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Laboratory Animals
2015, Vol. 49(S1) 21–29
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DOI: 10.1177/0023677215573040
Thioacetamide model in mice and rats la.sagepub.com

MC Wallace1, K Hamesch2, M Lunova2, Y Kim5, R Weiskirchen3,

P Strnad2,4 and SL Friedman1

Abstract
In addition to carbon tetrachloride (CCl4), thioacetamide (TAA) represents a second widely used model for the
induction of experimental liver fibrosis, but can also be employed for the development of acute liver failure
and liver tumours. While TAA itself is not hepatotoxic, its reactive metabolites covalently bind to proteins and
lipids thereby causing oxidative stress and centrilobular necrosis. Compared with CCl4, TAA leads to more
periportal infiltrates and more pronounced ductal proliferation. While TAA has been shown to induce liver
fibrosis development in several different mouse strains, wide variations in the administration routes, doses
and treatment durations have been reported. Therefore, an adoption of a universal standard operating pro-
cedure for the administration of TAA is urgently needed. For that purpose, we are presenting here two TAA
models (intraperitoneal administration of 150 mg/kg of TAA three times per week for 11 weeks in rats, and
TAA administration in drinking water at 300 mg/L for 2–4 months in mice) with which we have had success in
reliably and reproducibly developing chronic liver injury and fibrosis.

Keywords
hepatotoxin, CYP2E1, liver fibrosis, cirrhosis, animal models

Historical background and current usage

The organosulfur compound thioacetamide (TAA;
molecular formula C2H5NS), that was formerly used
as a fungicide and was occasionally found as a contam-
inant in orange juice,1 was first identified as being hep-
atotoxic to rats in 1948. After two years of chronic
exposure in drinking water, experimental animals
developed advanced fibrosis, cirrhosis and liver
tumours in a dose-dependent fashion.2 It was not
until almost 40 years later that chronic TAA intoxica-
tion was established as a reliable and reproducible
experimental model of fibrosis and cirrhosis in rodents
by either the oral or intraperitoneal (IP) routes.3–5 In
addition, acute exposure to TAA at higher doses is rec-
ommended by the International Society for Hepatic
Encephalopathy and Nitrogen Metabolism (ISHEN)
as a well-characterized model of acute liver failure,
encephalopathy and other events (Table 1) but these
will not be discussed in detail here.6,7 In a cirrhosis
imaging study, chronic oral TAA exposure in rats has
been shown to produce cirrhosis more reliably than
subcutaneous carbon tetrachloride (CCl4); however,
this route of administration of CCl4 is uncommon
and thus the results must be interpreted with caution.8
A variable, weight-based oral regimen for TAA
1
Division of Liver Diseases, Icahn School of Medicine at Mount
Sinai, New York, New York, USA
2
Department of Internal Medicine III, RWTH University Hospital
Aachen, Aachen, Germany
3
Institute of Molecular Pathobiochemistry, Experimental Gene
Therapy and Clinical Chemistry, RWTH University Hospital
Aachen, Aachen, Germany
4
Interdisciplinary Center for Clinical Research (IZKF), RWTH
University Aachen, Aachen, Germany
5
Kyung Hee Medical Center, Seoul, Korea
Corresponding author:
Scott L Friedman, MD, Division of Liver Diseases, Box 1123, Mount
Sinai School of Medicine, 1425 Madison Ave, Room 11-70C,
New York, NY 10029, USA.
Email: scott.friedman@mssm.edu
22 Laboratory Animals 49(S1)

Table 1. Usage of thioacetamide-induced liver damage in rodents.

Disease model Application Advantages Disadvantages References

Liver fibrosis IP commonly used Most closely resembles Long latency of fibrogenic 18, 30, 33, 35, 36
IP: 100–200 mg/kg alcoholic fibrogenesis. effects.
body weight, Reproducible results, Heterogenous results due
PO: 200 mg/L drink- low mortality, to missing
ing water less toxic than other standardization
chemicals
Acute hepatic IP, single dose 200 to Produces hepatic enceph- Monitoring and supportive 7, 37, 38
failure 1200 mg/kg body alopathy, care due to hypother-
weight large time windows mia, hypglycaemia and
before death others
Hepatocellular car- IP commonly used, Reproducible results Better models available 39, 40
cinoma or cholan- IP: 100–200 mg/kg (e.g. diethyl-
gio-carcinoma body weight, nitrosamine)
PO: 200 mg/L drink
ing water
Iron overload IP: 100 mg/kg body First non-transgenic 41
weight model of chronic liver-
injury-related iron
overload
Overview of frequently used rodent disease models employing thioacetamide application. IP ¼ intraperitoneal; PO ¼ per oral (drinking water).

administration in rats has been developed9 and
refined10 and has been proposed to produce cirrhosis
and portal hypertension more reliably and with less
toxicity than a non-variable dosing regimen. Less com-
monly, TAA may be used in conjunction with another
hepatocarcinogen such as N-diethylnitrosamine (DEN)
in a two-stage model to induce hepatocellular carcin-
oma (HCC).11 Another variation is the combination of
TAA with alcohol to produce stronger fibrosis.12
The use of chronic TAA varies widely, particularly in
the route of administration; dosage, timing and length of
treatment; and animal used (Table 2). In the first half of
2014, there have been 18 papers published in peer-
reviewed journals that utilized chronic TAA to produce
fibrosis or cirrhosis experimentally (references available
upon request). Thirteen studies used rats of various gen-
etic backgrounds (Sprague–Dawley, Wistar, F344, and
albino) and five used mice of either C57BL/6 or
Kunming backgrounds. The majority delivered TAA
by the IP route (12); the remainder (5) used the oral
route, three of which used a fixed dose (300 mg/L in
drinking water), the others (2) a variable, weight-based
dose. One paper did not report the route of administra-
tion. The oral and IP routes were used in both mice and
rats. Dosages for the IP route ranged from 10 mg/kg to
400 mg/kg, two to three times per week from between 5
and 20 weeks while recently most researches have used
dosages of between 100 mg/kg and 200 mg/kg, three
times per week for at least six weeks. Most used TAA
as the only model of fibrosis and cirrhosis (12); the
remainder (6) used TAA in conjunction with other
models such as CCl4 or bile duct ligation, for example.
Unfortunately, neither the proportion of experimental
animals which developed advanced fibrosis or cirrhosis,
nor the mortality rates were reported in these studies. It
is therefore difficult to make recommendations regard-
ing the most appropriate regimen based on recently
published studies. Local expertise and personal experi-
ence with the model is likely to be an important factor
when planning an experiment with TAA. Here we pre-
sent our experience with the TAA model, which
includes chronic oral exposure in C57BL/6 mice at a
fixed dose (300 mg/L in drinking water) for 2–4
months,13 IP injection of TAA in Sprague–Dawley
rats for 11 weeks with or without an experimental
drug,14 and a time-course pilot study of TAA by IP
injection in Sprague–Dawley rats for 12 weeks
(Figure 1). We report the rate and degree of fibrosis
and cirrhosis development, changes in portal pressures
and liver collagen content, as well as the mortality rate
in the experimental animals.
Mechanisms and pattern of liver injury
Despite the hepatotoxic properties of TAA being well
known since 1948, the mechanisms by which it causes
liver injury are complex and remain poorly understood.
TAA requires oxidative bioactivation of its toxic
metabolites to cause liver injury that affects both hep-
atocytes and cholangiocytes.15,16 With regard to the
Wallace et al. 23

Table 2. Representative examples of thioacetamide usage in mouse models of experimental liver fibrosis.

Application Mice References

IP: 100 mg/kg body weight C57BL/6 J and FVB/N background, 18, 41
3/wk, 8 wk old, mainly female
6 wk course
IP: 150 mg/kg body weight C57BL/6 J background, 36, 42
3/wk, 6–8 wk old, male
6 wk course
IP: 200 mg/kg body weight C57BL/6 background, 35
3/wk, 6 wk old, male
4–8 wk course
IP: 200 mg/kg body weight BALB/c background, 32
3/wk, 8–10 wk old, male
4 wk course
PO: 200 mg/L drinking water C57BL/6 J background, 33
18 wk course 6 wk old, unknown gender
PO: 200 mg/L drinking water C3H/He background, 30
6–16 wk course 10 wk old, unknown gender
The main application routes for thioacetamide treatment are intraperitoneal injection or administration in drinking water.
IP ¼ intraperitoneal; PO ¼ per oral (drinking water), wk ¼ week.

latter, an enlargement of portal tracts without signs of
cholestasis is frequently observed.17–19 TAA is first oxi-
dized to TAA-S-oxide and then to TAA-S,S-oxide
(Figure 2); this process can be carried out by hepatic
cytochrome P450 enzymes or FAD-containing mono-
oxygenases.20 The lethal dose (LD50) in rats and mice
after both oral and IP administration was determined
to be approximately 300 mg/kg body weight.21 Even at
high doses, TAA is not toxic to cultured hepatocytes
while TAA-S-oxide and TAA-S,S-oxide cause toxicity
as demonstrated by increases in cytosolic lactate
dehydrogenase (LDH) release.20 The oxidation of TAA
appears to be mediated primarily through CYP2E1,
although under varying physiological conditions, the
FAD-containing monooxygenases may play a more
important role.20,22,23 When Cyp2e1-null mice were
acutely exposed to TAA, they were protected from liver
injury, elevation in markers of inflammation, and oxida-
tive stress. By contrast, wild-type mice showed markedly
increased mRNA levels of tumour necrosis factor
(TNF)-a, interleukin (IL)-6 and glutathione peroxidase
plus developed centrilobular necrosis and inflamma-
tion.22 In addition to causing oxidative stress, glutathi-
one depletion and thus cellular injury, the reactive
metabolites of TAA covalently bind to cellular proteins
and lipids. Moreover, this results in alterations in cell
cycle-related proteins, protein folding, enzyme inhibition
and mitochondrial/chaperone function, all of which
leads to significant cytotoxicity.24–26
The administration of TAA causes a predictable and
reliable dose- and time-dependent pattern of liver injury
that resembles human fibrosis and cirrhosis.10,27 With an
increased length of intoxication, rodent liver microscop-
ically demonstrates more clearly a mixed inflammatory
infiltrate of the portal tract with bile duct proliferation
and some hepatocyte necrosis, than a typical pattern of
fibrosis with portal–portal and portal–central septa pro-
gressing to eventual features of macronodular cirrhosis
with bridging fibrosis and regenerative nodules.
Although both CCl4 and TAA result in centrilobular
liver damage, several differences between the models
should be noted.18,28 TAA displays a longer latency
between the exposure to the drug and the development
of liver injury.3,29 TAA also leads to more periportal
infiltrates and more pronounced ductal proliferation,
appearance of acidophilic bodies, and vacuolated
nuclei with prominent nucleoli, as well as more pro-
nounced hepatocyte vacuolization.18,19 Bile duct ligation
and repeated application of CCl4 have traditionally been
employed as the most widely used experimental model of
fibrosis. Common to all these three models is the devel-
opment of hepatic fibrosis that can be most easily visua-
lized with Sirius Red staining (Figure 3).
With prolonged treatment using TAA (18 weeks and
longer), cellular atypia may become evident and mitotic
figures may be observed.10 As demonstrated in the
original TAA publication,2 chronic intoxication fre-
quently results in malignancy, although TAA is cur-
rently rarely used in isolation as a model of HCC or
cholangiocarcinoma. In an oral, weight-based TAA
model, rats developed portal hypertension and
splenomegaly after 12 weeks of exposure, indicating a
strong functional relationship to human cirrhosis.10
Additionally, and perhaps most importantly, rodent
24 Laboratory Animals 49(S1)

Portal Pressure
(a) AST (c)
20 Control
1250
TAA
Control
1000 15
TAA

mm H2O
750
IU/L 10
500
5
250

0 0
2 4 6 8 10 12 2 4 6 8 10 12
Week Week

(d) Collagen Content

ALT
20 Control
750
TAA
Control
15
TAA

% Collagen
500
IU/L

10

250
5

0 0
2 4 6 8 10 12 2 4 6 8 10 12
Week Week

(b)
mRNA
Fold change relative to GAPDH

10.0

Col1A1
7.5
Asma
Pdgrfb
5.0 Mmp2
Timp2
2.5

0.0
0 2 4 6 8 10 12
Week

Figure 1. Time course experiment of thioacetamide (TAA) by intraperitoneal (IP) injection and phosphate-buffered saline
(PBS) controls in rats. (a) Aspartate aminotransferase (AST) (upper panel) and alanine aminotransferase (ALT) (lower
panel) levels spiked initially and then returned to normal at week 4. (b) Several fibrosis related genes demonstrated
consistent elevation throughout the course of the experiment. (c, d) Portal pressures (c) and collagen content in liver
tissue (d) plateaued at week 8. Col1A1: collagen type 1a1; Asma: a-smooth muscle actin; Pdgrfb: Platelet-derived growth
factor receptor type b; Mmp2: matrix metalloproteinase-2; Timp2: tissue inhibitor of matrix metalloproteinases 2; GAPDH:
glyceraldehyde phosphate dehydrogenase.

hepatic stellate cell activation in TAA intoxication is [PBS]-injected animal) was sacrificed every 12 weeks
central to the observed fibrosis and cirrhosis and assessment was performed of various fibrosis asso-
development.30 ciated genes by quantitative real-time reverse transcrip-
To validate the model, we performed a time-course tion-polymerase chain reaction (qRT-PCR), portal
experiment in which Sprague–Dawley rats were pressures by portal vein cannulation, biochemical ana-
exposed to TAA by IP injection (150 mg/kg, three lysis of liver injury by serum alanine aminotransferase
times/week) for 12 weeks. One group (2 experimental (ALT) and aspartate aminotransferase (AST) measure-
animals and one control phosphate-buffered saline ments, and quantification of collagen content by the
Wallace et al. 25

Figure 2. Simplified model for the formation of noxious thioacetamide (TAA) metabolites and their effects in hepatocytes.
In a first biotransformation step, TAA is reversibly metabolized to TAASO. In a second oxidation step, the highly reactive
species TAASO2 is formed. Subsequently, TAASO2 can directly react with amine groups (R-NH2) on cellular proteins or
lipids resulting in dysfunction. In addition, TAA gives rise to glutathione depletion and elevated levels of reactive oxygen
species (ROS), lipid peroxides, cytotoxicity and mitochondrial injury. Together the events may lead to apoptosis, necrosis
and the formation of hepatocellular carcinoma and cholangiocarcinoma. Details of the sequence of chemical reactions
induced by TAA can be found elsewhere.20,34

Figure 3. Histological characterization of the most widely used experimental liver fibrosis models. To visualize the most
widely used experimental models, liver sections were prepared from untreated animals, mice subjected to bile duct
ligation (BDL) for three weeks or administered carbon tetrachloride (CCl4) (2/week, 0.2 mL/kg for 12 weeks) and
thioacetamide (TAA) (3/week 100 mg/kg for 15 weeks), respectively. Haematoxylin and eosin (H&E) staining was
employed for the overall histological evaluation whereas Sirius Red staining was carried out to evaluate the extent of liver
fibrosis. Scale bar: 100 mm (top), 200 mm (bottom).
26
BIO-QUANT Life Science morphometry systemÕ
(BIOQUANT Image Analysis Corp, Nashville, TN,
USA). The results are presented in Figure 1. After an
initial spike in inflammation and liver injury at two
weeks as assessed by serum ALT and AST, these
values returned to normal at week 4. Sustained
mRNA elevation of fibrosis associated genes including
collagen 1A1 (Col1A1), a-smooth muscle actin (Asma),
platelet-derived growth factor receptor b (Pdgfrb),
matrix metalloproteinase 2 (Mmp2) and tissue inhibitor
of metalloproteinase 2 (Timp2) were demonstrated.
Both portal pressures and collagen content of liver
tissue reached maximal levels in TAA rats at week 8
and then plateaued. These results reinforce the efficacy
of the TAA IP injection strategy for the production of
significant fibrosis between 8 and 12 weeks.
General considerations
. Safety concerns: Based on the high toxicity of TAA,
personal protective equipment including eyeglasses,
protective gloves, appropriate clothing and respira-
tory mask are necessary whenever handling this hep-
atotoxin. To avoid chemical decomposition, TAA
should be stored in a cool, dry and safe place to
which only authorized staff have access (e.g. poison
cabinet).
. Genetic background: While the susceptibility to liver
fibrosis development in different experimental
models has been shown to display significant strain
differences,31 no systematic analyses on strain-speci-
fic susceptibility to TAA have been reported. In fact,
the successful induction of TAA-mediated liver
fibrosis has been described in a variety of genetic
backgrounds of both mice and rats.18,32
. Route, dosage, timing and duration of treatment: As
discussed above in ‘Historic background and current
usage’, there is wide variation in currently used TAA
models and it is beyond the scope of this standard
operating procedure (SOP) to discuss the differences.
As a guide to planning a TAA experiment, we pre-
sent here two models with which we have both per-
sonal experience and confidence in their safety and
efficacy for fibrosis and cirrhosis induction.
. Experimental uses and design: A typical TAA experi-
ment involves the comparison of the rate or degree
of fibrosis or cirrhosis development between a con-
trol group and a treatment or intervention group. In
this era of rapidly increasing interest in anti-fibrotic
therapies, it is a convenient and reliable tool for the
relatively rapid in vivo screening of potential thera-
pies, as it allows for the generation of results within
4–5 months. The timing of the introduction of the
experimental therapies can be modified to assess
Laboratory Animals 49(S1)
their effectiveness in modulating different phases of
fibrosis. The TAA model may also be used to study
functional consequences of cirrhosis such as portal
hypertension, and as previously mentioned, acute
TAA exposure in high doses can be used to study
acute liver failure and encephalopathy. Finally, TAA
administration may also be used to study the biology
of fibrosis, as it provides a reliable stimulus for stel-
late cell activation and fibrosis development. Genetic
knockdown and overexpression techniques are best
utilized in this way. The remainder of this SOP will
focus on the use of TAA to produce fibrosis and
cirrhosis.
. Analysis of treatment and control cohorts: Selection of
readouts will depend on the aims of the project. In a
study in which the degree of fibrosis development is
the major outcome being assessed, a thorough ana-
lysis of liver histology plus a fibrosis quantification
technique should be used. Techniques used will
depend upon local expertise, but important consider-
ations are the use of an experienced liver histopath-
ologist to assess random sections in a blinded fashion.
We suggest formalin-fixed, paraffin embedded liver
sections should be stained with haematoxylin and
eosin for histological assessment of fibrosis/cirrhosis
grade plus assessment of any ductular reaction, cellu-
lar atypia or steatosis, for example. For fibrosis quan-
tification, four sections should be stained with Sirius
Red with nine random images taken per slide for a
total of 36 images that can be analysed by the
BIO-QUANT Life Science morphometry systemÕ .
Alternatively, hydroxyproline quantification can be
performed. Col1A1 mRNA can be assessed by qRT-
PCR and protein by Western blot. Portal pressures
can be assessed at the time of sacrifice by inserting a
16 G angiocatheter into the portal vein and measur-
ing the height of a water column.
. Biometric assessment In a typical experiment an
experimental drug is concomitantly administered
along with TAA. A possible readout system to
assess the efficacy of the drug is the mRNA expres-
sion of Col1A1 which may change at the end of the
experiment by a factor of 8 (with an inter-individual
variance of 25%) (cf. Figure 1d). The control group
will receive only TAA. It can be estimated that the
drug may reduce the expression of collagen by a
factor of 2. In addition, experience in our labora-
tories has shown that a difference of less than 30%
in collagen mRNA expression does not necessarily
impact intrahepatic collagen deposition and scar-
ring. For such an experiment, in our view it is advis-
able to use nine animals per group. If the mortality
rate of TAA when applied for eight weeks is further
assumed to be approximately 10%, this experiment
should be planned with 10 animals in each group.
Wallace et al.
For details on how to calculate biometric param-
eters, please refer to Scholten et al.’s article ‘The
carbon tetrachloride model in mice’ published in
this special issue of Laboratory Animals.
Practical implementation
We provide here two TAA protocols, by IP injection in
Sprague–Dawley rats and oral administration in
C57BL/6 mice. We have found these approaches to
produce reliable and reproducible fibrosis and cirrhosis
that allows for an easy distinction between control and
treatment groups.
TAA via IP injection
Our experience with IP injection of rats with TAA was
recently published14 and is summarized below as a
standard approach for reference:
Animals: Sprague–Dawley rats between 280–300 g pur-
chased from The Jackson Laboratory (Bar Harbor,
ME, USA) are kept in a 12 h light–dark cycle with
free access to water. It should be noted that recent
guidelines from the US National Institutes of Health
(NIH) suggest equal numbers of male and female ani-
mals be used in all future experiments.
TAA preparation and administration: TAA purchased
from Sigma Chemical Co (St Louis, MO, USA) is
dissolved in 0.9% normal saline to a total volume of
1–1.5 mL per rat approximately one hour before injec-
tion. Treatment groups receive 150 mg/kg of TAA
three times per week for a total of 11 weeks while
control groups receive the same volume of 0.9%
normal saline.
General handling: Rats are examined regularly for
signs of distress or ill health and weighed prior to each
injection to accurately calculate the dosage of TAA.
Injection technique: Rats are handled at all times with
care, as the experience can be distressing. We recom-
mend that lab personnel with specific training in animal
handling be responsible for the IP injections. Rats are
scuffed behind the neck between the middle and index
fingers with the tail hooked between the little and ring
fingers to expose the abdomen. A sterile, disposable
syringe (25–27 G) is used to inject the TAA into the
lower side of the abdomen with care taken to avoid
injection into the bladder or abdominal organs. The
solution should be at room temperature at time of
injection. The routine administration of anaesthesia is
generally not recommended as it results in unnecessary
stress to the animals.
Animal sacrifice: At the end of the study period animals
are placed under inhalation anaesthesia with 1–5% by
27
volume isoflurane plus analgesia and laparotomy per-
formed. Portal pressures can be measured as described
prior to the removal of organs.
Side-effects: TAA administration can lead to cirrhosis
and its associated complications; however, these are
rarely the source of any discomfort to the animals.
Mild body weight loss is likely to be observed.
Mortality of up to 15% can be expected in the treat-
ment groups. This should be reported in the Methods
section of any publications.
Efficacy of treatment: After 11 weeks of treatment at the
dose of 150 mg/kg three times per week, virtually all of
the animals will have developed the histological fea-
tures of cirrhosis.
TAA via oral administration
Our experience with orally administered TAA was
recently published13 and is summarized below as a
standard for reference:
1. Animals: 8–10-week-old C57BL/6 mice purchased
from The Jackson Laboratory are kept in a 12 h
light–dark cycle with free access to water. It should
be noted that recent guidelines from the US NIH
suggest equal numbers of male and female animals be
used in all future experiments. Of note, male
mice have been reported to be more susceptible to
development of TAA-induced liver fibrosis,33 which
is in line with similar findings in other fibrosis models.
2. TAA preparation and administration: TAA pur-
chased from Sigma Chemical Co (St Louis, MO,
USA) is dissolved in drinking water at 300 mg/L
and administered for 2–4 months. Control animals
are given normal water with the same volume. In
order to reduce the risk of infection, drinking
water should be filtered and acidified (pH around
4.0). The water volume should be measured on a
daily basis and changed twice weekly.
3. General handling: Mice are examined daily for signs
of distress or ill health. The body weight is deter-
mined twice a week starting at the beginning of the
experiment.
4. Animal sacrifice: At the end of the study period ani-
mals are placed under inhalation anaesthesia with
1–5% by volume isoflurane plus analgesia and lapar-
otomy is performed. Portal pressures can be measured
as described prior to removal of organs.
5. Side-effects: TAA administration can lead to cirrho-
sis and its associated complications; however, these
are rarely the source of any discomfort to the ani-
mals. Mild body weight loss is likely to be observed.
Low mortality can be expected in the treatment
groups. This should be reported in the Methods
28
section of any publications. In general, oral adminis-
tration of TAA leads to less extrahepatic toxicity than
IP administration due to the first-pass effect. In con-
trast to the intraperitoneal treatment, mice are not
known to experience pain and a more continuous
dosing is achieved. Because of these advantages, we
prefer oral administration to IP dosage, although the
latter has been used more frequently in the literature.
6. Efficacy of treatment: There is a progressive develop-
ment of fibrosis and cirrhosis development across the
study period. At the end of four months virtually all
of the treatment animals will have developed the
histological features of cirrhosis.
Classification of severity of procedure
According to Article 15 of the EU Directive 2010/63
(http://eur-lex.europa.eu/LexUriServ/
LexUriServ.do?uri¼OJ:L:2010:276:0033:0079:en:PDF)
the estimated degree of pain, suffering, distress or last-
ing harm to the animals subjected to TAA application
should be classified according to a scoring system that
consists of ‘non-recovery’, ‘mild’, ‘moderate’ or
‘severe’. The classification criteria that underpin this
assessment have been established by the Expert
Working Group on severity classification of scientific
procedures performed on animals. Further details can
be found at: http://ec.europa.eu/environment/chem-
icals/lab_animals/pdf/report_ewg.pdf.
The severity of the procedure depends on the TAA
dose and duration of the experiment. TAA application
to induce fibrosis with no major impairment of liver
function is classified as a moderate procedure accord-
ing to Article 15 of the aforementioned EU Directive
2010/63. The application of TAA to induce liver failure
with associated complications such as cerebral oedema
and encephalopathy, coagulopathy, renal, haemo-
dynamic and cardiorespiratory failure is classified as
a severe procedure. According to the 3 R principle,
the procedure should undergo a refinement, and
humane endpoints must be implemented with frequent
observation points to restrict pain, suffering, distress or
lasting harm to the animals, at which point the proced-
ure can be reclassified as moderate.
Concluding remarks
The administration of TAA via the oral or IP route is a
safe and effective technique for the study of fibrosis in
rodents. There is a wide variation in the route, dose,
timing and animal used. The scientific community
would benefit from the adoption of a universal SOP
for the administration of TAA with the intent of study-
ing fibrosis and its associated complications. Here we
Laboratory Animals 49(S1)
have presented two TAA models with which we have
had success in reliably and reproducibly induce chronic
liver injury and fibrosis in rodents.
Ethical statement
All animal experiments were performed in accordance with
the United States Animal Welfare Act and the Guide for the
Care and Use of Laboratory Animals of the National
Institutes of Health. The Institutional Animal Care and Use
Committee (IACUC) of the Icahn School of Medicine at
Mount Sinai approved all the animal experiments performed.
Funding
Work in Dr. Friedman’s laboratory is supported by NIH
Grants RO1DK56621 and RO1AA020709.
Conflict of interest statement
The authors have no conflicts of interest to declare.
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