutations in the mouse lymphoma L5178Y cell line at the
thymidine kinase (TK) gene. This assay is based on the fact
that mutant TK cells are resistant to the addition of the pyri-
midine analogue, trifluorothymidine (TFT) and synthesize
their DNA de novo. Non-mutant cells die in the presence of TFT.
After exposure of cells to the test article or to positive con-
trols, the mutation is given time to express and then the cells
are placed in TFT. Only mutant cells survive and are counted.
In vivo mouse micronucleus assay. This in vivo assay detects
chromosome damage in the cells of the bone marrow of mice.
Polychromatic erythrocytes (PCE) – precursor cells to circu-
lating red blood cells – are assessed for the presence of
nuclear material (micronuclei) that may be present after
genotoxic interaction with the test article or positive control.
These micronuclei represent parts of chromosomes or whole
chromosomes left behind as the PCE ejects its nucleus in the
normal progression of development to a mature erythrocyte.
In vivo unscheduled DNA synthesis (UDS) assay. This in vivo
assay detects any type of DNA damage in hepatocytes in rats.
Normally, liver cells do not undergo DNA synthesis or division.
Using a 14C-thymidine label, no significant incorporation of
label in liver cells will occur unless the test article or positive
control damages the DNA and thus stimulates the repair system.
Carcinogenicity
The potential carcinogenicity of amotosalen and its photo-
products was evaluated in p53 transgenic mice. The p53
tumour-suppressor gene has received attention because of its
frequent alteration in a wide variety of human tumours.
Fifty-per cent or more of all human tumours have detectable
p53 genetic lesions [34]. A number of animal models have
been developed and studied for the role of p53 in mammalian
tumorigenesis [35]. These gene-knockout models consist of
mice that are heterozygous for the p53 gene. In a study
assessing the spontaneous tumorigenesis in p53-deficient
mice, 60 p53 homozygous and 97 heterozygous mice were
examined, twice weekly, for tumours [36]. By the age of
4·5 months, ≈ 50% of the homozygotes had developed
tumours and, by 10 months of age, all of the mice had died
or developed tumours (Fig. 2). In contrast, the p53 heterozy-
gotes remained healthy for a longer period of time. By
12 months of age, only 8% had tumours. However, after
12 months of age the rate of tumour development accelerated
176 V. Ciaravino et al.
Fig. 2 Tumour incidence in p53-deficient mice
, surviving wild type mice (p53+/+); , heterozygous mice (p53+/–); , homozygous mice (p53–/–). (Taken
from Harvey et al. [36]).
and, by 18 months of age, > 50% of the mice had developed
tumours. Control mice of the wild-type allele did not develop
tumours over the 18-month period. Therefore, in this assay,
heterozygous mice were used between the ages of 7–10 weeks
at the initiation of dosing. At the end of the 26-week dosing
period, spontaneous tumorigenesis is low, thus avoiding
interference in the interpretation of the results of the study.
In the assay, designed to assess the potential of amotosalen
to develop tumours, groups of 20 mice/gender were given
high doses of i.v. amotosalen and its photoproducts (up to
250 times the anticipated clinical exposure in a 1-l FFP trans-
fusion) three times weekly for 6 months. The mice were then
euthanized and their tissues examined histologically.
Vein irritation
Because plasma is administered intravenously, information
on the vascular irritation potential of photochemically treated
100% plasma (without CAD treatment to have maximal
exposure to residual amotosalen and its free photoproducts)
was examined in a conventional model system in the rabbit.
Vein irritation was assessed by histopathological evaluation
of the vascular tissue.
Phototoxicity
Psoralens are known, in certain circumstances, to be capable
of eliciting dermal and ocular phototoxicity in the presence
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171–182
of UV light, including sunlight [27,37,38]. These responses
were studied in animals administered photochemically treated
100% plasma. The phototoxicity studies employed exagger-
ated conditions in that simulated sunlight exposure was
intense and was given at about the time of the peak plasma
level of the test compound; in addition, albino animals were
employed because they are more sensitive than pigmented
animals.
Results
Acute and repeated-dose toxicity
Several acute and repeated-dose studies were conducted in
rats using 100% homologous plasma. A summary of the studies
is presented in (Table 2). The results were consistent with,
and supplemented the data obtained using, photochemically
treated 35% plasma. Neither acute exposure nor prolonged
90-day daily dosing of photochemically treated 100% plasma
resulted in any treatment-related toxicity in the studies.
Safety pharmacology
Treatment with photochemically treated 100% plasma did
not result in any functional or gross pathological changes in
the renal system of the rat, which was consistent with, and

Table 2 Single- and repeated-dose toxicity studies
Test Species Solutions used
Single-dose Rat Photochemically treated
100% plasma with CAD
treatment
28-day Rat Photochemically treated
100% plasma with CAD
treatment
13-week Rat Photochemically treated
100% plasma, with and
without CAD treatment
CAD, compound adsorption device (used to remove residual amotosalen and free photoproducts).
supplemented the data obtained with, photochemically treated
35% plasma.
Administration of photochemically treated 100% plasma
did not result in any electrocardiographic or haemodynamic
effects on the cardiovascular system of the dog. These results
were consistent with, and supplemented the data obtained
with, photochemically treated 35% plasma.
Although no specific studies were conducted to evaluate
respiratory or gastrointestinal effects, no effects were seen in
acute rat toxicity studies following i.v. infusion of 80 ml/kg
of photochemically treated 100% plasma, with or without
CAD treatment.
Reproductive toxicity
Administration of photochemically treated 100% plasma did
not cause maternal or fetal developmental toxicity. These
results were consistent with, and supplemented the teratology
study conducted with, photochemically treated 35% plasma
[29,30]. Additionally, for the photochemically treated 35%
plasma studies, a complete series of reproductive toxicology
studies was conducted, which included male and female
fertility studies in rats, an additional teratology study in the
rabbit, and a peri- postnatal reproductive development study
in the rat, none of which resulted in any reproductive toxicity
as a result of administration of photochemically treated 35%
plasma administered at 25 ml/kg.
Genotoxicity testing
Photochemically treated human platelets processed as in clin-
ical use, but without CAD treatment, indicated no genotoxicity
in any in vitro or in vivo assay (Table 3). In these studies, the
residual amotosalen levels were far below the threshold con-
centrations for the positive effects observed with amotosalen
alone (≥ 5 µg/ml). To increase residual amotosalen and photo-
products in a platelet concentrate unit, amotosalen addition
and UVA illumination were repeated 25 times in a single
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171– 182
Preclinical safety profile 177
Dosing Findings
25 and 80 ml/kg Not toxic, all rats survived, and no treatment-
for 2 h related clinical or gross pathologic changes
were observed after 14 days
20 and 60 ml/kg Not toxic, all rats survived, and no treatment-
for 2 h daily related clinical, clinical, gross or
histopathologic changes were observed
40 ml/kg for No abnormal findings were observed at the
2 h daily end of dosing or in groups of rats after
a 1-month observation period
sample. These human platelets, multiply processed by photo-
chemical treatment without CAD treatment, were genotoxic in
both the Ames assay (Salmonella typhimurium strain TA1537;
in the absence of metabolic activation) and in the CHO cell assay.
These effects were attributed solely to the high concentration
of residual amotosalen and do not reflect clinical exposures.
Amotosalen alone was genotoxic in vitro in the Ames
assay in Sal. typhimurium strain TA1537, in the mouse lym-
phoma TK assay, and in the CHO cell assay for chromosome
aberrations. In the Ames assay, amotosalen was not genotoxic
in any other strain tested, including Sal. typhimurium TA98,
TA100 and TA1535 and E. coli WP2 uvrA (pKM101) and WP2
(pKM101). These are the expected results for an intercalator
in an in vitro assay (intercalation is the mechanism for amo-
tosalen’s specificity for targeting nucleic acids).
Metabolic activation with a hepatic S9 fraction reduced the
genotoxic potential of both amotosalen and the multiply
processed photochemically treated human platelets without
CAD treatment, in all in vitro genotoxicity studies. These
results are consistent with those obtained from in vivo
genotoxicity assays (the hepatic unscheduled DNA synthesis
assay in rats and the micronucleus assay in mice), in which
mammalian metabolic pathways were intact and functional,
and high doses of i.v. amotosalen were not genotoxic.
The ratios between the doses of amotosalen that were
genotoxic in a given assay and expected clinical peak plasma
levels (12 ng/ml) were very high in all the assays carried out
(Table 4). Among the in vitro assays, in which a positive result
was obtained, the lowest ratios were found in the CHO cell
studies (> 167-fold without metabolic activation and > 2000-
fold with metabolic activation). The in vivo assay results were
negative; the ratio of the highest non-genotoxic dose to the
expected clinical exposure level were ≥ 3000-fold.
Carcinogenicity
There was no evidence of an increased incidence of tumours in
any of the mice treated with amotosalen or photochemically
178 V. Ciaravino et al.

Table 3 Genotoxicity studies

Test Test system Solutions used Findings

Ames assay Salmonella typhimurium Photochemically treated human Not genotoxic at the highest dose tested:
and Escherichia coli a platelets without CAD treatment 2–2·5 µg/ml residual amotosalen and
(± S9) 8–10 µg/ml photoproducts

Ames assay Salmonella typhimurium
and Escherichia coli a
Photochemically treated human
platelets exposed to 25 cycles
of amotosalen addition and
illumination (± metabolic activation)
In the presence of metabolic activation;
not genotoxic at the highest dose tested:
62 µg/ml residual amotosalen
In the absence of metabolic activation; positive
in strain TA1537 at mean concentrations of
residual amotosalen of 35 µg/ml

Ames assay Salmonella typhimurium Psoralen amotosalen In the presence of metabolic activation;
and Escherichia coli a positive for strain TA1527 at 44 µg/ml

Chromosome aberration CHOb cells Photochemically treated human In the absence of metabolic activation;
assay platelets without CAD treatment positive at 103 µg/ml
(± metabolic activation) Not genotoxic at the highest dose tested; 2–
2·5 µg/ml residual amotosalen and 8–10 µg/ml
photoproducts

Chromosome aberration CHOb cells Psoralen amotosalen In the presence of metabolic activation;
assay positive at averag econcentrations of 46 µg/ml
In the absence of metabolic activation;
positive at ≥ 5 µg/ml

Chromosome aberration CHOb cells Photochemically treated human In the presence of metabolic activation;
assay platelets exposed to 25 cycles of positive at > 15 µg/ml residual amotosalen
amotosalen addition and In the absence of metabolic activation;
illumination (± metabolic activation) positive at 7·5 µg/ml residual amotosalen

Mouse lymphoma assay L1578Y/TK+/– cells Photochemically treated human Not genotoxic at the highest dose tested;
platelets without CAD treatment 2–2·5 µg/ml residual amotosalen and
(± metabolic activation) 8–10 µg/ml photoproducts

Mouse lymphoma assay L1578Y/TK+/– cells Psoralen amotosalen In the presence of metabolic activation;
positive at > 7·5 µg/ml
In the absence of metabolic activation;
negative at > 65 µg/ml

In vivo UDSc assay Rat Photochemically treated human Not genotoxic at 200 µg/kg residual amotosalen
platelets without CAD treatment

In vivo UDSc assay Rat Psoralen amotosalen Not genotoxic at doses as high as 34 mg/kg

In vivo micronucleus assay Mouse Photochemically treated human Not genotoxic at 200 µg/kg residual amotosalen
platelets without CAD treatment

In vivo micronucleus assay Mouse Psoralen amotosalen Not genotoxic at doses as high as 66 mg/kg

a
Salmonella strains TA98, TA100, TA1535 and TA1537; Escherichia coli strains WP2 uvrA [pKM101] and WP2 [pKM101].
b
Chinese hamster ovary cells.
c
Unscheduled DNA synthesis.
CAD, compound adsorption device (used to remove residual amotosalen and free photoproducts).

treated 35% plasma [31]. These results, like those of the

Venous irritation
genotoxicity studies, support the conclusion that plasma
prepared using the INTERCEPT Blood System does not In a specific study designed to assess vascular irritation
present a carcinogenic risk to patients. potential, photochemically treated 100% plasma, without

© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171–182

 Preclinical safety profile 179

Table 4 Relationship of clinical exposure to exposures in genotoxicity

Phototoxicity
studies
Two studies were conducted to evaluate the phototoxic
Multiples of potential of photochemically treated 100% plasma: a single-
Study type clinical exposurea
dose study and a 1-month study. In the single-dose study,
male and female rats were administered 2-h i.v. infusions
Photochemically treated 35% plasma
of 20 or 80 ml/kg/dose of photochemically treated 100%
Ames
Non-activated/activated (all strains) > 208b
plasma. The control group was given 2-h infusions of 80 ml/
Ames (multidose illuminate) kg/dose of 100% plasma that had not received photochemical
Non-activated (TA 1537) 2000 treatment. At ≈ 15 min after dosing (control) or at ≈ 15 min,
Non-activated (all other strains) > 5167b 6 h or 24 h after dosing, a depilated, dorsal skin site and both
Activated (all strains) > 5167b eyes were exposed to an estimated 0·5 minimal erythema
Mouse lymphoma dose (MED) of ultraviolet radiation (UVR) provided by a
Non-activated/activated > 167b xenon-arc solar simulator. No evidence of dermal or ocular
CHO cell phototoxicity occurred in the study.
Non-activated/activated > 167b
In the 1-month study, up to 50 ml/kg photochemically
CHO Cell (multidose illuminate)
treated 100% plasma, administered over 2 h, was given three
Non-activated 250
times/week for 4 weeks to male and female rats. The rats were
Activated > 1083c
Rat hepatocyte UDS (i.v. bolus) > 50d
exposed to simulated sunlight following the third dose each
Mouse micronucleus (i.v. bolus) > 50d week. Additionally, groups of rats were administered photo-
Amotosalen chemically treated 100% plasma that was partially CAD treated,
Ames such that residual amotosalen levels were as high as 10 µM,
Non-activated (TA1537) 1417 10 times higher than the clinical concentration of ≈ 1 µM in
Activated (TA1537) 3750 a plasma unit. No evidence of dermal or ocular phototoxicity
Non-activated/activated > 595 250 occurred in the study. In both studies, evidence of dermal photo-
(all other strains) toxicity was present in the positive control, 8-methoxypsoralen
Mouse lymphoma
(8-MOP)-treated females and males (10 µg/kg). 8-MOP is cur-
Non-activated 417
rently used for therapy of chronic refractory psoriasis and
Activated > 5417b
cutaneous T-cell lymphoma.
CHO cell
Non-activated 167
Activated 2000 Neoantigenicity
Rat hepatocyte UDS (i.v. bolus) > 3000b
Mouse micronucleus (i.v. bolus) > 3000b The photochemical treatment of plasma leads to the formation
of covalent adducts with plasma macromolecules. Because
a
Ratio of highest non-genotoxic amotosalen dose level (in vitro studies) or the adducts are primarily associated with lipids instead of
peak amotosalen plasma level (in vivo studies) to the clinical peak plasma proteins, their neoantigenic potential is considered low.
level of amotosalen (12 ng/ml) after transfusion of 1-l of fresh-frozen
Nevertheless, the neoantigenic potential of photochemically
plasma (FFP).
b treated plasma has been extensively studied in preclinical
Negative at the highest doses tested in all assays.
c and clinical studies. Toxicology studies (especially the 1-
Negative at the highest dose tested in one or more assays.
d
Negative at the highest feasible intravenous (i.v.) dose level. and 3-month toxicity studies) also provided information
relevant to neoantigenicity. In these toxicity studies, there
was no clinical evidence of sensitization resulting in his-
CAD treatment, was non-irritating to the ear vein of the tamine release. Histopathologically, there was no evidence
rabbit when administered intravenously by infusion for 2 h in any study of lymphocyte proliferation, immune-tissue
at a dose volume of 20 ml/kg. stimulation or immune-complex deposition. There was also
In a 13-week toxicity study in rats, there was no evidence no evidence of any immunological response to photochemi-
of infusion-site venous irritation when photochemically cally treated plasma or coagulation factors, as activated
treated 100% plasma, with or without CAD treatment, was partial thromboplastin time (aPPT) and prothrombin time
administered intravenously via the tail vein at 40 ml/kg for (PT) values were unaffected in animals that were dosed
2 h daily for 13 weeks. daily for up to 3 months with photochemically treated
In these single- and repeat-dose studies, the daily infusion 100% plasma. These data further support the absence of a
volume in all species was up to a 3·5-fold multiple of the neoantigenic response to photochemically treated 100%
clinical infusion volume of 1 l (≈ 17 ml/kg body weight). plasma.

© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171– 182
180 V. Ciaravino et al.
Fig. 3 Safety margins in toxicology studies obtained with the INTERCEPT Blood System for plasma, presented as multiples of the clinical exposure of amotosalen
(4 µg/kg).
Discussion and conclusions
The toxicology studies conducted with photochemically
treated 100% and 35% plasma used the same amotosalen con-
centration (150 µM) and UVA exposure (3 J/cm2). Analytical
characterization high pressure liquid chromatography (HPLC)
and liquid chromatography/mass spectroscopy/mass spectro-
scopy (LC/MS/MS) of 100% and 35% plasma after amotosalen
photochemical treatment indicated that the same residual com-
pounds (amotosalen and free photoproducts) were present in
both preparations, and no materials were characterized in
one preparation but not in the other. Toxicology studies with
photochemically treated 35% plasma are therefore applicable
to the preclinical safety evaluation of photochemically treated
100% plasma. To supplement the studies carried out with 35%
plasma, the following toxicology studies were conducted
with photochemically treated 100% plasma: acute systemic
toxicity; 1- and 3-month toxicity; safety pharmacology
(a cardiovascular study in dogs and a renal study in rats);
developmental toxicity; vein irritation; and single-dose
and 4-week multiple-dose phototoxicity studies. Toxicology
studies completed with photochemically treated 100% plasma,
as well as photochemically treated 35% plasma and amoto-
salen alone, are presented in Table 1.
The potential toxicity of amotosalen and photochemically
treated 35% plasma was extensively studied in > 80 studies,
using the same approach as a new pharmaceutical study and
according to ICH guidelines [31,32]. In toxicology studies con-
ducted with illuminated 100% plasma, the levels of amoto-
salen administered were well above the exposure a patient
would receive in clinical applications (≈ 4 µg/kg of residual
amotosalen in a 1-l FFP transfusion based on a body weight
of 60 kg). Pharmacokinetic data from toxicology studies
© 2003 Blackwell Publishing Ltd. Vox Sanguinis (2003) 85, 171–182
showed a half-life of amotosalen ranging from 6 to 9·1 h
(from studies conducted with 100% and 35% plasma) com-
pared to 8·9 h in humans (determined in a phase 1 clinical
trial). Safety margins in the toxicology studies are shown in
Fig. 3 and are represented by the types of studies conducted
and the multiples of clinical exposure in these studies. No
specific target-organ toxicity (clinical pathology or patho-
logy) occurred in any study conducted with 100% or 35%
plasma. The principal findings for amotosalen alone were
CNS observations (used at concentrations 6250-fold than the
estimated clinical exposure of 4 µg/kg), electrocardiographic
observations (10 000-fold), and phototoxicity (250-fold). With
respect to mutagenicity, the ratios of the highest non-mutagenic
dose levels to clinical exposures were 167- and 2000-fold
higher for the in vitro studies, without and with metabolic
activation, respectively. For in vivo assays (which were all
negative), the ratios were at least 3000-fold. Based on the ext-
remely large safety margins (up to 10 000 times the estimated
clinical exposure to amotosalen), the CNS and electrocardio-
graphic observations are not considered to be of any physio-
logical relevance for the proposed use of the CAD-treated,
photochemically treated 100% plasma. Based on the absence
of any phototoxicity with photochemically treated 100%
plasma, and the large safety margin for amotosalen alone
(1000-fold), photochemically treated 100% plasma is not
considered to be phototoxic for its proposed use. Based on
the mechanism for in vitro mutagenicity, the extremely large
ratios for in vitro mutagenicity and the absence of any muta-
genicity for in vivo assays, the in vitro mutagenicity findings
are not considered to be of any toxicological significance for
the proposed use of photochemically treated 100% plasma.
Furthermore, the possible concern regarding genotoxicity
relates to the potential for a carcinogenic risk. No evidence

of carcinogenicity occurred in a p53 carcinogenicity assay,
which is a highly sensitive model for genotoxic carcinogens.
In that study, p53 transgenic mice were dosed intravenously
for 6 months with very high doses of amotosalen and its
photoproducts, the highest dose being 250-fold the clinical
amotosalen exposure.
The results reviewed in this article show that treatment
with the combination of the psoralen, amotosalen, plus UVA
light, in the INTERCEPT Blood System for plasma, yields
plasma preparations with a very low potential for toxicity in
patients. Findings from previous studies have demonstrated
the ability of the INTERCEPT Blood System for plasma to
inactivate a wide range of viruses, bacteria and protozoa, as
well as leucocytes, in platelet concentrates [13,14], without
compromising haemostatic efficacy [15–22] thus potentially
increasing transfusion safety. The results of the preclinical
safety studies indicate that there are no toxicologically relevant
effects associated with plasma prepared using this new system.
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